Rabinowitch (1951) PhotosynthVol II Part1

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PHOTOSYNTHESIS
AND RELATED PROCESSES
VOLUME II
Part 1
J/. rO
PHOTOSYNTHESIS
and Related Processes
^:(
By EUGENE I. RABINO WITCH

Research Professor, Photosynthesis Research Labora-
tory, Department of Botany, University of Illinois.
Formerly Research Associate, Solar Energy Research
Project, Massachusetts Institute of Technology.
VOLUME II . Part 1
Spectroscopy and FInoreseence
of Photosynthetie Pigments;
Kinetics of Photosvntiiesis
1951
INTERSCIENCE PUBLISHERS, INC., NEW YORK

Interscience Publishers Ltd., London
Copyright, 1951, by Interscience Publishers, Inc.
ALL RIGHTS RESERVED. This book or any part thereof must not be reproduced
in any form without permission of the publisher in writing. This applies specifically to
photostat and microfilm reproductions.
INTERSCIENCE PUBLISHERS, Inc., 250 Fifth Avenue, New York 1, N. Y.
For Great Britain and Northern Ireland:

INTERSCIENCE PUBLISHERS Ltd., 2a Southampton Row, London W. C. I.
PRINTED IN THE UNITED STATES OF AMERICA

BY MACK PRINTING COMPANY, EASTON, PA.
PREFACE
to Volume II, Part 1
The manuscript of ^'oUlme II of this monograph was ready in draft

foi-m when the first vokime was dehvered for pubHcation in 1944. After
the intermption caused by war, I felt reluctant to publish the second
volume without thorough revision in the light of new research data and
of my own better understanding of some of the phenomena discussed.
I began, in 1947, a revision of the manuscript; while I was revising the
material, research in the field of photos^Tithesis picked up after the war-

time slack, and the writing became something of an Achilles vs. turtle race.
When, finally, the text achieved a temporary completeness, the count of
the galleys revealed that it had become too long to be published under
one cover. It was therefore divided into two half-volumes. The division
cut through the part dealing with the kinetics of photosynthesis. Because
of this, it seemed inadvisable to provide this half-volume with a separate
subject index; an index for the whole work will be found at the end of the
second part.* The latter will complete the treatment of the kinetics of
photosjmthesis (temperature effects, flashing light experiments, induction
phenomena, and the function of the pigments, especially the energy transfer
between them). The last three chapters will constitute an addition to
Volume I, and will deal particularly with the new work on photochemistry
of pigments (in solution and in chloroplasts) and with studies of the ,
chemistry of carbon dioxide reduction by means of radioactive carbon.

The hopeful advance in these two fields has changed the appearance
of the whole field of photosjTithesis. The analysis of kinetic data, to
which many pages in this half -volume are devoted, now seems somewhat
like an attempt to reach a treasure chamber by drilling through steel walls
while keys have been found to unlock the door. However, photosjTithesis
is not only a biochemical process in which all we want to learn is the
chemical composition of the intennediates and the nature of the enzymes

involved. It isa most interesting physicochemical phenomenon;
also
its kinetics will be worth continued study even after organic chemists
* As in Volume I, an index of the most important investigations described is pro-

vided at the end of this half-volume.
VI PREFACE
and biochemists have disentangled its chemical processes as thoroughly

as they did those of respiration —
which is still a long way to go. As a
matter of fact, the kinetic aspects of respiration themselves are not ade-
quately known, and will have to undergo hard study sooner or later.
Mr. Earl E. Jacobs not only kindly checked the derivation of kinetic
equations in Chapters 26, 27, and 28, but has contributed much original
thought and work to their development and interpretation it is a pleasure
;
to thank him for his unstinted assistance.

Much of the work on this volume was carried out while I was a member
of the Solar Energy Research Project at the Massachusetts Institute of
Technology. My thanks are due to the Project Committee and its chair-
man, Professor Hoyt C. Hottel, for generous assistance. I am equally
indebted to the Photosynthesis Research Project, Department of Botany,
University of Illinois, and Professor Robert Emerson, whose understand-
ing and help have made the termination of the work possible.
Mrs. Carolyn Baer, Mrs. Marjorie Goodrich, and Mr. T. R. Punnett
have given me valuable aid in the reading of the proofs and the checking
of the bibliography.
Eugene I. Rabinowitch
Urhana
June 1951
. . ..
CONTENTS
Page
Preface V
PART THREE— SPECTROSCOPY AND FLUORESCENCE

OF PHOTOSYNTHETIC PIGMENTS
Chap. 21. Absorption Spectra of Pigments in Vitro 603
A. Absorption Spectra of Chlorophyll and Its Derivatives 603
1. Absorption Spectra of Chlorophylls a and b 603
2. Absorption Spectra of Chlorophylls c and d, Bacteriochlorophyll
and Protochlorophyll 614
3. Relation between Absorption Spectrum and Molecular Struc-
ture of Porphin Derivatives 619
4. Some Theoretical Remarks on the Spectrum of Chlorophyll. . 630
(a) The Term System 630
(b) Life-Time of the Excited States of Chlorophyll 633
B. Influence of JVIedium on Absorption Spectrum of Chlorophyll and
Bacteriochlorophyll 635
1. Solvent Effect in the Spectra of Chlorophyll and Bacterio-
chlorophyll 637
2. Absorption Spectrum of Colloidal and Adsorbed Chlorophyll. 649
C. Absorption Spectra of the Carotenoids 656
1 Experimental Results 656
2. Theoretical Considerations 662
D. Absorption Spectra of the Phycobilins 664
Bibliography 668
Chap. 22. Light Absorption by Pigments in the Living Cell 672

A. Light Absorption by Plants 673
1. General Remarks 673
2. Average Transmittance and Reflectance of Leaves and Thalli in
White Light. Intensity Adaptation and Movements of Chloro-
plasts 677
3. Absorption by Nonplastid Pigments 684
B. Spectral Properties of Plants : 686
1. Empirical Plant Spectra 686
2. Band Maxima of Chlorophyll and Bacteriochlorophyll in the
Spectra of Living Plants 697

(a) Red Band of Chlorophyll a 697
(b) Red Band of Chlorophyll b 701
(c) Red and Infrared Bands of Bacteriochlorophyll 702
(d) Blue-Violet Bands of Chlorophyll 705
(e) Protochlorophyll 705
vii
.
Vlll CONTENTS
Chapter 22, contd. PaGE

3. Absorption Bands of Accessory Pigments in Live Cells 705
4. True Absorption Spectrum of the Pigment Mixture in the Liv-
ing Cell 709
C. Distribution of Absorbed Energy among Pigments 717
L Effects of Spatial Distribution of Pigments in the Cell 717
2. Apportionment of Absorption in Uniform Mixture 718
3. Absorption by Chlorophylls a, 6, c and d 719
4. Absorption by Carotenoids in Green Plants 721
5.Absorption by Carotenoids in Brown Algae 723
6. Absorption by Carotenoids and Phycobilins in Blue-Green
Algae 728
Appendix. Natural Light Fields 730
Bibliography 736
Chap. 23. Fluorescence of Pigments in Vitro 740
A. Fluorescence of Chlorophyll in Vitro 740
1. Fluorescence Spectra of Chlorophyll and Its Derivatives in
Solution 740
2. Yield of Fluorescence and Life-Time of the Excited States of
Chlorophyll 751
3. Factors Limiting the Yield of Fluorescence 755
(a) Internal Conversion (Physical Dissipation of Excitation
Energy) 756
(b) Isomerization or Dissociation ("Mononiolecular" Chemical
Quenching) 756
(c) Reaction with Foreign Molecules ("Bimolecular" Chemical
Quenching) 756
(d) Bulk Transfer of Electronic Energy 757
(e) Self-Quenching 759
4. Influence of Solvent on Yield of Chlorophyll Fluorescence 763
5. Influence of Concentration on Yield of Fluorescence. Self-
Quenching. Fluorescence of Chlorophyll in Colloids and
Adsorbates 772
6. Quenching and Activation of Chlorophyll Fluorescence by
Admixtures 777
7. Long-Lived Active States and Afterglow of Chlorophyll 790
8. Summary —
A Scheme of Fluorescence and Sensitization 795
B. Fluorescence of Carotenoids and Phycobilins in Vitro 798
1. Fluorescence of Carotenoids 798
2. Fluorescence of Phycobilins 799
Bibliography 801
Chap. 24. Fluorescence of Pigments in Vivo 805
1 Fluorescence Spectra of Plants 806
2. Fluorescence Yield and Sensitized Fluorescence in Vivo 812
3. Effects of Heat and Humidity on Chlorophyll Fluorescence
in Vivo 817
4. Variations of Chlorophyll Fluorescence Related to Photo-
synthesis 819
Bibliography 826
CONTENTS 1>^
PART FOUR—KINETICS OF PHOTOSYNTHESIS

Page
Introduction ^^^
Methods of Kinetic Measurements 833

Chap. 25.
1. Material 833
Light Measurements 837
2.
Measurements of Oxygen Evolution 844
3.
Measurements of Carbon Dioxide Consumption 851
4.
5. Measurements of Carbohydrate Production and Energy Con-

°'^'^
version
854
6. Application of Isotopic Indicators
Bibliography 855
External and Internal Factors in Photosynthesis

858
Chap. 26.
1. The "Cardinal Points" and the "Luniting Factors" 858

Photosynthesis Not a Homogeneous Reaction 864
2.
Some General Kinetic Considerations 866
3.
(a) Sources of Saturation in Photosynthesis

866
(b) Origin of Kinetic Curve Systems of Different Types 868
4. Internal Factors and the "Physiological Concept" of Photo-
synthesis 8< 2
5. Rate of Photosynthesis under Constant

Conditions. Midday
Depression and Adaptation Phenomena 873
880
6. Aging and Self-Inhibition
Bibhography ^^
Concentration Factors 886
Chap. 27.
886
A. Experimental Carbon Dioxide Curves
1. Carbon Dioxide Molecules and Carbonic
Acid Ions 886
891
2. General Review of Carbon Dioxide Curves
Carbon Dioxide Compensation Point 898
3.
Carbon Dioxide Fertilization and Inhibition 901
4.
External Supply and Exhaustion Effects 903
5.
Role of the Stomata 910

6.
Interpretation of Carbon Dioxide Curves 916

7.
The Carboxylation Equilibrium 917
(a)
921
(b) Diffusion Factors
Slow Carboxylation 924
(c)
(d) Nondissociable ACO2 Compound. The Franck-Herzfeld
Theory 927
Back Reactions in the, Photosensitive Complex 930
(e)
Acceptor "Blockade" • 933
(f)
(g) Calculation of Carboxylation Constant

from Carbon Di-
oxide Curves
934
937
(h) Are Experimental Carbon Dioxide Curves Hyperbolic?
939
B. Carbon Dioxide Concentration and Fluorescence
943
C. Concentration of Reductants
Bacteria 943
1. Effect on Rate of Carbon Dioxide Reduction in
949
2. Effect on Yield of Fluorescence
X CONTENTS
Chapter S7, contd. PaGE

D. Concentration of Inhibitors 951
1. Inorganic Ions 951
2. Poisons and Narcotics 954
Bibliography 960
Chap. 28. The Light Factor. I. Intensity 964
A. Light Curves of Photosynthesis 964

1. General Review 965
2. Linear Range 979
3. Compensation Point 981
4. Saturating Light Intensity 985
5. Maximum Rate
Absolute 989
6. Maximum Rate and Average Rate of Photosynthesis under
Natural Conditions 996
7. Interpretation of Light Curves 1007
(a) Influence of Inhomogeneity of Light Absorption 1007
(b) General Shape of Light Curves 1012
(c) Analytical Formulation: Effect of Preparatory Dark Re-
actions 1017
(d) Analytical Formulation: Effect of Processes in the Photo-
sensitive Complex 1020
(e) Analytical Formulation: Effect of "Finishing" Dark Re-
actions 1033
(f) Analytical Formulation: Narcotization 1041
B. Light Curves of Fluorescence I 1047
1. Relation between Light Curves of Photosynthesis and Fluo-
rescence 1047
2. Effect of Various Factors on Light Curves of Fluorescence. . . . 1051
(a) Carbon Dioxide 1051
(b) Reductants 1052
(c) Temperature 1055
(d) Cyanide 1057
(e) Hydroxylamine and Azide 1062
(f ) Ion Concentration 1063
(g) Narcotics 1063
(h) Oxygen 1063
3. Interpretation of Light Curves of Fluorescence 1067
Bibliography 1078
Chap. 29. The Light Factor. II. Maximum Quantum Yield of Photosynthesis 1083
1. Quantum Yield Measurements by the Manometric Method. . . . 1085

2. Nonmanometric Measurements of Quantum Yield 1118
(a) Chemical Methods 1118
(b) Polarographic Methods 1120
(c) Calorimetric Method 1123
3. Quantum Yield of Bacterial and Algal Photoreduction 1125
4. Quantum Yield of Oxygen Liberation by Isolated Chloroplasts 1129
CONTENTS XI
Chapter 29, Contd. PaQE

5. Maximum Quantum Yield in Relation to Lighit Curves as a
Whole 1132
(a) Extrapolation of Maximum Quantum Yield from Measure-
ments at Higher Light Intensities 1132
(b) Quantum Yields in Strong Light 1136
6. Theoretical and Actual Maximum Quantum Yield 1137
Bibliography 1139
Chap. 30. The Light Factor. IIL Photosynthesis and Light QuaUty; Role of
Accessory Pigments 1142
1. Action Spectrum 1142
2. Quantum Yield and Wave Length in Green Plants. Role of
Carotenoids 1147
3. Photosynthesis of Green Plants in Ultraviolet and Infrared .... 1152
4. Monochromatic Light Curves, and the Action Spectrum of
Photosynthesis in Strong Light 1158
5. Quantum Yield and Action Spectrum of Photosynthesis in
Brown Algae 1168
6. Quantum Yield and Action Spectrum of Red and Blue Algae.
Role of PhycobUins 1178
7. Action Spectrum of Purple Bacteria 1187
Bibliography 1188
Index 1193
PART THREE
SPECTROSCOPY AND FLUORESCENCE
OF PHOTOSYNTHETIC PIGMENTS
Chapter 21
ABSORPTION SPECTRA OF PIGMENTS IN VITRO
A. Absorption Spectra of Chlorophyll

AND Its Derivatives*
1. Absorption Spectra of Chlorophylls a and b
The spectra of chlorophylls a and b have been studied in detail because

of their theoretical interest, as well as because of their usefulness for the
spectrophotometric determination of these pigments. Until recently, the
results of different authors did not agree ver}^ well, either in the exact posi-
tions of the band maxima, or in the values of the extinction coefficients.
Lately, improved chromatographic purification methods have enabled
Zscheile and co-workers (cf. Zscheile 1934, 1935; Zscheile and Comar 1941;
Comar and Zscheile 1941; Harris and Zscheile 1943) and Mackinney
(1938, 1940, 1941) to obtain preparations of chlorophylls a and b that could
meet high standards of spectroscopic purity and reproducibilitj'.
Zscheile,Comar and Mackinney (1942) studied samples of chlorophyll prepared by

the two investigators at Purdue and by the third one at Berkeley, measuring the
first
extinction coefficients by means of two different photoelectric spectrophotometers.

For Zscheile and Comar's "wet" preparation of chlorophyll a (cf. page 60-1), the
two instruments gave practically identical extinction curves. (In the region between 430
and 660 m/x, all deviations were within 2%.) This shows how successfully large photo-
metric errors (which are common in visual and photographic determinations of absorption
curves) can be eliminated by the use of photoelectric devices.
The spectra of the solutions of chlorophylls a and b prepared by Mackinney in the
dry state were, on the whole, similar to that of Zscheile's moist preparation; but dif-
ferences up to 10% in chlorophyll a and 15% in chlorophyll b did occur between the ex-
tinction curves determined in the two laboratories, as well as between these two curves
and that of Zscheile's preparation. The deviations varied irregularly with wave length,
indicating the probable presence, in Mackinney's preparation, of an admixture of colored
components. One may regret that no measurements were made below 430 m^, since
earlier experiments have shown a particularly strong variability of the absorption curve
in this spectral region [cf. page 607).
The differences between the absorption curves of Zscheile and Mackinney appear
minor when compared with the discrepancies that existed between the curves published
by earlier investigators {cf. Table 2LI). These discrepancies must have been due to the
use of less reliable photometric devices, and to the inferior purity of the earlier chloro-
* Bibliography, page 668.
603
604 ABSORPTION SPECTRA OF PIGMENTS IN VITRO CHAP. 21
phyll preparations —the latter clearly indicated by the relatively high absorption in the
green {cf. last column in Table 21. IB).
A spectroscopically important impurity likely to be present in many

chlorophyll preparations is the magnesium-free pheophytin, formed from
chlorophyll whenever the latter comes in contact with acids. Elimination
of magnesium from chlorophyll may take place even in living plants, e. g.,
under the influence of acid fumes (cf. Stern 1935, 1938; and Tiegs 1938);
it can easily occur during extraction, when the pigments are exposed to the
action of acids contained in the cell sap. (Harris and Zscheile added mag-
nesium carbonate to the extracting solvent to neutralize these acids.)
This "primary" pheophytin is removed during chromatographic separation
(according to Zscheile 1941, a pheoph>'tin layer in the chromatogram was
responsible for his earlier belief that leaf extracts contain a "chlorophyll
c," cf. Vol. I, p. 402); but some pheophytin can again be formed afterward,
e. g., under the influence of atmospheric carbon dioxide.
As shown in figures 21.18 and 21.19, the pheophorbides (and this ap-
plies to pheophytins as well) have rather strong absorption bands in the
green. Zscheile and Comar (1941) and Harris and Zscheile (1943) found
that the ratios of the extinction coefficients in the maxima of the red
chlorophyll bands (660 mn component a in ethyl ether and 642.5 mfx
for
for component b in the same solvent) and of the green bands of pheophytin
(505 and 520 niyu, respectively) reach 52 in solutions of the purest prepara-
tions of chlorophyll a, and 19 in similar preparations of chlorophyll b,
but may
drop to as low as 20 and 4.5, respectively, after these preparations
have been allowed to stand for as little as a single day in the dry state.
Zscheile and Comar (1941) recommended therefore that drying be avoided
altogether in the preparation of spectroscopically pure chlorophyll solu-
tions. More recently, Zscheile, Comar and Mackinney (1942) succeeded
in preparing dry chlorophyll a which could be stored and still showed,
upon dissolution, the high ratio of extinctions in the red and in the green
indicative of high purity; but no standard procedure for obtaining such
stable preparations could be given.
Zscheile, Comar and Harris (1944) found that the spectra of ethereal
solutions of pure preparations of chlorophyll a show signs of deterioration
after about one week storage at 0-5° C. in darkness.
Crude ether extracts
from leaves, on the other hand, proved to be comparatively stable some —
gave no evidence of spectroscopic change even after 14 weeks storage (at
— 20° C). Fresh corn leaves could be stored at —20° C, for a whole
month without deterioration of chlorophj'-ll.
Another problem of chlorophyll purification is the elimination of traces
of chlorophyll a from chlorophyll b. According to Zscheile, supposedly
"pure" chlorophyll b, used by many earlier observers, did contain up to
CHLOROPHYLLS tt AND h 605
10% of chlorophyll a. can easily be recognized by increased

Its presence
light absorption at 614 mju. According to Biermacher (1939), the fluores-
cence spectrum of chlorophyll b is even more senstive to contamination with
chlorophyll a than the absorption spectmm (cf. chapter 23, page 744).
He recommended extraction with hexane (which dissolves chlorophyll a

much more easily than chlorophyll b) as a means of final purification of the
6-component Extraction is repeated until the fluorescence spectrum
.
of the
residue no longer shows the chlorophyll b band.
Meyer (1939) asserted that the band at 535 ni/u, which is noticeable in most if not
all extinction curves of pure chlorophyll a {cf. fig. 21. IB, and Table 21. lA), is not found
in the spectra of fresh leaf extracts, and concluded that a mixture of the purified chloro-
phylls a and not identical with what he designated as "native" chlorophyll (a
b is b). +
This conclusion was criticized by Mackinney (1940, 1941), who found, to the contrary,
that by mixing the two pure chlorophyll components one can reproduce the spectrum of
a fresh leaf extract in all its details (except, of course, for the blue-violet region, where
the absorption of the extracts is partly due to the carotenoids).
Fjgiu-es 21.1 A and B show the extinction curves of the two pure chloro-
phyll components in ethyl ether, according to Zscheile and Comar (1941).
(The second figure is an enlarged detail of the first one.)
180
160
140
120
100
80
60
40
20-
The absorption curves given by Mackinney (1938, 1940, 1941) and

by Winterstein and Stein (1933), although less detailed, agree satisfactorily
with figure 21.1; but those of Sprecher von Bernegg, Heierle and Almasy
(1935) and of Hagenbach, Auerbacher and Wiedemann (1936) show con-
siderable deviations, illustrated by Tables 21.1.
Table 21.1. Chlorophyll Solutions in Ethyl Ether
A. Absorption Maxima. Main bands in italics.

. — — —
CHLOROPHYLLS a AND 6 607
Table 21.1 Continued
Chlorophyll b in ethj'l ether
Observers (643 m/i) (453 m>i) (510 m^)
Sprecher von Bernegg et al.
(1935) 4.7 9.4 0.28 2.0 17

HagenbacheâZ. (1936).. 7.10 20.9 0.34 2.91 21
Mackinney (1940) 5.00 13.6 0.26 2.72 19
Zscheile and Comar (1941) 5.15 15.5 0.24 3.01 21
Zscheile, Comar and Mac-
kinney (1942)^ 4.80 12.9 0.24 2.65 20
Same'' 4.98 13.9 0.24 2.79 21
Harris a nd Zscheile (1943) -- — — 2.98 —
" Band "axes" i. e., arithmetic means of the wave lengths of the hmits of blacken-
ing on a photographic plate (all other data in this table were obtained by photoelectric
photometry)
''
and Comar's moist preparation measured by Mackinney.
Zscheile
" Mackinney's dry preparation measured by Zscheile and Comar.
<*
Mackinney's dry preparation measured by Mackinney.
The reproducibility of the red band encourages its use for the spectrophotometric
assay of the two chlorophylls. This method was developed by Ghosh and Sen-Gupta
(1931), Zscheile (1934, 1935), Sprecher von Bernegg, Heierle and Almasy (1935), Haskin
(1942), Comar and Zscheile (1942), Comar (1942) and Comar, Benne and Buteyn
(1943). Measurements at two different wave lengths are required to calculate the
concentrations of the two components. The use of the absorption maxima at 642.5
and 660 mix permits the most sensitive determination, but requires precise work, since
the extinction values in the sharp absorption peaks are very sensitive to variations in
the width of the spectrometer slit or to sUght errors in the adjustment of the monochro-
mator. Cross-checks at other wave lengths are therefore desirable. All errors could be
eliminated by the use of monochromatic Ught; but the spectrum of the mercury arc
the usual source of monochromatic light in the laboratory — does not contain suitable
Unes in the red and orange regions.
As pointed out once before, particularly wide discrepancies can be

noted between the different extinction curves in the blue-violet region {cf.
Table 21. IB). was noted by Albers (1941) that the subsidiary violet
It
band (situated, in ethereal solution, at 410 mju in chlorophyll a and 430
m/i in chlorophyll b) sometimes appears as a slight hump on the main
band, and sometimes as a separate peak, almost as prominent as the main

one. The observed variations in the maximum height of the violet peak
may be caused by the more or less complete separation of this doublet
structure.
Differences of this type cannot be attributed to the presence of caro-
tenoids, or other impurities. A
renewed photometric study of this spectral
region is desirable. If the deviations will not disappear upon further puri-
fication of the material and improvement of the photoelectric methods, one
may have to consider, as a possible explanation, the existence of tautomers.
V
608 ABSORPTION SPECTRA OF PIGMENTS 7.V VITRO CHAP. 21
H2C=CH CH3* HT rn
.C, ,C C i H I
C N N C V ., \. _/
H,c-c< El I ir |c-CH, „^ o< m \ \
m\
/\ \
H \
c c ^c'
H I lio 9. u
CH2 HC C. Hî
^ 1.
1
I,,".:
.iio
.
9
CH2 HC
^^^y'°'^
H39C20OOC-CH2
I I
COOCH
\ (Phytol)
H r
H39C20OOC mr
— CH2 LI
'
T
i
COOCH
X
C.
A B
H2C=CH CHj*
' H I
H3C-C(r^ C
I jj >C-C2H,
J
C N N C
HC S Mg fl CH
C N N C
" \/ ^J<
CHg HC C
(Phytol)
H39C20OOC— CHj
I I
\
COOCH
Fig. 21.2. Chlorophyll a structure according to Hans Fischer. A, B and C are three
isomeric or tautomeric (or mesomeric) structures, distinguished by the routing of the
all-roimd conjugated ring system (heavy line) and the positions of the "semi-isolated"
double bond and of the Mg—
N bonds (all of which depend on this routing). The
asterisk designates the position of a carbonyl group in chlorophyll h. A, semi-isolated
double bond in nucleus III Mg bound to nuclei I and II. B, semi-isolated double bond
;
in nucleus II; Mg bound to nuclei I and III. C, semi-isolated double bond in nucleus
I; Mg bound to nuclei II and III.
We recall that the three structures of chlorophyll, A, B and C, described

in chapter 16, Vol. I, (c/. fig. 21.2), were characterized there as probably
tautomeric rather than mesomeric. We also recall that Strain and Mann-
ing found {cf. page 403) in the chromatograms of leaf extracts, two new
CHLOROPHYLLS a AND h 609
chlorophylls, which they called a' and b' and interpreted as tautomers of
chlorophylls a and b. The forms A, B and C have different double bond ar-
rangements in the nonhydrogenated pyrrole nuclei, but the same hydro-
genated nucleus IV. Since the red absorption band is somehow associated
with the hydrogenation of this nucleus (cf. page 621), modifications A, B
and C may have identical red bands. They could, however, differ in the
positions or shapes of the blue-violet bands, associated with the conjugated
porphin system as a whole.
Erdman and Corwin (1946) noted the spectroscopic similarity of etio-
porphyrin and A'"-methyl etioporphyrin, and deduced from this that the
two "central" H-atoms in the porphin system must be fixed at definite
nitrogen atoms for »
10"* sec. This supports the hypothesis that struc-
tures such as those represented by the three formulas in figure 21.2 are
tautomeric rather than mesomeric.
We have used so far only data obtained with ethyl ether as solvent,
since they alone offered the possibility of comparison between the results
of several observers. Determinations of extinction curves of chlorophyll
in solvents other than ether are Usted in Table 21.11.
Table 21.11
Chlorophyll Extinction Measurements in Various Solvents
Solvent Reference
Methanol, Rabinovvitch and Weiss (1937) (ethyl chlorophyl-

Ude); Harris and Zscheile (1943); Mc Brady and
Livingston (1948, 1949)
Ethanol Meyer (1939); Sprecher von Bernegg, Heierle and
Almasy (1935)
Butanol (71- and iso-) Harris and Zscheile ( 1943)"
Octanol Harris and Zscheile (1943)"
l-Propyl ether Harris and Zscheile (1943)"
Dioxane Harris and Zscheile (1943)"
Benzene .... Winterstein and Stein (1933); Hausser {cf. Fischer
and Stern, 1940; Harris and Zscheile (1943)
Cyclohexane Harris and Zscheile (1943)
Acetone Mackinney (1940); Harris and Zscheile (1943)
Carbon tetrachloride Harris and Zscheile (1943)"
Methyl oleate Harris and Zscheile (1943)"
Olive oil Harris and Zscheile (1943)"
" Several of the curves of Harris and Zscheile (1943) are reproduced in figure 21.26.
The absorption spectra of the two chlorophylls in the ultraviolet are

shown in figure 21.3. The absorption remains considerable all the way
down to 200 m/u; the most prominent band is a double band of chlorophyll
b at 310 and 335 mju. In the spectrmn of chlorophyll a, distinct maxima
appear at 325 and 375 mn. Below the region shown in figure 21.3, both
components have an absorption maximum at 250 m/i (asp. ^^ 30).
An absorption band at 330 mix was first noticed in the spectrum of alcoholic extracts
from nettle leaves by Lewkowitsch (1928).
The absorption spectra of chlorophyll, ethyl chlorophyllide and phytol

in the infrared were described by van Gulik (1914) and Stair and Coblentz
80
72
64
56 -
48
40
32
24 \ Chlorophyll b
16
\_''
260 280 300 320 340 360 380 400 420

WAVE LENGTH, m/i
Fig. 21.3. Ultraviolet spectrum of chlorophylls a and b in ethyl ether (after

Harris and Zscheile 1943). Specific extinction coefficients; c in g./l.
(1933). Chlorophyll is transparent between 0.7 and 3 n (this may be useful

in preventing the overheating of leaves in direct sunlight). It has several
absorption bands at 3-4, and 5.8 most of them are found also in the
yu;
spectrum of phytol, and are absent from that of ethyl chlorophyllide (c/.
fig. 21.4 and Table 21.IIA); they thus belong to the phytyl chain rather
611
90
Chlorophyll
80
70
z* 60
o
<" 50
<f) 40
z
c 30
I-
silt width: Rock solt
20
Fluorite
10
_l L. j u _j i_
10 II 12 13 14 15
(
90 Phytol CzoHjoOH
Copillary film
10 II 12 13 14 15
Ethyl Chlorophyllide
70
Fluorite prism
2-60 1-
o
<n 50
00
^ 40
^ 30
20
10
2 9
\ 3 4 t 6 7 8 iO li 12 13 14 15
WAVE LENGTH, m,i
Fig 21.4. Infrared spectra: top, chlorophyll (a h) layer prepared by evaporation +

on rock salt cleavage plate center, liquid phytol in cell 0.3 mm. thick
of ethanol solution ;
layer between two rock salt

with rock salt windows (curves B and C) and in capillary
cleavage plates (curve A); bottom, thin evaporated iilms of ethyl chlorophyllides (o +
C and D) (after Stair and Co-
h) on fluorite (curves A and B) and on rock salt (curves
blentz 1933).
than to the chlorophyhin ring system. The frequencies 1045, 1265, 1450.
1545 and 1610 cm.-^, on the other hand, seem to belong to chlorophylhn.
CHLOROPHYLLS a AND h 613
harm-
plete absorption of all visible light appears to be unnecessary, or even
ful. As stated before (compare Volume I, chapter 19), chlorophyll prob-
ably was adopted as sensitizer for photosynthesis not because of a particu-
larly favorable absorption spectnim, l)ut because of its peculiar photosensi-
Once having found a suitable sensitizer, natiu'e then
tizing properties.
made adjustments to improve the supply of light energy to species living
in unfavorable habitats —
by increasing the quantity of chlorophyll b in
shade plants, and by providing brown algae with fucoxanthol, and red
algae with phycobilins. The presence of the latter makes the red algae
capable of growth even under a thick layer of blue-green sea water.
</)
UJ
>
UJ
on
Normal
I-
z
UJ
o
u.
u.
UJ
o
o
Q.
tr
o
(n
m
<
390 430 470 510 550 590 630 670 710

WAVE LENGTH, m,i
Fig. 21. 4A. Al)sorption spectrum of allomerized chlorophyll a in methanol

(after Livingston 1948).
A word must be said about the absorption spectrum of allomerized chloro-

phyll. When chlorophyll in alcoholic solution is permitted to stand in air,
it is "allomerized," ?'.
e., according to Conant and Fischer, oxidized at the
r(10) iitom (cf. Vol. I, page 4(i()). This reaction is catalyzed, according to
Livingston and co-workers, by salts such as LaCls and BaCl2. Spectro-
scopic evidence indicates that a similar, or identical, oxidation occurs also
under the influence of iodine or bromine even in the absence of air:
Fischer's chemical observations indicate that quinone has the same effect.
According to Livingston (1948, 1949), allomerization of chlorophyll is
characterized by a spectral change, illustrated by figure 21. 4A. Tlu^ two
curves in this figure were obtained by chromatographic separation of a

partially allomerized solution; a curve identical with curve h is obtained
by leaving a methanolic solution of chlorophyll a stand in air at 80°C. for
two days, or by adding to it 2-3 equivalents of iodine, or by adding traces
(10"^ ml.) of LaCls or CaCls. Standing in air, or addition of traces of
iodine, has no on the spectrum of chlorophyll a in ether or carbon
effect
tetrachloride, except for slow general bleaching. If some methanol is added
to the nonpolar solvent, allomerization proceeds more slowly, but ends
with being as complete as in pure methanol; transfer of allomerized pro-
duct into pure ether or carbon tetrachloride does not reverse the change.
In cresol, allomerization also seems to occur, but is complicated by other
changes, probably due to the acid nature of this solvent.
Livingston and co-workers also have measured the absorption spectrum
of the yellow and brown unstable intermediates in the reactions of chloro-
phyll (in methanol) with FeCls (Vol. I, page 464) and with alkali (see
"phase test," Vol. I, page 459). The results will be described in chapter 37,
in the section dealing with new observations on the chemistry of chloro-
phyll.
2. Absorption Spectra of Chlorophylls c and cl, Bacteriochlorophyll and

Protochlorophyll
The absorption spectrum of "chlorophyll c," also called chlorofucin (cf.
Vol. I, page 406), is characterized, according to the earlier authors (e. g.,
Tswett), by a band in the region of 630 m/x. Strain and Manning (1942)
determined the extinction curve of this compound, first by subtraction of
the extinction curve of pure chlorophyll a from that of the chlorophyll ex-
tract from brown algae, and later by direct spectrophotometry of isolated
chlorophyll c. Figures 21. 5A and B show that the results of the two meth-
ods are in approximate agreement. Two "chlorofucin" bands are situated
in the orange and red —with peaks at 575.5 and 627 m/i, respectively, in
methanol, and at 581 and 631 m/x, respectively, in 80% acetone. Figure
21. 5C alsoshows a band in the blue (at 446 mû) almost ten times stronger.
The ratio between the intensities of the bands in blue and red is thus much
larger than in chlorophyll b (where it is about 3), not to speak of chlorophyll
a (where the two bands are approximately equal in intensity, cf. Tables
21. IB and 21.VIII), but the general pattern of the spectrum is similar.
Wa.ssink and Kersten (1946) and Tanada (1951) gave similar absorp-
tion curves for a "chlorophyll c" fraction from chromatographic frac-
tionation of the pigments of diatoms. The three absorption peaks ap-
pear, in methanol, at about 450, 590 and 635 m/u, with the first band about
ten times more intense than the other two (cf. p. 623).
CHLOROPHYLLS C AND d, BACTERIO- AND PROTOCHLOROPHYLL 615
Egregia
Extract
c
o
u Extract
minus a
I I I I
580 610
(A)
61G ABSORPTION SPECTRA OF PIGMENTS 7.V VITRO CHAP. 21
The absorption spectrum of the '' chlorophyll d" of red algae also was
determined by Manning and Strain (1943). Since this investigation was
j
only briefly mentioned in Vokime I, a few words may be said here about
this newly discovered pigment. Its presence is revealed by a bulge on the
red side of the chlorophyll a band, observed in the spectra of methanol
extracts from red algae. In pure chlorophyll a solution in methanol, the
ratio of extinction coefficients at 665 and 700 mû is 90; in extracts from
twenty red algae, this ratio was between 15 and 65. Short extraction leads
to products with even lower I'atios r. g.
; wo minutes extraction of Gigarlina
, t
agardhii gave a product Math a ratio of only 10:1. Apparently, chlorophyll d

is much more easily extracted by methanol than chlorophyll a. Chromato-
graphic purification can be used for the preparation of pin-e chlorophyll d.
Figure 21.51) shows the spectrum of this pigment in methanol. In ethyl
ether, the band maxima of chlorophyll d lie at 686 and 445 myu, and below
395 niju.
In spectrum as well as in solubility and other chemical properties,
chlorophyll d resembles chlorophyll a more than chlorophyll h. Careful
search for chlorophjdls h and c in the chromatograms of pigments from red
algae gave negative results (the upper limit foi- tlie content of chlorophyll h
is 0.3% of that of chlorophyll a).
The isornerization of chlorophyll d was mentioned in chapter 16 (Vol.
I). It occurs in the methanolic solution upon standing in the dark, in the
presence or absence of air, and leads to three new pigments, which seem
to be interconvertible. They were designated chlorophyll d', isochloro-
phyll d and isochlorophyll d'. The spectra of the components d and d',
respectively, are very similar, and the same is true of those of the isomers
iso-d and iso-c?'. The latter two spectra are almost identical with those of
the chlorophylls a and a' (cf. fig. 21. 5E) ; but the maximum of chlorophyll
iso-d and iso-d' lies about 5 mfu. further toward the blue (cf. fig. 21.5 F).
The red band of chlorophyll d (and d') lies about 37 m^ further toward the
infrared than that of chlorophylls a and a' (cf. fig. 21.5F).
Isochlorophylls d and d' are not found in fresh extracts from algae.
The conversion d -^ iso-d appears to be slower than the conversions d-^ d'
and iso-d -^ iso-d'.
Four interconvertible pheophytins with diffei'ent spectra were pro-
duced by the action of acids on the four d-pigments; but successive treat-
ment with alkali and acid led to spectroscopically identical products for
all four d-pigments. These products are distinct from the compounds ob-
tained by a similar treatment of either of the two a-pigments. Table
21. Ill illustrates the relationships between the six pigments a, a', d, d',
iso-d and iso-d' and their transformation products.
The main absorption band of the bacteriocMorophyll of purple bacteria
CHLOROPHYLLS C AND d, BACTEKIO- AND PROTOCHLOKOPHYLL 017
Table 21. Ill
"
ISOMERIZATION AND OtHER REACTIONS OF CHLOROPHYLLS d AND a
Chlorophyll d' Isochlorophyll d' Chlorophyll u'
It
Chlorophyll d Gochlorophyll d Chlorophyll a

,.
CH-Mgl HCI CHjMgl
Pheophytin d — Isopheophytin d Pheophytin a
KOH KOH KOH KOH KOH KOH
681 m^ 698 m;i 658 m^ 636 m^ 662 m^ 649 my.
CHjCOOH CHjCOOH CHjCOOH
672 m^ 691 Wfi 660 m/i 672 m/i 667 m^ 652 m^

HCI HCI HCI HCI
——
HCI
f
Ihci
SSOmjii 667 m^
" After Maiming and Strain (1943).
0.20
Fig. 21.6. Relative absorption curve

of green pigment from bacterium Spi-
rillum rubrum in methanol (after French
1937). 0.2 ml. moist cells extracted in
the dark at 0° with 5.2 ml. absolute
methanol Extract kept in dark and
measured at room temperature with
very weak light. A similar curve was
given by Vermeulen, Wassink and
Reman (1937) for alcoholic extract from
Chromaiium (major peak at 790 m/n,
minor peaks at 705, 600, 510, 470 and
440 mM).
600 800
WAVE LENGTH, m/i
lies in the near infrared and has not yet been measured precisely; its gen-
eral shape is shown by figure 21.(). According to this figure, the absorp-
tion spectrum of bacteriochlorophyll contains
three main bands one in the —
infrared (770 m/i in methanol, shown also in fig. 21.21), one in the orange
(005 m/x) and one in the violet (~400 ni/i). One may assume {cf. below,
page 622) that the two latter bauds are analogous to the red and the blue-
violet bands of chlorophyll a, respectively (the whole system having been

shifted by about 60 m^t toward shorter waves), while the band in the near
infrared has no analogue in the spectrum of ordinary chlorophyll.
The status of the "orange" bacteriochlorophyll band needs additional
clarification. Our interpretation is based on figure 21.6. A strong band
on the short-wave side of the main red band is also recognizable in the spec-
trum of bacteriopheophytin as observed by French (fig. 21.21), but is
situated much further toward the green, at 530 m^; in addition, there is a
weaker band at 680 m/x and indications of a still weaker one at about 630
m/n. Dutch observers state (see page 702, chapter 22) that "alcoholic
-
1.0-
r;0.5-
c
o
u
SPECTRUM AND STRUCTURE OF PORPHIN DERIVATIVES 619
alcoholic extracts from ChJorcUa (green alga), Chlorobium limicola (green

bacteria) and Oscillatoria (blue alga). The differencebetween the curves
1 and 3 can be attributed to the absence of chlorophyll h in blue algae;
while the larger difference between the curves 3 and 2 indicates a chemical
distinction between chlorophyll a and bacterioviridin. The absorption
peak of the latter pigment in ethanol lies at 668 m/x, while that of the former
one is situated at about 662 m/x.
The absorption spectrum of protochlorophyU (from squash seeds) was
described by Noack and Kiessling (1929, 1930, 1931) as well as by Rudolph
(1933), Seybold (1937) and Koski and Smith (1948). The long-wave
bands are listed in Table 21.IV; the whole spectrum is shown in figure
21.8.
Table 21. IV
Absorption Bands of Protochlorophyll
In chloroform-pyridine
020 l.bxiu^
1
The conjugated double bond system of porphin (which is the basic

structure of all chlorophyll pigments as well as of the porphyrins) is a
chromophore, capable of pi'txlucing sti'ong absorption bands in the visible
and near ultraviolet. This is iUustrated b}' the absorption spectrum of the
parent substance of the group, porphin (c/. fig. 21.9). This spectrum has a
typical pattern of four bands between 480 and 700 mn, generally increasing
in intensity' toward the violet, but with the third band from the red weaker
than the second one. Stern and Wenderlein (1936) called this pattern the
"phjdlo type" (fig. 21.10, 3); it is exhibited by many porphyrins. Other
Electronic
States
Vibrational
States
upon the from the prophin system to the dihydroporphin {chlorin

transition
or rhodin) system. ("Rhoclins" are chlorins derived from chlorophyll 6;
c/. page 447, Vol. I.) Chlorins are green (as their name implies) and this
Table 21.V
PoRPHiN Bands
X (him)
V (cm."0
;
prolochlorophyll, a green compound which, according to H. Fischer {cf.

Vol. page 445), nevertheless is a prophyrin rather than a chlorin or phor-
I,
bin. The spectrum of protochlorophyll (cf. fig. 21.8 and Table 21. IV)
does not show the predominance of the red band over the bands in yellow
and green to the same extent as do the typical spectra of dehydro- or tetra-
hydroporphin derivatives; but it resembles these spectra somewhat more
than it does the typical porphyrin spectra in figure 21.10. A re-examina-
tion of the structure of the protochlorophyll molecule is therefore desirable.
It is noteworthy that "protopheophytin," obtained from protochlorophyll
by the action of acids, was found to have a typical porphyrin spectrum.
A similar case is presented by chlorophyll c. As stated on p. 014,
this pigment has an arrangement of bands similar to those of the chloro-
phylls a and b, but the red ))and is very weak compared to the Soret band
in fact, the spectra of chlorophyll c (insert in fig. 21. 5C) and protochloro-
phyll (fig. 21.8) are extremely similar. It is therefore significant that
Granick concluded, from chemical evidence, that chlorophyll c, too, is a

porphin rather than a chlorin derivative (cf. chapter 37).
Another interesting problem of the same character was raised by an
investigation of Aronoff and Calvin (1943). They prepared (by condensa-
tion ofbenzaldehyde with pyrrole) several compounds that tliey interpreted
as isomeric hexaphenylporphins. Some of these compounds had porphyrin
spectra of the "etio type" (fig. 21.10, 1), but others had spectra with a pre-
dominant sharp band in the red (fig. 21.13, curve B). The authors
thought at first that these green isomers may contain one pyrrole nucleus
turned around, placing its N
atom on an outside corner.
Rabinowitch (1944) suggested they could perhaps be interpreted as
chlorins: Chlorins isomeric with hexaphenylporphin could be formed, e. g.,
by attachment of one phenyl group to a pyrrole nucleus, and shifting of the
two liberated hydrogen atoms to another pyrrole nucleus, thus:
Tetrophenylporphin Tetrophenylchlorin
624 ABSORPTION SPECTRA OF PIGMENTS 7.V VITRO CHAP. 21
Subsequently, Calvin, Ball and Aronoff (1943) found indications that

the two "isomers" whose spectra are shown in figure 21.13 actually l)elong
to two different reckiction levels of the porphin system. Thus, in this case
at least, the dominant red band was confirmed as an indicator of partial
hydrogenation.
3.0 X 10
H
2.5
2.0
« 1.5
50Q 550 600 650

WAVE LENGTH, m^
Fig. 21.13. Molar absorption spectra of two tetraphenylporphin "iso-
mers" (after Aronoff and Calvin 1943).
No such explanation can as yet be given to another observation of

Aronoff and Calvin —
that addition of hydrochloric acid to tetraphenylpor-
phin solutions with spectra of the etio type causes a reversible transition
to chlorin type (fig. 21.14). Aionoff and Calvin attributed this to salt
formation: porphin + 2HC1-^ (porphin H2)++(C1~)2. The addition of 2
hydrogen ions thus appears to have the same effect on the porphin spectrum
as does the addition of two hydrogen atoms. If this is true, the problem of
the spectroscopic difference between prophins and chlorins becomes cognate

to the proljlem of acid-base color (;hanges (concerning the latter, see

Epstein, Kariish and Rabinowitch 1941, and Lewis and Bigeleisen 1943).
According to Pruckner (1942), imidoporphyrins (which differ from por-
phin derivatives by the substitution of an XH gi-oiip for a bridge carbon)
0.800
0.700
0.600
0.500
3
0.400
0.300
0.200
0.100
500 550 600 650 700

WAVE LENGTH, m/x
Fig. 21.14. Effect of increased acidity on spectrum of a tetraphenylporphin (after

Aronoff and Calvin 1943). 10 ml. alcohol, containing 5 ml. 3 X 10~' ^^ solution of free
base and variable amounts of hydrochloric acid. (1) no acid, (2) 0.0204 A'' HCl, 0.5 ml.,
(5) same, 1 ml, (,/,) same, 2 ml, (5) same, 3.50 ml, (6) 6.3 A HCl, 5 ml.
also have a strong absorption band in the red. She suggested tliat the
appearance of this band is generally associated with increased molecular
symmetry; but it is not quite clear why hydrogenation of one or two
pyrrole nuclei, or substitution of NIT groups for (' atoms, should lead to
higher symmetry.
The "c'hlorin type" spectrum remains almost unaffected by all trans-

formations leading from the parent substance chlorin to chlorophyll a.
This is in accordance with general experience as far as the introduction of
methyl and ethyl groups is concerned. It is noteworthy, however, that
the introduction of an unsaturated (vinyl) substituent in nucleus I also
causes only a slight shift of the bands, as illustrated by figure 21.15. In
other words, a difference of two hydrogen atoms in a side chain is almost
without influence on the spectiaim, while a similar difference in the nucleus
has a strong effect.
The spectroscopic effect of carboxyl groups in chlorophyll also is small.
The alcohols esterifying these carboxyls have a certain influence on the
6.4 X 10*
62 Rhodochlorin dimethyl ester
Mesorhodoctilorin dimethyl
5.6 ester
(both in dioxane)
o
o
E
470 490 520 550 580 610 640 670

WAVE LENGTH, m^
Fig. 21.15. Effect of vinyl group on chlorin spectrum in di-
oxane (after Stern and Molvig 1937).
bands (but none on their position) The shorter

intensity of the absorption :
the alcohol, the sharper the absorption peak. As an example, figure 21.16
shows the extinction curve of phytyl pheophorbide (pheoph^^tin), together
with that of methyl pheophorbide.
noteworthy that closure of the carbocyclic ring, i. e., the
It is further
transition from chlorin to phorbin, also hardly affects the spectrum at all,
as shown by figure 21.17. The carhonyl group in nucleus II, whose pres-
ence distinguishes chlorophyll b and its derivatives from the corresponding
compounds of the a series, has a much stronger effect on the spectrum:
The two chlorophylls have distinctly different colors one blue-green and —
the other yellow-green. Figure 21.1 shows that this difference is caused by
SPECTRUM AND STRTICTItrE OF PORPIIIN DERIVATIVES 027
different positions of the bhie-violet bands: That of chlorophyll a is con-

fined to the violet and ultraviolet regions, allowing free transmission of blue
light, whereas thea])S()rption peak of the 6 compound is situated in the
blue, so that this compound transmits only green light. The arrangement
of the weaker bands in the middle of the visible ii^gion is also affected by
the carbonyl in position 5, to such an extent that Stern classified the spectra
of chlorophyll b and its derivatives as a separate "rhodin" type (fig. 21.10,
5) distinct from the "chlorin" type (fig. 21.10, 4).
480 520 560 600 640 670 480 520 560 600 640 670
WAVE LENGTH, m>i WAVE LENGTH, m/i
Fig. 21.16. Effect of esterification on Fig. 21.17. Effect of closure of car-

chloriri spectrum (after Stern and Mol- bocyclic ring (transition chlorin —*
vig 1937). phorbin) on chlorin spectrum in di-
oxane (after Stern and Wenderlein
1935).
The other carbonyl group of chlorophyll, the one in the carbocyclic

ring (in position 9), has no pronounced influence on the spectrum of chlorin
derivatives. This is striking because, in the case of porphyrins, a carbonyl
group in a similar position affects the spectrum to a considerable degree.
Stern (c/. Fischer and Stei-n 1940, page 343) suggested an explanation of
this difference,based on Fischer's earlier assignment of the two extra hy-
drogen atoms to nucleus III. By this assignment, the double bond C=0
in position 9 was removed from conjugation with other double bonds in the
molecule (cf. Volume I, page 441), and this could explain why its effect
628 AnSORrTION SPECTRA OF PIGMENTS IN VITRO CHAP. 21
on the spectrum is less pronounced than that of the conjugated C=0 bond
in position 3. As stated on page 441, Vohime I, Fischer later concluded
from chemical degradation experiments that the two extra hydrogen atoms
are located in nucleus IV. A certain difference between the conjugation in
nuclei II and III exists, however, also in the latter structure (formula A,
—
page 608) namely, the double bond 3-4 in nucleus II is part of the all-
round "aromatic" ring system, while the double band 5-6 in nucleus III is
1 0.0x10^
9.0 Methyl chlorophyllide a
Methyl pheophorbide a
A
<
480 520 560 600 640 670

WAVE LENGTH, m^
Fig. 21.18. Effect of magnesium on a porphin spectrum (in
dioxane) (after Stern and Wenderlein 1936).
merely in "one-sided" conjugation with this system. In structure B, on

the other hand, bond 5-6 is part of the all-round conjugated system, and
bond 3-4 is in one-sided conjugation. Finally, in structure C, both bonds,
3-4 and 5-6, participate in all-round conjugation. Thus, a difference be-
tween the chromophoric effects of carboxyls in nuclei II and III appears
plausible in structures and B, but not in structure C. We note further
A
that, according to Stern and Pruckner (1939), a carbonyl group in nucleus
I also has no strong effect on the spectrum. This indicates that, with re-
—
SPECTRUM AND STRUCTURE OF rORPITIN DERIVATIVES 020
spect to conjugation, the role of nucleus I is similar to that of nucleus III

and this points to structure B as the tme structure of chlorophyll, in prefer-
ence to structure A, advocated by Fischer (page 443). (As mentioned on

page 444, Vol. I, structure B also has the advantage of providing a direct
link to bacteriochlorophyll.)
If this interpretation of the spectroscopic data is correct, it means
that the introduction of a carbonyl group has a stronger effect on the spec-
60
Chlorophyll a
7.0 Pheophorbide a
5.0
Dihydropheophorbide
Pheoporphyrin 05
60
4.0
30000 25000 20000 ^'
3.0 _i L
!
[ I
•
r •
I r I'
350 400 450 500 600
WAVE LENGTH, m/i
6.0
Chlorophyll b
Pheophorbide b
4.0-
30000 25000 20000 30000 25000 20000

' '
'I
350
'
I I'
400 450 500

I r 'I—
600
r- 3.0 __i i_ -1
350
-I I
400 450 500

'
1
1
1
•
]•
'
'\
600
. '
•
\
*
WAVE LENGTH, m^ WAVE LENGTH, m^
Fig. 2 1.1 9 A. Effect of porphin -> chlo- Fig. 21.19B. Extinction curves of
on the visible and ultraviolet
rin transition chlorophyll and pheophorbides in the
spectrum (after Hagenbach, Auerbacher visibleand near ultraviolet (after
and Wiedemann 1936). Hagenbach, Auerbacher and Wiede-
mann 1936).
trum if this group comes into conjugation with a C=^C bond, which is
merely in one-sided conjugation with the "aromatic" system, than if it is

attached directly to the latter system.
Introduction of magnesium, into the molecule (transition from phorbin
to phyllin) has the effect of enhancing further the main red absorption
band, and of weakening the bands in the green, as illustrated by figure 21.18.
The result is the beautiful pure green color of chlorophyll so different —
from the dull olive-green of pheophytin.
While the porphyrins, chlorins, phorbins and rhodins differ in their
absorption spectra in the green, yellow, orange and red, their spectra in the
G30 ABSORPTION SPKCTRA OF PIGMENTS IN VITRO CHAP. 21
violet and ultraviolet all show the same pattern. As pointed out by Stern
(1939), neither the transition from porphin to chlorin nor the introduction
of magnesium has much effect on the intensity of the blue-violet absorption
band. This is shown by figures 21.19A and B. The blue-violet band is
shifted by hydrogenation toward shorter waves, but suffers no appreciable
change of intensity.
Comparison of figure 21. 19 A and B shows that, while both magnesium
and the extra hydrogen atoms enhance the main red absorption band,
these two substituents have antagonistic effects on all the rest of the spec-
trum, below GOO m.fx.
4. Some Theoretical Remarks on the Spectrum of Chlorophyll
(a) The Term System
In section 3 (c/. fig. 21.11) we interpreted the four absorption bands of

porphin in yellow and green as vibrational bands belonging to the same band
system (electronic transition X
-^ A). A similar interpretation has been
suggested by Prins (1934) for the bands of chlorophyll in the red, orange,
yellow and green it is made plausible by the magnitude of the Ai* values
;
given in Table 21. VI. (c/. the infrared absorption frequencies in Table
21.IIA). The blue-violet and the two ultraviolet bands are best inter-
preted as separate electronic transitions.
Table 21. VI
Chlorophyll a Bands
v (cm.~0
:
ANALYSIS OF CHLOROPHYLL SPECTRUM 631
X -^ Ai, and the band X -^ Ao must be considered missing because of low

intensity. However, this interpretation is made untenable by the observa-
tion— to be discussed chapter 23—that both porphins and dihydro-
in in in
porphins the main fluorescence bands are close to the absorption band first
inthe red — whether this band the weakest of

is absorption bands, as all in
porphin, or the strongest one, as in chlorophyll. This shows that in both

cases, the first observed absorption bands lead to the vibration-free upper
level Ao. If the first X -> Ai (as tenta-
absorption band of porphin were
tively suggested above), we would expect to find the main fluorescence
«3
^1
Yo
o
>
a>
Fig. 21.20. Hypothetical term system of the chlorins.

Band X - Ao is submerged by band X —» Yi.
—
band some distance toward the red from it in the approximate position of
the "invisible" X -^ Ao absorption band (Rabinowitch 1944).
Since this is not the case, an alternative hypothesis must be considered.
It is represented in figure 21.20, and suggests that the hydrogenation of
one pyrrole nucleus creates a new low electronic excitation state Y, situated
a little lower than the level Ao, "inherited" from the nonhydrogenated
system.* Figure 21.12 makes one suspect that the second chlorin band
(612.5 m/i in chlorophyll), which is stronger than the corresponding band
in the nonhydrogenated compound, may also belong to the system X
—> F
(as a second band of this system, X Yi, ^
.), and that it masks the weak
. .
l)and A^ -^ Ao. A similar interpretation can be suggested for the infra-

red band of bacteriochlorophyll and other tetrahydroporphin dei'ivatives
We can attribute the strongest infrared band of bacteriochlorophyll (fig.
—
Another possibiHty suggostetl by the spectra of protochlorophyll and chloro-
*
phyll c —
iR that the band A' -* I'o exists also before hydrogenation, but is strongly
enhanced by the latter.

21.6) to anew electronic transition, X

—> Z, added (in consequence of the
hydrogenation of a second pyrrole nucleus) to the three transitions Y, X^
X —^ A and X
-^ B present in the spectrum of chlorophyll. Simultane-
ously with the creation of a new excited electronic level Z, the "old" levels
Y, A and B are shifted upward, thus accounting for the "violet shift"
of all bacteriochlorophyll bands "inherited" from chlorophyll. The in-
frared band X —> Z dominates
the spectrum of bacteriochlorophyll to an
even greater degree than the red band, X —> Y, dominates the visible spec-
trum of ordinary chlorophyll.
500 600 700 800 900

WAVE LENGTH, m/i
Fig. 21.21. Absorption spectrum of bacteriopheophytin from

Spirillum rubrum (after French 1940). Specific absorption coef-
ficients, c, in mg./i., d in cm.
Since the absolute extinction coefficients of bacteriochlorophyll are as yet unknown

(fig. 21.6 gives only the optical densities), it isnot certain whether the predominance
of the 770 niM band is eau.sed by its great absohite lieight, or tjy a relatively low intensity
of tlie otlier bands. A was given by
specific extinction curvt; of bacteriopheophyiin
PVench in a later paper (1940), and is reproduced in figure 21.21. It shows that the
molar absorption coefficient of bacteriopheophytin (in methanol) reaches 2.7 X 10^
:
ANALYSIS OF CHLOROPHYLL SPECTRUM 033
in the maximumof the orange band, while the infrared peak is almost exactly twice as
high. According to figure 21. Hi, tiic maxinmni absorption coefficient of ordinary pheo-
phytin a in the red is about 4.2 X IQ' (in dioxane, wliere the peaks are usually sharper
than in methanol). It thus seems that the dominant position of the infrared band is
due both to its own outstanding intensity and to the comparative weakness of all other
bands.
The addition of a new low olectronic level in consefiiienee of each hydro-

jienatioii step of Ihe poriihin system offer.s an iutei-estiMg inobleni for theo-
retical (liscussit)n. OlThand, one would cxpecl increased satui'ation lo de-
crease ratlier than to increase the niiniher of excited electronic states.
If the red bands of dihydroporphin (and the infrared bands of tetra-
hydropoiphin) are brought about (or enhanced) by the presence of electi'ons
associated with the ad(Htional carbon-hychogen bonds, it seems pkiusible
that hght could specifically activate the "extra" hydrogen atoms. This
would make excited chlorophyll an effective
(or bacterioehlorophyll)
—
hydrogen donor a property which may
be of decisive importance for the
photochemical function of this pigment. In Volume I (chapter 19, pages
552-554) the primary photochemical oxidation of chlorophyll was dis-
cussed as a possible mechanism of sensitization in photosynthesis. This
hypothesis would gain consideral)ly in plausi]:)ilit3^ if it could be j^roved that
absorption of light actual)}' activates chlorophyll as a hydrogen donor.
The effect of light on the chlorophyll-ferric iron equilibrium (c/. Vol. I,
page 488) is the only observation at present that lends experimental sup-
port to the concept of chlorophyll as a light-activated reductant.
Stoll (1936) thought that the excitation of chlorophyll by hght activates especially
its "odd" hydrogen atom in position 10.
Studies by Krasnovsky {c.j. chapter 35) indicate the capacity of chloro-

phyll to act also as a light-activated oxidant.
(b) Life-Time of the Excited States of ChloropMjll
The natural life-time of the state Y can be calculated from the integral
area of the red absorption band Xo -^ Fo.
Strictly speaking, one should take into account also the probabilities of transitions
from Fo to the vibrating states Xi,2. which could be derived from the relative intensi-
. .
,
ties of the successive bands in the fluorescence spectrum (c/. fig. 23.2); but we are con-
cerned here with orders of magnitude only.
Prins (1934), who made this integration, obtained for the number of
"absorption electrons" (i. e., the number of harmonic oscillators with the
charge e that could account for the observed intensity of absorption ac-
cording to classical electromagnetic theory)
:
0.24 per molecule chlorophyll a (in ethanol)

(21.1)
/-C 22 per molecule chlorophyll b (in ethanol)
In the quantum theory, / is the measure of the transition probability

between the states Xo and Fo; and the reciprocal of the transition probabil-
mean life-time, t, of the excited state (as far as the latter is limited
ity is the
only by the fluorescent transition Fo -^ Xo) The relation between / and
.
r is:
3 ^, mc 2 1 _ 1.96 X 10-
(21.2)
8 eîr
where m, c, e and tf have the usual meaning. The theoretical mean life-
time of chlorophyll molecules in the lowest excited state (reached liy ab-
sorption of red light) is therefore
8.2 X 10~8 sec. for chlorophyll a (in ethanol)

(21.3) = { 8.9 X 10~* sec. for chlorophyll h (in ethanol)
The higher intensity of the blue-violet absorption band (particularly in

chlorophyll b) indicates that the natural life-time of the excited state B,
is somewhat shorter than that of state F probably 5 —
10 ~^ sec. or less. X
NUCLEAR DISTANCE
Fig. 21.22. Crossing of potential curves.
The actual life-times of chlorophyll in states B, A and F are considerably

shorter than the "natural" life-times. This is indicated by the complete
absence of fluorescence originating in the levels B and A and the relative
weakness of fluorescence originating in level F.
From the complete absence of blue-violet fluorescence in chlorophyll
solutions page 748) it follows that the energy of state B must be dissi-
(c/.
pated within 5 X 10 ~'^ sec. or less. (With the natural life-time of 5 X

INFLUENCE OF MEDIUM 635
10~^ sec, energy dissipation within 5 X 10~^^ sec. would reduce the yield
of fluorescence to <0.01% and thus make it practically unobservable.)
Similar considerations apply to state A. The ease with which states
.4 and B are transformed into state Y (as shown by the excitation of red
fluorescence with yellow or blue light, cf. page 748) indicates that the po-
tential energies of states A, Y and B (plotted against some appropriate
"configiH'ation co-ordinate") give curves of the type shown in figure 21.22.
At point M, the electronic excitation energy of state B is easily transformed
into the (smaller) electronic excitation energy of state Y, plus a large
amount of vibrational energy.
The yield of red fluorescence of chlorophj'll in solution is of the order of
10% (cf. chapter 23). This shows that the actual life-time of state Y in
solution is of the order of one tenth of the above calculated "natural" life-
time, i. c, about 5 X 10"^ sec. The various "quenching" processes that
may contribute to this shortening of the life-time of excited molecules will
be discussed in chapter 23 (page 755).
B. Influence of Medium on Absorption Spectrum of

Chlorophyll and Bacteriochlorophyll*
We have spoken so far of the absorption spectra of chlorophyll antl its

derivatives in solution as though they were determined only by the chemi-
cal structure of these However, the absorption spectra also
compounds.
are affected by changes in the nature of the solvent, and even more strongly
by adsorption on solids, or by the formation of colloidal aggregates. These
spectroscopic changes are caused by interaction between the light-absorbing
molecules and their neighbors. Kundt had noticed as early as 1878 that
the absorption bands of many dyestuffs are shifted toward longer waves
with increasing refractivity of the solvent. This relation appears plausible
in the light of London'swhich establishes a connection between
theor}^,
the capacity of molecules to refract light and the intensity of intermolecular
forces —
both properties being determined by polanzability of the molecules.
Parallelism between molecular attraction and polarizability presumes
the absence of other, chemical or physical forces between the interacting
molecules. Such forces may arise if the molecules bear electric charges,
dipole moments, or possess incompletely saturated valencies; therefore,
we can expect Kundt 's rule to apply primarily only to neutral, nonpolar,
saturated molecules in nonpolar, saturated solvents. The rule may also
apply to series of polar or nonsaturated molecules in which the additional
interactions due to dipoles or residual valencies are approximately constant
(e. g., to a homologous series of alcohols).
London's theory of molecular attraction predicts that solvents with a

high refractive index (i. e., strong polarizability) should exercise strong at-
traction on solute molecules, thereby causing considerable deformation of

their electronic systems and considerable shifts of their energy levels.
The direction of the resulting displacement of the absorption bands depends
on the comparative polarizability of the solute molecules in the ground
state and in the excited state. Figure 21.23 shows that the excitation
SOT.VKNT EFFECT 037
(21.4a) ^"soi. - hv = AS + 8
the red shift thus being decreased by the amount 8.
Probably no other compound has been so often studied from the point
of view of Kundt's rule as chlorophyll. The origin of this interest was the
fact, first noticed by Hagenbach in 1870, that the maximum of the red band
of chlorophyll in living plants is displaced by about 20 mn toward tbe red
end of the spectmm. compai-ed to its position in solution, (lerland (1871)

found that a similar displacement occurs in the case of the absorption bands
of chlorophyll in the yellow and green. It was early suggested that this
position of the bands indicates a peculiar state of chlorophyll in the living

cell, and numerous attempts have been made to reproduce this state in
vitro. We however, in the following review of experimental data,
will see,
that the "red shift" is not a specific effect, and could be caused by various
types of aggregation or complexing.
We with chlorophyll solutions in different organic sol-
will first deal
vents, and then with colloidal solutions, complexes and adsorbates, in which
chlorophyll is associated with proteins, lipides or other carrier;-.
1. Solvent Effect in the Spectra of Chlorophyll and Bactericchlorcphyll
Chlorophyll was one of the dyestuffs whose stutiy caused Kundt (1878)
to postulate a relation between the refractive index of the solvent and the
position of the absorption bands, which became known as "Kundt's rule."
Since then, numerous observations have been made of the spectrum of
chlorophyll in different solvents, e. g., by Baas-Becking and Koning (1934),
Hubert (1935), Wakkie (1935), Katz and Wassink (1939), Bicrmacher
(1939), Egle (1939) and Harris and Zscheile (1943). The agreement be-
tween the different authors is not very satisfactory— for example, Hubert
found 662.5 mju for the position of the absorption peak of chlorophyll a in
methanol, and 604 m/x for its position in ether, while Katz and Wassink
obtained, for the same solvents, 604 and 661 m^, Harris and Zscheile 664
and 660 mfi, and Bass-Becking and Koning 656 and 666 m/x, respectively.
Many discrepancies probably have been caused by the use of poor
spectrophotometric equipment; Mackinney (1938) stressed, for example,
the errors inherent in the identification of the band maximum with the so-
calledband "axis" (cf. above, page 607, and chapter 23, page 744). Other,
and perhaps more important, differences may have been caused by the
—
preparations used often leaf extracts containing chlorophylls a and b
in unknown proportions. It is difficult enough to obtain spectroscopic
reproducibility even with purified preparations of a single chlorophyll com-

ponent! Small solvent impurities, too, may strongly change the spectrum
(c/. p. 647).
to
IS
>
a
o
w.
H
w 0)
X)
O O
o
M
o
o
Ph
o
o
H
«
o
n
o
03
o
CO
O
SOLVENT EFFECT 639
»fl
COS
PQ
05
CO
CO
05-0
J3
13
03
PQ
o
en
Tables 21. VII and 21. VIII contain a summary of the experimental re-
sults. Considering the former table as a whole, one finds only a very
—
rough confirmation of Kundt's rule it consists mainly in the fact that
^max. values greater than 670 ran are found only with solvents whose refrac-
tive index is greater than 1.5. Between Ud =
1.33 and 1.5, the variations
of Xmax. appear small and irregular.
However, if one sorts out solvents of the same chemical type, a more
regular shift at least of the red absorption maximum becomes apparent.
Figure 21.24 summarizes, as an example, the data of Katzand Wassinkand
Harris and Zscheile for nonpolar solvents (curve A) and for alcohols (curve
B).
672 Carbon disulfide.
670
NONPOLAR
I" 668 SOLVENTS
2-Et-l-hexanol
iLl
666
UJ POLAR SOLVENTS P l-Amylol Xylene
g JJ I'Butanol
Benzene
'^2-Me-l-
664 Pôpanol
"Ethanol ^Toluene
Methanol
662
Pentane
® ©iCyclohexane
660
Hexane 1.50 1.60
Fig. 21.24. Absorption maxima of chlorophyll a in solvents of different

refractivity.
The shift of the blue band remains irregular, even in selected series of
solvents. The
highest value of Xâx. of the blue band was found in the
solvent with the lowest refractive index, methanol. (This fa(;t may per-
haps be attributed to a specific sensitivity of the blue-violet band to tautom-
erization— a hypothesis discussed on page 607; tautomerization equilibra
are known to be strongly affected by solvent changes.)
Livingston and co-workers (1949) noted that, in the series of chlorophyll
a solutions in alcohols (from methanol to octanol), the relation between the
two peaks in the blue-violet region changes systematically. In methanol,
they are equally high and separated onl}' by a dip; in octanol, the short-
SOLVENT EFFECT 041
wave peak apiiears as a lower liiimp separated from the main peak by a
trough.
Wakkie (1935), too, divided solvents into classes and gave four
separate Xâx. = /(^) curves —one for nonpolar solvents, one for weakly
polar solvents (ethers, ketones), one for alcohols and one for colloidal solu-
tions in water and glycerol. However, the last curve is not directly com-
parable with the other three, since, as we shall see later, the position of the
X
»-
o
z
Ijj
I
;
for chlorophyll a —
the former being the more po'ar of the two compounds.
For example, according to Harris and Zscheile, the red band of chlorophyll
a lies at 660 mn in ether and 664 m^t in methanol (AX =
4 m^), while the
corresponding values for chlorophyll b are 642.5 and 651 m^t, respectively
(AX = 8.5 m/x). Hydrogen bonding, too, may have to be taken into con-
sideration.
If one compares the effects of varying refractivity of the solvents on the
spectra of different homologous solutes, one may expect the solute with the
stronger polarizability to exhibit the strongest shift. Polarizability in-
creases with the intensity and wave length of the first absorption band.
Thus, the spectra of dyestuffs should be more sensitive to solvent changes
than the spectra of noncolored substances, and the sensitivity of dyes of dif-
ferent color should increase with the shift of the main absorption band to-
ward longer waves. This is confirmed by the finding of Pruckner (1940)
that the solvent effect increases strongly from porphins through dihydro-
porphins (chlorins and phorbins) to tetrahydroporphins (bacteriochloro-
phyll). The first absorption band of chlorophyll is situated further toward
the red and is more intense than the first absorption band of the porphyrins
and the same is true of the first absorption band of bacteriochlorophyll
compared to that of chlorophyll. The two (or four) additional hydrogen
atoms contained in these compounds contribute electrons that are easily
excitable by light (thus giving rise to long-wave absorption bands) and
easily displaceable in electric fields (thus producing strong polarizability).
Figure 21.25 shows the shifts of the band maxima of bacteriochlorophyll
and ordinary chlorophyll in the same solvents. This figure indicates that
on the wâve length scale the solvent effect is about twice as strong for the
first band of bacteriochlorophyll as for the first band of ordinary chloro-
phyll.
Katz and Wassink (1939) extrapolated the curves in figure 21.25 to
vacuum (refractive index 1) and predicted that the absorption peak of free
chlorophyll molecules — if it can ever be determined — will be found at 648
± 5 m/i, and that of free bacteriochlorophyll molecvdes at about 740 m/x.
In piperidine solution, the absorption peak of chlorophyll lies at 642 niyu, i. e., be-
yond the extrapolated Hmit for the free molecule. This demonstrates the existence of
exceptions to Kundt's rule, probably caused by specific chemical interactions between
solvent and solute. Other (less striking) exceptions from Kundt's rule have been dis-
cussed by Mackinney (1938, 1940) and Egle (1939).
Theoretically, it would be more appropriate to plot, in figures 21.24
and 21.25, wave numbers (or frequencies) since these are proportional to
energies and therefore bear direct relationship to the terms in equation
(21.4). In a narrow spectral range, such as is used here for a given band of
a single pigment, linear extrapolation on a wave number scale would give
results not significantly different from those obtained by linear extrapola-
SOLVENT EFFECT 643
tion on a wave length scale. In the comparison of the shifts of bands in

different parts of the spectrum, on the other hand, the use of wave lengths
may be quite misleading; for example, a shift by 10 m/x at 440 m^ is
equivalent, on the energy scale, to a shift by 22.5 m^ at 660 mju.
The effect of solventson absorption maxima of chlorophyll other than

the main red peak has not been studied systematically, but it is known that
in general all of them experience shifts toward longer waves with increasing
refractivity of the solvent (see e. g., Egle 1939). Exact measurements of
this shift prove useful in the interpretation of the spectrum, since ab-
may
sorption bands that lead to the same electronic upper state can be expected,
according to page 636, to show the same shift. Krasnovsky et al. (1949)
found bands II and III to be shifted, in pyridine, by 30-35 m^x (to 643
and 622 m^), while other bands were shifted by 8-15 m/x only.
The solvent effect is not restricted to hand shifts, but also involves
changes in the width and shape of the bands and (perhaps as a conse-
quence of these changes) alterations in the absolute and relative intensities
of the band maxima. In this case, too, bands leading to the same excited
electronic level must show a similarity of behavior (cf. Pruckner 1940).
In the case of chlorophyll, the ratio between the intensities of the blue-
violet and the red peak is quite different in different solvents. While
Table 21. IB showed a value of 1.3 for ether solutions of chlorophyll a, the
ratio drops to 1methanol (Mackinney 1940, Albers 1941, Harris and
in
Zscheile 1943) and rises to approximately 1.5 in dioxane (Harris and
Zscheile 1943) and perhaps also in benzene (according to Hausser's meas-
urements, cf. Fischer and Stern 1940; not confirmed by Table 21. VIII).
Figure 21.26 shows the absorption curves of chlorophylls a and h in a
variety of solvents, according to Harris and Zscheile.
Table 21. VIII
Relative Intensity of Chlorophyll Absorption Peaks in Different Solvents

(after Harris and Zscheile 1943)
Blue max. /red max.
Solvent Chi. a Chi. b
Methanol 1.00 2.85

2-Ethvl-l-hexanol .00 —
2-Methyl-l-propanol
1
02 —
1-Butanol
1
1 03
.
—
Methyl oleate 1.25
.
—
Acetone 1.26 2.95
Olive oil 1.29 —
Isopropyl ether 1 29
Carbon tetrachloride 1 32. 2 54

.
Benzene 1 33.
2 45
.
Ethyl ether 1.33 2.98

Cyclohexane 1 36 —
1-n-Dioxane 1
.
.46 —
G44 ABSORPTION SPECTRA OF PIGMENTS IN VITRO CHAP. 21
SOLVENT EFFECT G45
Characteristic changes apparently occur in the "doublet structure" of

the blue-violet band. In chlorophyll a in alcohols, the two components of
this band and almost equal in prominence. (This sepa-
are well separated
ration of the band two almost equal components may be the cause
into
for the fact that the blue-violet peak is in this case not much higher than
the red peak.) In dioxane, on the other hand, the violet component is a
"satellite" only half as high as the main blue peak (the latter being in this
case almost 50% higher than the peak of the red band). It was mentioned
TT
I 1
Dry benzene
Benzylamine
Benzyl alcohol
A Benzene activated with benzylamine
or benzyl alcohol
o
o
CL
CE
O
cn
m
<
UJ
>
UJ
ir
450 550 650

WAVE LENGTH, m/i
Fig. 21.26A. Absorption spectra of chlorophyll a in pure hydrocarbon and hydro-

carbon containing an alcohol or amine (after Livingston et al. 1949).
on page 607 that these differences may perhaps be indicative of tautomeric

eciuilibria; but this is merely a conjecture.
It wôuld be interesting to evaluate the total areas under the different
curves to find whether the transition probabilities are changed by the sol-
vent, or whether the latter merely affects the shapes of the bands, without
changing their total areas.
An as yet httle investigated subject is the absorption spectrum of dye-
stuffs in general, and of chlorophyll in particular, in mixed solvents. Ob-
servations of this type could give information about the occurrence, and
SOLVENT EFFECT 647
energy, of complex formation of chlorophyll with different organic mole-

cules. Livingston, Watson and McArdle (1949), in a study devoted pri-
marily to the strong effect of small admixtures of polar solvents on the
fluorescence of chlorophyll solutions in hydrocarbons, noted that these ad-
mixtures also changed the absorption spectrum. As an example, figure
21.26 B shows the effect of traces of water on the absorption spectrum of
chlorophyll b in benzene. In the dry solution, both main peaks are lower
and a shoulder appears at about 670 m^t on the long-wave side of the red
LlJ
O
Ll.
Ll.
LlI
O
O
a.
cr
o
(/>
CD Wet
<
LlJ
>
J I I l_ _I I I I
I I
450 550 650

WAVE LENGTH, m/x
Fig. 21.26B. Absorption spectra of chlorophyll 6 in dry and wet benzene.
peak. Figure 21.26 A

shows the effects of two other polar solvents, benzyl-
amine and benzyl on the absorption spectrum of chlorophyll a in
alcohol,
dry benzene. In this case, the main absorption peaks are higher in dry
nonpolar solvent, and no "shoulder" appears on the long-wave end of the
spectrum. A remarkable fact shown by this figure is that the spectrum of
the activated solution (the term "activated" refers to fluorescence, which
is absent in pure benzene), is the same whether activation is due to amine
or to alcohol, although the absorption spectra of chlorophyll a in pure

benzylamine and in pure benzyl alcohol are quite different. This could
mean that polar molecules associate preferentially with a certain tautomeric
form of chlorophyll, and in this way stabilize it; the presence of a small
648 ABSORPTION SPECTRA OF PIGMENTS IiY VITRO CHAP. 21
number of such molecules thus converts the spectrum of "normal" chloro-

phyll into one of "tautomerized" chlorophyll. In pure polar solvent, on the
other hand, polar molecules surround the chlorophyll molecule from all
sides and thus cause a diffei-ent and more radical change in its absorption
spectrum.
The absorption spectrum of the "activated" solution is affected by in-
creasing temperature, indicating a shift toward dissociation of the equilib-
rium
(M+~ = polar molecule, tChl = tautomeric chlorophyll). The interpre-

tation of these interesting results will be discussed in chapter 23 (page 769)
after presentation of the corresponding fluorescence data.
Evstigneev, (xavrilo^'a and Krasnovsky (1949") noted that polar mole-
cules have no effect on absorption spectrum and fluorescence of magnesium-
free compounds (pheophytin and phthalocyanine) and therefore ascribed
this effect to the binding of these molecules bj^ the residual valencies of
magnesium.
A few words can be added here on the effect of dissolved gases on the absorption
spectrum of chlorophyll solutions. Padoa and Vita (1932) described changes in the ab-
sorption spectra of chlorophylls a and b (in benzene solutions) in contact with nitrogen,
oxygen, carbon monoxide and carbon dioxide. A strong effect was observed in the case
of carbon monoxide —
a result taken as an indication of the existence of a chlorophyll-
carbon monoxide complex, similar to carboxyhemoglobin. However, the spectra re-
produced in the paper of Padoa and Vita are so different from the true spectrum of
chlorophyll, that they must have been obtained with some decomposition products
rather than with the intact pigment. Katz and Wassink (1939) found practically iden-
tical extinction curves for colloidal aqueous bacteriochlorophyll extracts in atmospheres
of oxygen, hydrogen sulfide, nitrogen, hydrogen and air.
Evstigneev, Gavrilova and Krasnovsky (1949^) asserted that the pres-

ence of oxygen does have a certain effect on the spectrum of chlorophyll
(a + b, or pure h) in toluene. Upon evacuation, the absorption coefficient
decreased in both maxima, which were shifted slightly toward the red. In
chlorophyll b, a new maximum of absorption appeared, when air was re-
moved, at 670 mju. These changes were reversible but irreversible changes
;
were noted in the ultraviolet part of the spectrum. Similar changes were
observed in carbon tetrachloride and heptane, but not in pyridine, ethanol,
acetone or benzene. Addition of one drop of alcohol, pyridine or acetone
to 10 cc. toluene destroyed the effect of evacuation. Later (1949^) the
same investigators found that the effects they had ascribed to the admission
of airwere actually caused by the admission of water vapor.
These results obviously bear a relation to Livingston's conclusions that
chlorophyll is present, in nonpolar solvents, in a state different from that to
I
COLLOIDAL AND ADSORBED CHLOROPHYLL 049
which converted by the presence of even traces of an alcohol or water.

it is
Evstigneev et al. (19490 suggested that in the absence of polar molecules
chlorophyll is dimerized, and that the dimer is dissociated by oxygen

molecules, dimerization being due to unsaturated magnesium valencies,
which can also be saturated by oxygen. Their subsequent results (19492)
made this interpretation of ox.ygen action unnecessary.
In solvents of intermediate type, or in mixed (or simply not extremely
purified) solvents, both types of interaction may occur simultaneously.
Changes in the absorption spectra of chlorophyll can be caused, accord-
ing to the observations of Livingston and co-workers (1948, 1949), also
by small ciuantities of admixtures other than polar solvents. Examples are
iodine, bromine, ferric and (Rabinowitch and Weiss, 1937), and,
eerie salts
in the case of chlorophyll h (but not chlorophyll a!) phenylhydrazine. Some
,
effects of this type might be similar to those of polar solvents, i.e., they may
be caused by reversible formation of molecular complexes. Mostly, how-
ever, they are due to irreversible chemical changes such as allomerization
(or, more generally, oxidation), or reduction (which is likely in the case of
chlorophyll h and phenylhydrazine); these phenomena do not belong

under the heading of "effects of the medium on
the absorption spectrum of
chlorophyll," but rather under that of "chemical reactions of chlorophyll,
revealed by spectroscopic measurements" {cj. pages 450-467 in Vol. I, and
chapter 37 in this volume).
2. Absorption Spectrum of Colloidal and Adsorbed Chlorophyll
Colloidal aqueous solutions of chlorophyll are obtained by mixing a

molecular solution of the pigment (in alcohol or acetone) with water. The
spectrum of the resulting solution depends on the conditions of mixing;
this the reason that earlier investigators could not agree on the position
is
of the band maximum of colloidal chlorophyll. Herlitzka (1912), Will-

statter and Stoll (1918) and Baas-Becking and Koning (1934) reported
that this maximum
coincides with that of chlorophyll in leaves, i. e., lies
close to 680 Ivanovski (1907, 1913) and Hubert (1935), on the other
m^-
hand, found the maximum at 668 m/x, i. e., in the same region as in many
true solutions. Hubert noticed, however, that the position of the maxi-
mum was affected by the degree of dispersion of the colloidal sj^stem Ad- :
dition of magnesium chloride, which caused a growth of the particles (and

finally led to flocculation) shifted it by as much as 8 mn from 668 to —
676 m/z. Later, Wakkie (1935) and K. P. Meyer (1939) found that the es-
sential factor is not the .size of the colloidal ])articles, but their internal
density, i. e., the concentration of chlorophyll molecides in them.
A colloidal solution prepared by Meyer by rapid addition of 3 volumes

of water to 1 volume of a chlorophyll solution in ethanol was clear, trans-
parent and nonfluorescent. Its particles were 0.5 to 3 /i in diameter.
The band maximum was at 670 m/x, and the shape of the extinction curve
was similar to that of the original solution in ethanol. On the other hand,
a colloidal solution prepared by adding quickly 0.6 volume of water to 1
volume of ethanol and then diluting by 6.4 volumes of water, was turbid
and opalescent. Its particles had a diameter of 1-3 n, i. e., were not sub-
stantially largerthan those of the first, transparent colloid, but they con-
tained more pigment. The absorption maximum of this colloid was sit-
uated further toward the red, at 673 m^, and the whole shape of the extinc-
tion curve was more like that of the leaves, as shown by figure 21.27
(Meyer described the spectrum of this colloidal preparation as "identical"
with that of the leaves, but figure 21.27 does not justify this statement).
By counting the particles of the colloid, Meyer found that the concentra-
48
400 450 500 550 600 650 700

WAVE LENGTH, m/i
Fig. 21.27. Transmission curves of leaves (1 and 2) and of colloidal chlorophyll

solutions (3) (after K. P. Meyer, 1939).
tion of chlorophyll in the particles of the turbid solution was of the order of
0.13 mole/1., i. e., similar to that in the chlorophyll grana in the leaves (cf.
Vol. I, chapter 15, page 411). Even these ''densely packed" colloid par-
ticles are still far from "sohd," but contain up to 90% solvent.
Because of the intensity of the absorption bands, the extinction curves
of dyestuffs usually are measured with concentrations of the order of 10"^
to 10~'* mole/I. No deviations from Beer's law (i. e., no changes in the ex-
tinction curve with concentration) were observed in chlorophyll solutions
in this range of concentrations. With very thin glass cells (~0.1 mm.
deep), dyestuff solutions containing 10 ~^ mole/1, can be investigated, but
even this is a hundred times more dilute than the 0.1 mole/1, reached in
Meyer's colloidal particles and also present in the chlorophyll grana in
leaves. Wakkie (1935) emulsified a chlorophyll solution in ether in a satu-

rated water-ether mixture, bubbled air through it and watched the changes
of the absorption spectrum as the ether evaporated and the chlorophyll in
the drops grew more and more concentrated. At a certain concentration
(not estimated in the paper) the absorption band began to shift to the red,
from 666 to 676 m^. This in(Hcates that a shift similar to that caused by
the accumulation of chlorophyll in colloidal particles can be produced also
by an increase of its concentration in true molecular solution. When the
ether was completely evaporated, the remaining dry chlorophyll had an
absorption maximum at 679 mû. This agrees approximately with the
measurements of Hubert (1935), who gave 680.5 m/x for the absorption
maximum of solid chlorophyll (thin film of dried chlorophyll on glass).
The exchange between excited and normal chloro-

possibility of energy
phyll molecules and the spectroscopic effects of this exchange which must —
—
increase with increasing concentration of the pigment will be discussed in
chapter 32, in connection with the concept of the "photosynthetic unit"
and similar hypotheses.
A similarity between the absorption spectra of solid chlorophyll in suspension and

of chlorophyll in the living cell was first claimed by Ivanovski (1907, 1913).
thus appears that a shift of the red chlorophyll band toward the
It
longer wave lengths (approaching its position in the leaf spectrum) can be
achieved not only by interaction with a solvent of high polarity or polariz-
ability, but also by interaction with other chlorophyll molecules. This of-
fers several alternatives for the interpretation of the state of chlorophyll
in vivo. In the case of bacienochlorophyll, Katz and Wassink (1939)
noted that the absorption band of evaporated pigment was shifted by not
more than 2.5 m^ from its position in solution, while in live bacteria (and
in colloidal extracts from bacteria) the same band is shifted toward longer
waves by as much as 80-100 mn. In this case, the position of the band in
the spectrum of the living cells definitely indicates interaction with other
cell components and not merely close mutual proximity of the pigment
molecules.
A "red shift" of the absorption bands be ob-
of chlorophyll probably can
tained also by adsoi-plion on appropriate carriers: According to Seybold
and Egle (1940), the red absorption band of chlorophyll adsorbates on
starch is situated at 662 mî, i. e., in the same region as in solution. Figure
21.27A, taken from Seybold and Weissweiler (1942), shows the absorption
peaks of chlorophylls a and b, adsorbed on sugar, in the following positions:
Chlorophyll a, m/x, and chlorophyll b, 662 and 488 m^— values
670 and 450
that correspond to shifts by 10 and 20 m/x for the a-component and 19 and
35 mn for the 6-component (compared to band positions in ether). The
652 ABSOnrTION RPKCTRA OF TIOMENTS /,V VITRO CHAP. 21
100
Sugar
/ /
90 /a I
\ I I
\ I I
\i I
t I
i\
80 I
\t 1
\ I
\ I
70
2
O
1-
o
UJ
o
10
Ol 1 1 I 1 I I 1 I I I I r I
400 500 600 700

WAVE LENGTH, m/x
Fig. 21.27A. Tran.smission and reflection curves of chlorophylls a and h

adsorbed on sugar (after Seybold and Weissweiler 1942).
1.5
(A) Spinach leaf extract in water (B) Spinocti leaf extract in 2% digitonin,
diluted 1: 10 with water
0.0 1 1 ^
400 500 600 700 400 500 600 700
WAVE LENGTH, m^x WAVE LENGTH, m,i
Fig. 21.28. Absorption curves of spinach leaf extracts (after Smith 1941).
figures for the blue-violet bands are so high as to call for a recheck (see
chapter 22, page 705, for the position of the blue-violet bands in the living
cells). Eisler and Portheim (1922) and Noack (1927) stated that the ab-
sorption spectra of chlorophyll adsorbates on proteins are "similar to those
of the living cells," but gave no figures.
-(A) Distilled water

r rr
The relatively strong absorption of the crude extract in the violet may be
due to the presence of yellow pigments its stronger absorption in the far ;
red (>700 mû) was attributed by Smith to scattering. However, in-

creased absorption in the far red has also been observed by Noddack and
2.0
(a) Phosphate buffer,^ {b) Distilled water
pH6.6, 1/15 M
820
n
— —r— — —
-|
860 900
I I
940 740 780

1
820
r
r
T
860 900
— — — —940
1
i I
WAVE LENGTH, m/i WAVE LENGTH, m/i
(c) Culture medium No. 231 (.d) I. Cells in distilled

water
2. Alcoholic extract
1.5
o
o
1.6
860
— 1
1.4
— —900— 940
r" I
I I
740 780 820

—
900
1
— —940
I
740 780 820

WAVE LENGTH, vnfi wavE LENGTH, m/i
Fig. 2 1.30 A. Comparative absorption spectra of cell suspensions and pigment ex-
tracts of strain D purple sulfur bacteria in different media (after Katz and Wassink
1939). Curve 1, cells; curve 2, extract. See page 656.
Eichhoff in Chlorella suspensions (see fig. 22.21), although these investiga-

tors used an integrating method, which wâs supposed to give true absorp-
tion values, free of scattering effects.
Rabideau, French and Holt (1946) gave absorption curves (obtained
with an Ulbricht sphere) and transmission curves (obtained with a Beck-
man spectrophotometer) for chloroplast dispersions prepared by means
1 r
of supersonic waves. These curves, which can be found in figure 22.15.

indicate that enhanced extinction in the far red is characteristic of trans-
mission much more than of true absorption— thus supporting Smith's ex-
planation and contradicting the results of Noddack and Eichhoff.
2.0
(o) Phosphate buffer, AJH 6.6, \/\5M {b) Distilled water
ENERGY, e.v. ENERGY, e.v.

1.6 1.5 1.4 1.3
740 780
1.5
820
T
1
— ———
860 900
1.4
r I I
1.3.
r-^
940 740
I
780 820 860 900 940

WAVE LENGTH, m/i WAVE LENGTH, m^
2.0
(c) Culture medium No. 231 {.d) \. Cells in distilled water
2. Alcoholic extract
1
— ———
I
1
I I r—
860
— r—900— 940
I
820 860 900 940 740 780 820

WAVE LENGTH, m/i WAVE LENGTH, 'm/i
Fig. 21. SOB. Comparative absorption spectra of cell suspensions and pigment ex-
tracts of Rhodospirillum rubrum in different media (after Katz and Wassink 1939).
Curve 1, cells; curve 2, extract. Concerning the difference between the cell (and the
colloidal extract spectra), in A and B, see page 703.
According to Smith, the molar extinction coefficient of chlorophyll in

the maximum of the red band is approximately the same in the aqueous
extract containing digitonin and in ether or acetone solution. In other
words, the shift in the position of this band from GGO to G75 m^ occurs
without a change in its intensity.
Absorption curves of colloidal chlorophyll-protein extract were given

also by Katz and Wassink (1939). Figure 21.29 shows the red band in the
spectra of Chlorella suspensions, and in colloidal extracts from the same
cells (a) in phosphate buffer pH 6.0, (6) in distilled water, (c) in Knop's
culture medium and (d) in fresh egg albumen. The position of the maxima
are but slightly different in all these ciu'ves (approximately 080 m/x);
the shapes of the curves are, however, affected by the nature of the medium,
particularly in extracts a and b. The solution in egg albumen has an ex-
tinction curve practically identical with that of the living cells. Fig. 21.29e
shows, as a contrast, the strong shift occurring upon extraction of the pig-
ment with alcohol. Similar curves were given by Katz and Wassink (1939)
and French (1940) for colloidal extracts from purple bacteria (c/. figs. 21.30).
A transmission curve of a water extract of the blue-green alga Chroococ-
cus can be found in figure 22.48B. In these extracts, the phycocyanin-
protein forms a true colloidal solution, while the other pigments probably
are in the same state of dispersion as in extracts from green algae and leaves.
C. Absorption Spectra of the Carotenoids*

1 . Experimental Results
The extinction curves of carotenoids in organic solvents have been in-

vestigated by numerous authors, among whom we may mention Willstatter
and Stoll (1913), Pummerer and Rebmann (1928), McNicholas (1931),
Smakula (1934), Gillam (1935), Sprecher von Bernegg, Heierle and Almasy
(1935), Miller (1935, 1937), Strain and co-workers (1938, 1942, 1943, 1944),
French (1941), Beadle, Zscheile and co-workers (1942, 1944, 1945) and
Zechmeister and co-workers (cf. review by Zechmeister 1944).
Recently, a number of absorption curves were determined by Karrer
and co-workers, and were reproduced in a monograph by Karrer and Jucker
(1948).
The absorption spectra of carotene and "leaf xanthophyll" in the in-
frared were observed by Stair and Coblentz (1933). They show a series
of absorption bands characteristic of long unsaturated carbon chains;
many of them coincide closely with the absorption bands of phytol (cf.
Table 21.IIA).
The spectra of all carotenoids in the visible are characterized by two or
three intense bands near the violet end of the spectrum. Depending on
how bands extend into the blue and green, the color of the pig-
far these
ments may be yellow, orange or even red. The position of the absorption
bands depends, often even more strongly than in the case of chlorophyll,
— —
ABSORPTION SPECTRA OF THE CAROTENOIDS 657
on the state of the pigment and the surrounding medium. Table 21. IX
shows that the direction of the band shift in different solvents is the same
as for chlorophyll i. e., they are displaced toward the longer waves with
increasing polarity and polarizability of the solvent. From ether to carbon
disulfide, the "red shift" amounts to 43 m/x for carotene /3 and 35 m/x for
luteol —
as compared to only 10 m/z for chlorophyll. However, the effect of
transition from a nonpolar to a polar solvent of approximately the same
polarizability e. —
g.,hom ether to ethanol seems to be smaller for the carot-
enoids than for the more polar chlorophylls (c/. Table 22. IX).
A still stronger displacement sometimes occurs in aqueous colloidal
solutions. According to Karrer and Strauss (1938), the maximum of
band I of carotene is shifted, from 480 m^ in hexane and ether, to 510 m/x,
or even 535 m^, in hydrosols. This, too, is a much wider shift than was ob-
served in chlorophyll colloids. (Because of this shift, some carotene sols
are red, while their molecular solutions are jêllow.)

In addition to a shift of the band maxima, changes of medium may also
cause a broadening of the carotenoid bands. This effect appears to be
particularly strong in the case of some algal carotenoids. For example,
the curves given for fucoxanthol spectrum by Strain, Manning and Hardin
(1944) show a considerable flattening of the absorption peaks and extension
of the absorption band toward the longer waves in ethanol as compared to
petroleum ether. The spectrum of peridinol, a pigment of the dinoflagel-
lates, shows a similarity strong solvent effect.
The spreading of the absorption bands of the carotenoids into the green,
rather than a shift of their peaks, probably explains the color of broA\Ti algae
and diatoms. The striking difference between their color and that of green
plants appears inexplicable if one considers only the solution spectra of
fucoxanthol 21.35A) or peridinol, since these are almost identical

{cf. fig.
with the spectra of the carotenols of green plants (e. g., luteol and zeaxan-
thol).
Recently, Karrer and co-workers (1943, 1948) published an absorption
curve of fucoxanthol in hexane (fig. 21.36) which shows a comparatively
slow decline of absorption toward the longer waves. The absorption re-
mains marked up to 550 mju, while that of most other carotenoids drops to
zero at 500 mû. The reason for the difference between this curve (which
togethei- with that of chlorophyll could explain the brown color of fucoxan-
thinol-bearing algae) and that given l)y Wald for thesame solvent, remains
unexplained.
The curves given by Wassinkand Kersten (1946) for the absorption spec-
trum "yellow" and the "orange" fraction of the carotenoids from the
of the
diatom Nitzschia dissipata also show an extension of the absorption in the
second fraction (in methanol) to about 550 m/z, the peaks being situated
I I - I
CO
CO
GO Ci
O (M
1
CO
CO
1 — o
^-H
^-^ C^
-—
"-^
S —, CO
q2
^
02
.2 "o _
^
&|
3 03
a;
O ^ 2 'o
c
-G S
li' 0)
Q) 3 >^ o 3 rt
^
o
C!
C3 O
lO
05 O GO «D
»0 lO lO -* '^ CO t^
I
O
PQ
c
I—
o
CO
K
X 00 (N 00
I
ci
O 00 t-
(M
* Tjl Tfl Tfi Tf* ^ TjH
_
lO
. .
H O
b -3
m O
<! ni I—
3
y.
a
<1 a
X
O
M
H
«
O
10
n
<:
ABSORPTION SPECTRA OF THE CAROTEN
05
e
S3
C
G
o3
1^
O O
a
Xi
a
&
o
>
o3
03
2^
o
n
GfiO ABSORPTION SrECTRA OF PIGMENTS IN VITRO CHAP. 21
300
c
o
o
400 420 440 460 480 500 520 540 400 450 500
WAVE LENGTH, n\^ WAVE LENGTH, m,i
Fig. 2 1.35 A. Alisorption spectrum of Fig. 21.35B. Absorption spectra of

fucoxanthol in hexane (after Wald 1942). diatoxanthol, diadinoxanthol, neodia-
(For a different curve for the same solution dinoxanthol, zeaxanthol and luteol (after
see fig. 21.36.) Strain, Manning and Hardin 1944).
3.2-
o
S 2.7
a
o
o
2.2
L7
at 426, 450 and 476 mn (Table 21. IX). The curves given by them for
Hve diatoms, colloidal extracts and pigment solutions in organic solvents
indicate a considerably increased absorption, in vivo, in the region 500-
560 mju.
Table 21. IX shows that the absorption bands of many bacterial carote-
noids are situated further toward the red than those of green plants and
—
algae a relation that reminds one of that between chlorophyll and bac-
teriochlorophyll.
Figures 21.31 and 32 show the extinction curves of three carotene iso-
mers and figures 21.33 and 21.34 those of luteol the most common carote- —
nol of green plants (c/. Vol. I, page 415). Figures 2 1.35 A and B repre-
sent the spectra of several carotenols of broAvn algae, diatoms and dino-
flagellates.
300
260 4510 A
220 -o4780
4680
180
140 >4920
100
60
J I L J I L
4 8 12 16 20 24 28
SPECTRAL REGION ISOLATED, m^
Fig. 21.37. Effect of width of spectral region isolated on the

specific absorption coefficient of /3-carotene at selected wave lengths
White, Beadle and Roach 1942). The changes in a
(after Zscheile,
observed by Miller with single monochromator (•) were not found in
the work with a double monochromator (O).
The absorption bands of the carotenoids are much broader than those of chloro-
phyll; they are therefore less sensitive to changes in the width of the slit, as illustrated
by figure 2i.37.
2. Theoretical Considerations
Carotenoids are 'polyene dyestuffs, i. e., their color is due to the chromo-
phoric properties of a straight chain of conjugated double bonds. Syn-
thetic polyenes, and the cyanine dyestuffs used in photographic sensitiza-
tion, are other examples of the same type. With increasing length of the
conjugated chain, the absorption bands are shifted regularly toward longer
wave length, and their intensity becomes greater.
These simple relations between color and molecular structure make polyene dyes
This problem was treated by Paul-
particularly suitable objects for theoretical studies.
and the concept of resonance, and by
ing (1939), who used the method of atomic orbitals
Mulliken (1939, 1941), who used the method of molecular orbitals. According to Paul-
ing, thefundamental resonance possibilities of polyene molecules are provided by the
which results in the transfer of an electron from one end of
shifting of the double bonds,
the molecule to the other. If we consider, e. g., a straight conjugated chain with an
even number of carbon atoms {A), the shift of all double bonds to the left will produce
structure B and a shift to the right, structure C:
{A) CH3CH=CHCH CHCH=CHCH3

(B) +CH3=CHCH=CH CH=CHCHCH3
(C) CHaCHCH^CH CH=CHCH=CH3 +
Each of the states B and C has a large dipole moment; but, since the two moments
have opposite directions, and B and C have equal probabilities, the molecule will show
no dipole moment at all, both in the ground state of the molecule and in the lowest ex-
cited states formed by resonance between the same three structures. However, the
transition from the normal state to an excited state of this type lias a "transition mo-
ment" whose order of magnitude is that of the dipole moment of the individual structures
B and C This is a very large moment, and it increases with length of the chain. In
wave mechanics, the probability of a spectroscopic transition between two states {i. e.,
the intensity of the corresponding absorption hne or band) is determined by the magni-
tude of the "transition moment." This explains why polyene molecules have strong
absorption bands, and why their intensity increases with the greater chain length.
The second approach to the same problem is that of the theory of "molecular orbi-
tals." It considers the actual state of the molecule without decomposing it into imagi-
nary resonating components. It tries to assign the electrons not to definite atoms or
bonds, but to definite lA-functions (orbitals) of the molecule as a whole. In a long chain
of conjugated double bonds, some of these orbitals include the nuclei of all atoms in the
chain, and electrons assigned to them can be considered as moving freely through the
whole chain (this being the counterpart to the "shifting of double bonds" in the reso-
nance theory). A conjugated double bond chain has, in this theory, a certain similarity
to a metallic wire.
An investigation of a molecule by this theory consists in the determination of the
qualitative characteristics of available orbitals and the evaluation of the relative ener-
gies of the states obtained by different assignments of the electrons to the orbitals.
Let us consider (Mulliken 1939) a straight chain of n carbon atoms (n = even num-
ber) and n/2 double bonds (the presence of symmetrical end groups on both ends of this
— —
conjugated chain which is common in carotenoids does not alter the problem). It
contains n "unsaturation orbitals," sweeping over the whole conjugated chain, of which
n/2 are "bonding" (i. e., electrons assigned to them stabilize the molecule), and n/2
"antibonding." Each orbital can, as usual, hold two electrons, so that the n available
"unsaturation electrons" are just enough to fill the n/2 bonding orbitals, thus giving a
singlet normal state. The transfer of any one of these electrons into any one of the n/2
antibonding orbitals leads to an excited state; there are therefore nV4 groups of ex-
cited states. Each group consists (because of the interaction of orbitals with the elec-
tron spin) of one triplet and one singlet state; however, because of the prohibition of
singlet-triplet transitions in light atoms, only the n^/4 singlet states are of importance
for absorption. The more numerous are the excited states. It
longer the chain, the
can be shown that the "center of gravity" of these states in the energy diagram remains
more or less unchanged, while the lowest excited states shift closer and closer to the
ground state as the chain grows longer. This is shown schematically in figure 21.38.
At the left we have the energy diagram of an A2-molecule, at the opposite end, that of an
infinite chain of A nuclei; the long- wave absorption limits are represented by the ar-
rows; they become shorter and shorter, i. e., the absorption shifts further and further
to the red, with increasing chain length, until it
extends into the infrared.

MuUiken has shown that, if the chain is a
straight as possible {i. e., if all carbon atoms
in the chain are in trann positions), the transi-
tion to the lowest excited state (arrow in fig.
21.38) more piobable than all the other transi-

is
tions together. This means that the intensity

of the absorption band with the lowest fre-
quency must increase steadily with increasing
chain length. We have thus obtained theo-
retical interpretations, both of the gradual shift
of the absorption band to longer waves, and of
the increase in its intensity with the growing
Fig. 21.38. Shift of first absorp- length of the chain.
tion band with increasing length of
conjugated chain (thickness of ar-
The two or three separate maxima
row indicates intensity). observed in carotenoid spectra may
mean as many distinct electronic transi-
tions; but more probably they correspond to coexcitation of one or several
vibrational quanta. The distance between maxima (ca. 1500 cm.~0 is
of the order of magnitude of vibrational quanta in organic molecules.
D. Absorption Spectra of the Phycobilins*
The absorption spectra of the phj'cobilins have been observed in living

algae, in aqueous colloidal extracts of chromoproteids and in organic solu-
tions of chromophores. The results are somewhat confused because both
phycocyanin and phycoerythrin apparently occiu* in several modifications
of slightly different color. (These modifications might be due either to
minor variations in the structure of the chromophores, or to the association
of the same chromophore with different proteins.)
The first extensive data on the absorption spectra of the phycochromo-
proteids were given by Schiitt (1888). Among the more recent papers on
this subject are those of Lemberg (1928, 1930), Svedberg and Lewis (1928),
Svedberg and Katsurai (1929), Dhere and Fontaine (1931), Svedberg and
Eriksson (1932), Roche (1933), Katz and Wassink (1939) and French and
co-workers (1948,1951).
Bibliography, page 67 1.
.
ABSORPTION SPECTRA OF THE PHYCOBILINS 065
The only data on the absorption spectra of the isolated (^. e.,
availal)le
protein-free)chromophores are those of Lemberg (1930). He gave the
following wave lengths for the maxima of the absorption bands:
Compound Medium Wave length
Cyanobilin (from Porphyra tenera) HCl (cone.) 598 m/i

Acid CHCI3 606 mju
Erythrobilin (from Porphyra tenera) HCl (cone.) 498 niM
Combination of the pigments with protein shifts the bands toward the
red; l)ut the amoimt of this shiftis not adequately described by compari-
son of the above-cjuoted figures with the positions of the absorption maxima
of aqueous from algae, because the data on free pigments refer to
exti'acts
strongly acid solutions, while those on chromoproteids relate to neutral or
only weakly acid solutions. The main absorption maximum of the phyco-
cyanin-protein complex lies at 615 mû in the pH range between 3.5 and
7, but is displaced, in concentrated hydrochloric acid, by as much as 41
ran toward the red (to 656 m/x). Comparing this strongly acid solution of
the chromoproteid with an equally strongly acid solution of the chromo-
phore, we find a "red shift" by as much as 58 mû; comparison with a neu-
tral chromoproteid solution would indicate a shift of only 17 m/x.
Similar figures were given more recently by Wassink (1948) for cyano-
bilin from blue-green Oscillatoria (Xmax. = 620 mî for the chromoproteid,
610 mn for the solution of the cyanobilin in chloroform, and 600 mju for its
solution in HCl)
Extinction curves of aqueous chromoproteid colloids were given by
Svedberg and Lewis (1928), Svedberg and Katsurai (1929), Svedberg and
Eriksson (1932), and French and co-workers (1948,1951).
Figure 21.39 shows the extinction curves of the phycoerythrins from
five different algae. Three maxima (566, 540 and 498 mix) are always
present, but with variable relative intensities, pointing to the existence of
three different forms of the pigment (perhaps the same chromophore linked
to different proteins). Van Norman et al. (1948) found only two absorp-
tion peaks (550 and 495 m/x) in aqueous extract from Iridaea. It also has
several bands in the ultraviolet.
Similar observations were made with phycocyanin. In phycocyanin
from a Rhodophycea (e. g., Ceramium rubrum. and Porphyra tenera), Sved-
berg and Katsurai (1929) found two bands in the visible, at about 615 and
550m)u and ultraviolet bands at 355, 271 and 240 m/x. In the phycocyanin
from a Cyanophycea (e. g., Aphanizomenon flos aquae) they found only one
visible band, at 615 mju, and ultraviolet bands at 368 and 277 mix.
GG6 ABSORPTION SPECTRA OF PIGMENTS IN VITRO CHAP. 21
Ceramium
450 500 550 600 650 450 500 550 600 650 450 500 550 600 650
WAVE LENGTH, m/i WAVE LENGTH, m/i WAVE LENGTH, m/i
Fig. 21.39. Specific absorption spectra of phycoerythrins from different algae
(after Svedberg and Eriksson 1932).
80
-
60
» 40
20
X X
ABSORPTION SPECTRA OF THE PHYCOBILINS 667
(1930) himself noted that the specific extinction of the phycobiHns is ten
—
times stronger than that of hemoglobin while his analysis indicated the
presence of only one half mole pigment per Svedberg unit of protein in
phycobilins, as against one mole pigment per unit of protein in hemoglobin.
If Lemberg's analysis is in error, and the content of phycobilins in the
chromoproteids is as high or even higher than that of hemin in hemoglobin,
the molar extinction coefficients of the phycobilins, given in Table 21,
will have to be proportionally reduced.
Table 21.
Estimated Molar Extinction Coefficients of Phycobilins

(in the Band Maxima)
Pigment
Bibliography to Chapter 21
Absorption Spectra of Pigments in Vitro
A. Absorption Spectra of Chlorophyll and Its Derivatives
1913 Willstatter, R., and StoU, A., U titer suchungen ilber Chlorophyll. Springer
Berlin, 1913.
1914 van Gulik, D., Ann. Physik, 46, 147.
1918 Willstatter, R., and StoU, A., Unter suchungen iiber die Assimilation der
Kohlensdure. Springer, Berlin, 1918.
1922 Metzner, P., Ber. deut. botan. Ges., 40, 125.
1928 Lewkowitsch, E., Biocheni. J., 22, 777.

1929 Noack, K., and Kiessling, W., Z. physiol. Chem., 182, 13.
1930 Noack, K., and Kiessling, W., ibid., 193, 97.

1931 Noack, K., and Kiessling, W., Z. angew. Chem., 44, 93.
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1933 Winterstein, A., and Stein, G., Z. physiol. Chem., 220, 263.
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1934 Prins, J. A., Nature, 134, 457.
Zscheile, F. P., /. Phys. Chem., 38, 95.
Zscheile, F. P., Botan. Gaz., 95, 529.
1935 Zscheile, F. P., Cold Spring Harbor Symposia Quant. Biol., 3, 108.
Stern, M., Kleine Mitt. Mitglied. Ver. Wasser-, Baden-, u. Lujthyg., 11, 47.
Sprecher von Bernegg, A., Heierle, E., and Almasy, F., Biochem. Z., 283
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Stern, A., and Wenderlein, H., Z. physik. Chem., A174, 81.
1936 Stern, A., and Wenderlein, H., ibid., A176, 81.
StoU, A., Naturwissenschaften, 24, 53.
Hagenbach, A., F., and Wiedemann, E., Helv. Phys. Acta, 9,
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Stern, A., Wenderlein, H., and Molvig, H., Z. physik. Chem., A177, 40.
1937 Stern, A., and Molvig, H., ibid., A178, 161.
Seybold, A., Pla7ita, 26, 712.
Vermeulen, D., Wassink, E. C., and Reman, G. H., Enzymologia, 4, 254.
French, C. S., J. Gen. Physiol., 21, 71.
Rabinowitch, E., and Weiss, J., Proc. Roy. Soc. London, A162, 251.
1938 Stern, M., Kleine Mitt. Mitglied. Ver. Wasser, Boden- u. Lufthyg., 14, 39.
Tiegs, E., ibid., 14, 34.
Mackinney, G., Plant Physiol., 13, 123.
Stern, A., Z. physik. Chem., A182, 18G.
1939 Seybold, A., and Egle, K., Sitzber. Heidtlberg. Akad. Wiss. Math, naturw.
Klasse, 1939, No. 1, 7.
Katz, E., and Wassink, E. C., Enzymolgia, 7, 97.

Stern, A., and Pruckner, F., ibid., A185, 140.
Biermacher, 0., Thesis, Univ. Fiil)ourg (Switzeiiand).
Egle, K., Sitzber. Heidelberg. Ak. Wiss. Math.-Nat. Klasse, 1939, No. 1, 19.
Meyer, K. P., Hclv. Phys. Acta, 12, 349.

EIELIOGRAPHY TO f'TT APTF.R 21 600
1940 Mackinney, G., J. Biol. Chem., 132, 91.

French, C. S., /. Gen. Physiol, 23, 483.
Fischer, H., and Stern, A., in Fischer and Orth, Die Chemie des Pyrrols,
Vol. II. 2, Akad. Verlagsges., Leipzig, 1940.

1941 Mackinney, G., /. Biol. Chem., 140, 315.
Zscheile, F. P., and Comar, C. L., Botan. Gnz., 102, 463.
Comar, C. L., and Zsclicile, F. P., Plant Physiol, 16, 651.
Albers, V. INI., Gibson Island A. A. A. S. Symposiion, on Photosynthesis
(unpubhshed).
Epstein, L. F., Karush, F., and Rabinowitch, E., /. Opt. Soc. Am., 31, 77.
1942 Strain, H. H., and Manning, W. M., /. Biol. Chem., 144, 625.
Comar, C. L., and Zscheile, F. D., Plant Phjsiol, 17, 198.
Haskin, H. H., /. Biol Chem., 144, 149.
Zscheile, F. P., Comar, C. L., and Mackinney, G., Plant Physiol, 17, 666.
Comar, C. L., Ind. Eng. Chem., Anal. Ed., 14, 877.

Pruckner, F., Z. phydk. Chem., A190, 101.
1943 Comar, C. L., Benne, E. J., and Buteyn, E. K., ibid., 15, 524.
Manning, W. M., and Strain, H. H., /. Biol Chem., 151, 1.
Harris, D. G., and Zscheile, F. P., Botan. Gaz., 104, 515.
Aronoff, and Calvin, M., J. Org. Chem., 8, 205.
S.,
Calvin, M., Ball, R. H., and Aronoff, S., /. Am. Chem. Soc., 65, 2259.
Lewis, G. N., and Bigeleisen, J., ibid., 65, 1144,
1944 Zscheile, F. P., Comar, C. L., and Ilariis, D. G., PlaJit Physiol, 19, 627,
Rabinowitch, E., Rev. Modern Phys., 16, 226.
1946 Wassink, E. C, and Kersten, J. A. H., Enzymologia, 12, 3.
Erdman, J. G., and Corwin, A. H., J. Am. Chem. Soc, 68, 1885.
1948 McBrady, J., and Livingston, R., J. Phys. & Colloid Chem., 52, 662.
Koski, V. M., and Smith, J. H. C, J. Am. Chem. Soc, 70, 3558.
Livingston, R., et al, First Annual Report on ONR Project 059028.
1949 Livingston, R., in Photosynthesis in Plants. Iowa State College Press,
Ames, 1949, pp. 179-196.
Kuhn, H., /. Chem. Physics, 17, 1198.
Simpson, W. T., ibid., 17, 1218.
1950 Piatt, J. R., ibid., 18, 1168.
Longuet-Higgins, H. C, Rector, C. W., and Piatt, J. R., ibid., 18, 1174.
1951 Tanada, T., A7n. J. Botany, 38, 276.
B. Influence of Medium on Absorption Spectrum of Chlorophyll and Bacterio-

chlorophyll
1870 Hagenbach, A., Ann. Phys. (Poggendorf) , 141, 245.

1871 Gerland, E., ibid., 143, 585.
1878 Kundt, A., Ann. Phys. {Wiedemann), 4, 34.
1907 Ivanovski, D., Ber. deut. botan. Ges., 25, 416.
1912 Herlitzka, A., Biochem. Z., 38, 321.
1913 Ivanovski, D., Ber. deut. botan. Ges., 31, 600.
Ivanovski, D., Biochem. Z., 48, 328.
1918 Willstatter, R., and Stoll, A., Untersuchungen iiber die Assimilation der
Kohlensaure. Springer, Berlin, 1918.
1922 Eisler, M., and Portheim, L., Biochem. Z., 130, 497.
1927 Noack, K., Biochem. Z., 183, 135.
1932 Padoa, M., and Vita, N., ibid., 244, 296.
1934 Baas-Becking, L. G. M., and Koning, H. C, Proc. Ac. Set. Amst., 37, 674.
1935 Hubert, B., Rec. trav. botan. neerland., 32, 323.

Wakkie, J. G, Proc. Acad. Sci. Amsterdam, 38, 1082.
1937 Rabinowitch, E., and Weiss, J., Proc. Roy. Soc. London, Al62j 251.
1938 Smith, E. L., Science, 88, 170.
Mackinney, G., Plant Physiol, 13, 427.

1939 Katz, E., and Wassink, E. C., Enzymologia, 7, 97.
Biermacher, 0., Thesis, Univ. Fribourg (Switzerland).

Egle, K., Sitzber. Heidelberg. Ak. Wiss. Math.-nat. Klasse, 1939, No. 1, 19.
Meyer, K. P., Helv. Phys. Acta, 12, 349.

1940 Mackinney, G., Plant Physiol., 15, 359.
Fischer, H., and Stern, A., in Fischer and Orth, Die Chemie des Pyrrols,
Vol. II.2, Akad. Verlagsges., Leipzig, 1940.
Pruckner, F., Z. physik. Chem., A187, 257.
Seybold, A., and Egle, K., Botan. Arch., 41, 578.
1941 Smith, E. L., /. Gen. Physiol, 24, 565.
Albers, V. M., Gibson Island A. A. A. S. Symposium on Photosynthesis
(unpublished).
1942 Seybold, A., and Weissweiler, A., Botan. Arch., 43, 252.
1943 Harris, D. G., and Zscheile, F. P., Botan. Gaz., 104, 515.
1946 Rabideau, G. S., French, C. S., and Holt, A. S., Am. J. Bot., 33, 769.
1948 Livingston, R., et Annual Report on ONR Project 059028.

al, First
1949 Livingston, R., et al, Second Annual Report on ONR Project 059028.
Livingston, R., Watson, W. F., and McArdle, J., /. Ayn. Chem. Soc, 71,
1542.
Evstigneev, V. B., Gavrilova, V. A., and Krasnovsky, A. A., Compt. rend.
(Doklady) acad. sci. USSR, 66, 1133.
Evstigneev, V. B., Gavrilova, V. A., and Krasnovsky, A. A., ibid., 70, 261.
Krasnovsky, A. A., Brin, G. P., and Vojnovskaja, K. K., ibid., 69, 393.
C. Absorption Spectra of the Carotenoids
1913 Willstatter, R., and StoU, A., Untersuchungen ilber Chlorophyll. Springer,
Berlin, 1913.
1928 Pummerer, R., and Rebmann, L., Ber. deut. chem. Ges., 61, 1099
1930 Kuhn, H., Winterstein, A., and Kaufmann, W., ibid., 63, 1489.
1931 McNicholas, H. J., /. Research Natl Bur. Standards, 7, 171.
1932 Kuhn, R., and Brockmann, H., Z. physiol Chem., 206, 41.
von Euler, H., Karrer, P., Klussman, E., and Morf, R., Helv. Chim. Acta,
15, 502.
1933 Kuhn, R., and Brockmann, H., Ber. deut. chem. Ges., 66, 407.
Rudolph, H., Planta, 21, 104.
Stair, R., and Coblentz, W. W., /. Research Natl. Bur. Standards, 11, 703.
BIBLIOGRAPHY TO CHAPTER 21 671
1934 Smakula, A., Angew. Chem., 47, 657.

1935 Gillam, A. E., Biochem. J., 29, 1831.
Karrer, P., and Solmssen, U., Helv. Chim. Acta, 18, 1306.
Sprecher von Bernegg, A. S., Heierle, E., and Almasj^ F., Biochem. Z.
283, 45.
Miller, E. S., Botan. Gaz., 96, 447.
1937 Miller, E. S., Plant Physiol, 12, 667.
1938 Karrer, P., and Strauss, W., Helv. Chim. Acta, 21, 1624.
Strain, H. H., Leaf Xanthophylls, Carnegie Inst. Wash. Publ. No. 490.
1939 Mulliken, R. S., /. Chem. Phys., 7, 121, 364, 570.
Pauling, L., Proc. Natl. Acad. Set. U. S., 25, 577.

1940 French, C. S., Botan. Gaz., 102, 406.
1941 Mulliken, R. S., and Rieke, C. A., Phys. Soc. of London, Reports on Prog-
ress in Physics, 8, 231.
1942 Zscheile, F. P., White, J. W., Beadle, B. W., and Roach, J. R., Plaiit
Physiol, 17, 331.

Beadle, B. W., and Zscheile, F. P., J. Biol Chem., 144, 21.
Wald, G., unpublished.
Strain, H. H., and Manning, W. M., J. Am. Chem. Soc, 64, 1235.
1943 Strain, H. H., and Manning, W. M., ibid., 65, 2258.
Strain, H. H., and Manning, W. M., Carnegie Inst. Wash. Yearbook, 42,
79.
Karrer, P., and Wiirgler, E., Helv. Chim. Acta, 26, 116.
1944 Strain, H. H., Manning, W. M., and Hardin, G., Biol Bull, 86, 169.
Zechmeister, L., Chem. Revs., 34, 267.
Nash, H. A., and Zscheile, F. P., Arch. Biocliem., 5, 77.
1945 Nash, H. A., and Zscheile, F. P., ibid., 7, 305.
1946 Wassink, E. C., and Kersten, J. A. H., Enzijmologia, 12, 3.
1948 Karrer, P., and Jucker, E., Carotinoide, Birkhauser, Basel, 1948.
D. Absorption Spectra of the Phycobilins
1888 Schiitt, F., Ber. deut. botan. Ges., 6, 36, 305.

1928 Lemberg, R., Ann. Chem. (Liebig's), 461, 46.
Svedberg, T., and Lewis, N. B., /. Ain. Cliem. Soc, 50, 525.
1929 Svedberg, T., and Katsurai, T., ibid., 51, 3573.
1930 Lemberg, R., Biochem. Z., 219, 255.
1931 Dhere, C, and Fontaine, M., Co7npL rend., 192, 1131.
Dhere, C., and Fontaine, M., Ann. inst. oc'anog., 10, 245.
1932 Svedberg, T., and Eriksson, L B., /. Am. CJiem. Soc, 54, 3998.
1933 Roche, J., Arch. phys. biol, 10, 91.
1939 Katz, E., and Wassink, E. C., Enzymologia, 7, 97.
1948 Wassink, E. C., Enzymologia, 12, 362.
Van Norman, R. W., French, C. S., and Macdowall, F. D. H., Plant
Physiol, 23, 455.
1951 French, C. S., Proc. Soc. Exptl Biol (in print).
— — : —
Chapter 22
LIGHT ABSORPTION BY PIGMENTS IN THE LIVING CELL
The determination of light absorption in sokitions or other homogeneous

media is a routine measurement, and the results permit a simple interpre-
tation (based on Beer's law) in terms of molecular absorption coefficients
(also called "extinction coefficients" —
since attempts to discriminate be-
tween these two terms have not been successful in practice). The experi-
mental determination of the absorptive power of plants is less simple, and
often the exact meaning of the results is problematical. The measurement
of light energy absorbed by leaves, algal thalli or cell suspensions is com-
plicated by scattering, which is significant not only in multicellular tissues,
but even in suspensions of single cells (because the dimensions of the cells,
~10~^ cm., are larger than the wave length of visible light, ~5 X 10~^
cm.) .The interpretation of the results in terms of the absorption constants
of the pigments is complicated, not only by the light scattering on phase
boundaries, but also by inhomogcneous distribution of pigments in cells and
tissues, and by the shifting and deformation of the absorption bands caused
by adsorption and complexing. Let us assume, for example, that we have
measured the energy, /, of a beam of light falling on a vegetable object
leaf, thallus or cell suspension —
and the energy, /', emerging from this
object, taking care to integrate the latter over all directions so as to include
both the light transmitted forward (T) and the light reflected backward
(R), and thus to avoid the "gross" errors that may be caused by scattering.
If now we try to apply to the results Beer's law
(22.1) I' (= T + R) = 7 X 10-"^''
with the intention of calculating an absorption coefficient, a, we find, first

of all, that scattering has made the length of the path of the light in the ab-
sorbing medium d in eq. 22.1 —indefinite (even its average — Mestre's "de-
tour factor" —
not constant, but depends on wavelength, cf. Kok 1948).
is
In the second place, we note that the local accumulation of pigments in the
chloroplasts has made the concentration of absorbing molecules in the path
of the individual light beams c in equation (22.1) — variable: Some light
l)eams pass between the chloroplasts and encounter no pigment molecules
at all (a phenomenon to which we will refer later as the "sieve effect").
G72
: :
GENERAL REMARKS ON LIGHT ABSORPTION BY PLANTS 073
In the overcoming these two difficulties, we succeed in obtain-

last place, if,
ing a reliable value of a, it is an average absorption coefficient of a mixture

of several pigments, whose individual absorption spectra in solution we may-
know, but whose bands are variously shifted and deformed, in the living
cell, by adsorption and complexing. The task of apportioning the total
absorption at a given wave length to the component pigments (which re-
(juires the knowledge of their individual absorption coefficients and of their
distribution in the cell) often proves impossible of achievement, except by
gross simplifications.
We shall deal first, in part A, with the determination of the amount of
light energy absorbed })y plants, and then, in part B, with the spectroscopic
properties of individual pigments in vivo and their contribution to the total
absorption.
A. Light Absorption by Plants*
1. General Remarks
In working with solutions in plane-parallel glass cells, the detennination

of the absorbed light energy (A) requires two measurements: Either one
measures the incident light flux (/) and the transmitted light flux (T), or,
more commonly, one compares T with the flux To transmitted by a blank
cell containing pure solvent. A is calculated by one of the following:
(22.2a) A = I - T or
(22.2b) A = To - T
Both Sive first approximations. Equation (22.2a) neglects all reflections; a

second approximation can in this case be obtained by subtracting from /
the light flux reflected from the front wall of the absorption cell
(22.3) A = 1(1 - r) - T
where r is the reflection coefficient of the cell material. However, reflec-
tion from the front wall is only part of the total reflection in the cell; to
make our equation exact, we should write, in place of (22.3)
(22.4) A = I - T - R
meaning by R the total reflected flux.

Equation (22.2b) is a better first approximation than (22.2a), because
it neglects only the difference between the reflections from the solution cell
and the blank cell. The fluxes reflected from the front walls of both cells
are identical, but those reflected from the back walls are different (because
:
674 LIGHT ABSORPTION BY PIGMENTS IN VIVO CHAP. 22
of the weakening that hght suffers in passing through the absorbing me-
dium). A second approximation, which can be substituted for (22.2b), is:
(22.5) A = To - T + I{1 - r)[r(l - lO-^^-^j]
where a is the absorption coefficient of the soUition and d the thickness

of the cell. In this equation, consideration has been given to one reflection
from the front wall (factor 1 — r) and one reflection from the back wall
(factor in brackets) However, these reflections are only the beginning of
.
an infinite series (as with two mirrors on opposite walls) The exact equa- .
tion for A in terms of the properties of the blank cell is
(22.6) A = {To - T) + {Ro - R)
where Ro and R are the total light fluxes reflected by the blank cell and the
solution cell, respectively.
If a and r are known, exact values of To, T, Ro and R can be obtained
by the summation of infinite power series.
"trapped" between the walls leaves the cell after an odd number of pas-
If the light
sages, it added to the transmitted ilux; if it escapes after an even number of passages,
is
it is added to the reflected flux. Consequently, the series for T contains only even
powers of r and odd powers of 10""'^, and the series for R only odd powers of r and even
powers of lO""''. (The ratio of the sums for T and To is given in equation 22.12.)
If a and r are R
(and Ro) must be determined experimentally.
unknown,
Since repeated reflection lengthens the average path of the light in the
absorption cell, it increases absorption. In the case of homogeneous solu-
tions in plane-parallel glass cells, this increase represents only a minor cor-
rection (to be estimated on page 711); we mention merely to illustrate
it
the complications in the measurement of light absorption that arise from

the presence of phase boundaries. In nonhomogeneous systems, the com-
plications are similar in principle, but much more important quantita-
tively. more numerous, but also stronger (because
Reflections are not only
of the varying angles with which the light strikes the interfaces) and they ;
are supplemented by refractions and total inner reflections, which all af-
fect the length of the path of the light beam and the direction in which it
leaves the medium.
Leaves and thalli are heterogeneous systems, with numerous phase
boundaries between ah- channels, cell w'alls, cytoplasm, vacuoles, plastids
and starch grains and the passage of light through plants or plant organs
;
is, therefore, a very complicated phenomenon. It has been repeatedly dis-

cussed—by Willstatter and Stoll (1918), Briggs (1929), Mestre (1935),
Seybold and co-workers (19321-2, 1933'-'-, 1934, 1943), Schanderl and
Kaempfert (1933), Meyer (1939) and Loomis (1941, 1949), among others—
but these discussions have not gone far beyond the qualitative stage.
: ;
GENERAL REMARKS ON LIGHT ABSORPTION BY PLANTS 675
A suitable statistical theory (c/. page 713) may permit the calculation
of A from measurements of light transmission in one direction, made with
two or more different optical densities of the scattering material (e. g.,
with a series of several leaves, or with several cell suspensions of different

concentration or layer thickness) . However, it is better not to rely on such
theoretical equations, but, particularly in working with leaves or thalli,
actually to measure the light fluxes transmitted and reflected in all direc-
tions. Having determined experimentally both T and R, one can use the
exact equation (22.4) for the evaluation of A. The time to use theoretical
equations for combined absorption and scattering comes when one is not
satisfied with the knowledge of the amount of absorbed energy, but wants
also to know the absorption coefficients, e. g., as indicators of the molecular
state of the pigment in the living cell.
Attempts have been made to use "blanks," for example, white parts of variegated
leaves (cf. Linsbaur, 1901, Brown and Escombe 1905, Weigert 1911, Meyer 1939,
1932i'2, 1933i, 1934), or algal thalli from which the pigments had been ex-
and Seybold
tracted {cf. Reinke 1886), or tissues bleached by long exposure to light (c/. Wurmser
1926), and to imitate in this way the method usually applied to transparent media. In
the latter case, the blanks provide an automatic correction for reflection (cf. page 673)
in the case of plants, they were intended to provide a correction also for scattering.
However, the approximation (22.2b), which is generally satisfactory in work with trans-
parent media, may give entirely erroneous results when applied to optically inhomogene-
ous systems. This was pointed out by Willstatter and Stoll (1918) and Warburg (1925)
when they criticized the absorption calculations of Weigert (1911). The error is caused
by the large difference between the fluxes R and Ra {cf. equation 22.6) reflected by the
green and the colorless leaf. A green leaf may transnut about 10% and reflect another
10% of incident white light, while a similar, pigment-free leaf may transmit 50% and
reflect the other 50%. If the absorption of the green leaf is calculated from these figures
by means of equation (22.2b), the result is A = 40%, which is only one half the correct
value (80%)!
Therefore, if one wants to determine absorption, A, by comparison of a green leaf
with a pigment-free leaf, one has to use the complete equation (22.6), i. e., to measure
the four quantities To, Ro,T and R, while measurement of only three quantities, /, T and
R, is sufficient to the same determination with a single leaf, according to equation
make
(22.4). Furthermore, in plant work, one is never certain whether the "blank" is entirely
free of pigments: Accordmg to Seybold and co-workers (1933S 1942), so-called "white"
leaves of Acer negundo absorb10-20% of incident white light; this absorption may be
caused by nonplastid pigments, or by a small quantity of residual chlorophyll or caro-
tenoids.
The transmitted light flux (T) and the reflected light flux (R) can both
contain a coUimated component, T, or R^ (light transmitted in the direc-
tion of the incident beam, or reflected according to the laws of specular re-
flection),and a diffuse component, T^ or Ra, so that equation (22.4) can
be written more explicitly as follows
(22.7) A = I - {r. + Ti) - {R. + Ri)

G76 LIGHT ABSORPTION BY PIGMENTS IN VIVO CHAP. 22
Measurements of T and R must include both the colhmated and the diffuse
components.
If the leaf is sufficiently thick, and not glossy, it acts as an ideal scatterer,
i. e., the intensity of light scattered in a given direction is proportional to
=*'**;S^.
o
UJ
u.
UJ
o
a:
LiJ
I-
UJ
<
<
TRANSMITTANCE AND REFLECTANCE OF LEAVES 677
whereas thick leaves with glossy surfaces may show a completely diffuse
transmission, but a marked specular reflection.
Figure 22.2 shows the forward and back scattering of light by a small
vessel containing a suspension of Chlorella cells, as observed by Noddack
and Eichhoff (1939). The sharp peak (C) at 180° is caused by specular
reflection from the glass wall.
In practical work, one can often di'op the distinction between T and R
and measure the total scattered flux .S' ^ {T -{- R) b\' means of some inte-
grating device:
(22.8) A = I - S
An example is the study by Rabideau, French and Holt (1946) of the ab-
sorption spectra of leaves and pigment extracts.
2. Average Transmittance and Reflectance of Leaves and Thalli in

White Light. Intensity Adaptation and Movements of Chloroplasts
The first measurements of the proportion of white light transmitted by
leaves were carried out by Sachs in 1861. Later this magnitude was meas-
ured by Detlefson (1888), Linsbauer (1901), Brown and Escombe (1905),
Purevich (Purjewitsch) (1914), Schanderl and Kaempfert (1933), Seybold
(1932i'2, 19331-2, The
1943), Loomis, Carr and Randall (1941,1947,1949).
transmittance oj algae was investigated by Reinke (1886), Wurmser (1921)
and Seybold and co-workers (1934, 1942).
The first measurements of the reflectance of leaves were made by Co-
blentz in 1912, and were followed by those of Pokrovski (1925), Shull
(1929), Seybold and co-workers
(19322,1933i'2,1942,1943) and Loomis, Carr
and Randall (1941,1949). The only data on the reflectance of algae are
those of Seybold and co-workers (1934, 1942, 1943).
Brown and Escombe (1905) and Purevich (1914) found comparatively
high values — of the order of 20% —for the transmission of (infrared-free)
white light by average leaves. Seybold (1932) suggested that these re-
sults were falsified by the inclusion, in the measured transmitted flux, of
the thermal radiation of the leaves. In agreement with Pokrovski (1925),
Seybold found that an average fully green leaf transmits not more than
10% of infrared-free white light. Leaves are almost transparent in the
far redand near infrared (c/. figs. 22.30 and 31). Therefore, transmission
values obtained by means of thermopiles (or other infrared-sensitive in-
struments) are deceivingly large if the light used for the measurements
contains a large proportion of infrared radiations. According to Loomis,
and co-workers (1941,1949), an average leaf transmits 30% of total sun-
light, including the infrared. With artificial light sources of lower tempera-
ture, the over-all transmission may be much greater. Selenium barrier

layer photocells ("photronic cells"), because of their low sensitivity in the
infrared and in the far red, indicate, in direct sunlight, a transmission of
only 5 to 10%, depending on the thickness of the leaf (c/. Seybold 1932^,
and Egle 1937).
The light absorption by a leaf depends on its thickness and the concen-
tration of the pigments. As mentioned above, fully green leaves transmit
and reflect only 10 or 15% of incident visible light, and absorb as much as
85 or 90%; on the other hand, green onion skins, one cell thick, transmit
85%, reflect 10% and absorb less than 5% (Seybold 19322). In chapter 15
70 -
I 80
OC
O
</)
5 90
400
MOVEMENTS OF CHLOROPLASTS 679
shade leaf of Fagus, as compared to a sun leaf of the same species. The two
lowest curves in figure 22.14 indicate a sightly larger difference between the
spectral transmission curves of a dark-green and a light-green Hibiscus
leaf. Finally, figure 22.10 illustrates the effect of extreme variations in
chlorophyll content, such as occur in aurea leaves. Here, a few per cent
of the normal chlorophyll content (cf. Table 15.1, Vol. I) suffice to produce
from two thirds to nine tenths of normal absorption in the region between
520 and 700 m^.
Without varying the concentration of the pigments, many plants have
it in their power to adjust the light absorption by the displacement or re-
orientation of the chloroplasts. These tactic reactions, discovered by Bohm

in 1856, were investigated by Stahl (1880, 1909), Senn (1908, 1909, 1917,
1919), Liese (1922) and Voerkel (1933), among others. It was found that
each chloroplast moves independently, i. e., it is not carried by streaming

of the protoplasm. In moderate light, the chloroplasts gather on the illumi-
nated front walls and orient themselves so as to present their large cross-
1 I
Apostrophe Antistrophe Diastrophe Porastrophe
Fig. 22.4. Schematic representation of different chloroplast orientations (after

Benecke and Jost 1924). Arrows show direction of light incidence.
sections to the light ("antistrophe," "epistrophe" and "diastrophe" in

figure 22.4; the produced by one-sided, and the other two by
first one is
two-sided, illumination). In strong direct light, on the other hand, the

chloroplasts turn their axes parallel to the light beams and line the side
walls of the cells ("parastrophe" in fig. 22.4). During the night, they often
—
assume characteristic "night positions" congregate around the nuclei, or
disperse throughout the cytoplasm, or line the internal walls ("apostrophe"
in fig. 22.4). The largest variety of chloroplast movements has been ob-
served by Senn in green and brown algae, and in diatoms. In leaves, the
chloroplasts in the parenchyma cells assume positions similar to those
shown in figure 22.4, but the chloroplasts in the palisade tissue usually re-
main arrayed along the side walls, leaving the end walls free. Instead of
moving bodily these chloroplasts merely change their shape In strong dif- :
fuse light, they spread flat against the walls, whereas in weak light they
protrude into the cytoplasm, without losing contact with the walls. lUu-
mination with strong parallel light may, however, produce antistrophe in

these cells as well.
Obviously, the effect of parastrophe is to decrease, and that of epis-
trophe, antistrophe or diastrophe to increase the absorption of hght. How
130
120
no
100
90
en
en
80
en
70
<
a.
60
50
<
UJ
40
o
30
20
10
Fig. 22.5. Chloroplasts in Funaria

(after Voerkel 1933): above, in liglit
(epistrophe) ; below, in darkness (apos-

trophe).
MOVEMENTS OF CHLOROPLASTS 681
in Table This table indicates that in blue-violet light increase in

22.1.
transmittance may be by as much as one-third (from 19 to 25%).
Table 22.1
Transmittance of a Leaf of Adiantum cuneatum

(after Schanderl and Kaempfert 1933)
White
light Red Yellow Violet
(inchiding and and and
Light exposure infrared) infrared green blue
In diffuse room light, % 31 34 16 19

25
After 4 hr. exposure to sun, %. 37 42 18
With some plants {e. g., T radescantia ?>mrfis). Schanderl and Kaempfert
found a reversal of the effect after the first hour of illumination {cf.
half
fig. 22.6) ; they attributed it to increased scattering, caused by the forma-

tion of starch grains.
Schanderl and Kaempfeit did not prove that increased transparency of sun-exposed
leaves was due entirely to reorientation of the chloroplasts, and not, e. g., to a
partial
bleaching of the pigments. However, the results of Willstatter and StoU (1918), which
showed no change in chlorophyll concentration after strong illumination {cf. Vol. I,
chapter 19, page 549), argue against the second explanation.
. X««1(-X-)H( "X ^X-

100
90
80
»? 70 - BLUE LIGHT
/ /BLUE-GREEN LIGHT
YELLOW-GREEN LIGHT/
L5 20 2.5 3.0 3.5 4.0 45

LOG INTENSITY, lO^cal/cm.^ hr.
Fig. 22.7. Orientation of chloroplasts in Funaria in light of different color

(after Voerkel 1934).
The phototaxis caused mainly by blue-violet

of the chloroplasts is
Hght and is 22.7).
entirely absent in red light It must thus be sensi- (fig.
tized by the carotenoids rather than by chlorophyll. This is not a proof

(a) (b)
Fig. 22.8. Leaves of Naegelia zebrina (after Mecke and Baldwin 1937). Normal
leaves, left; water-filled leaves, right; photographed on (a) panchromatic plate and on
(6) infrared-sensitive plate (max. 850 mju)- Increase of transmittance by decreased
scattering best recognizable on the infrared photograph.
Fig. 22.9.Leaf of Tussilago farfara on black background (after Schan-

derland Kaempfert 1933). Dark sections imbibed with water, therefore
more transparent than the air-filled section.
TRANSMITTANCE AND REFLECTANCE OF LEAVES 683
that the whole phenomenon is unrelated to photosynthesis, since in natural
light fields, red light does not occur without the presence of some blue-
violet hght as well.
Detlefson (1888) and Purevich (1914) found that leaves become less transparent
in the presence of carbon dioxide, but Ursprung (1918) was unable to confirm this result.
This effect, if at all real, could be attributed to increased formation of starch grains.
Scattering of light in leaves can be decreased by injection of water into

the air channels (e. g., by evacuation under water; cf. figs. 22.8 and 22.9).
According to Seybold (1933-), water-filled leaves of land plants (as well as
those of aquatic plants of the type of Elodea) transmit about twice as much
light as air-filled leaves. For example, a submerged leaf of Potomageton
alpinus was found to transmit as much as 22% of white light (Seybold
1932); similar figures were obtained by Seybold (1934) for the transmis-
sion by algae {Chloj-ophyceae, Phaeophyceae and Rhodophyceae). In-
creased transparency of water-filled leaves or thalli is due to reduced dif-
fuse reflection (5% instead of >10%), rather than to weaker absorption.
As a rough approximation, it can be assumed that average land leaves
transmit 10% of (infrared-free) white light (400-700 m/x), reflect 10%o and
absorb 80% while average aquatic plants or algal thalh reflect 5%, trans-
;
mit 15% and absorb 80%.

Loomis (1947) found, for 28 species of leaves, transmissions between 2
and 9%, and reflections of the same order of magnitude. Yellow tobacco
leaves transmitted -^ 30% of visible light, and light yellow leaves, 36% of
visible and 53% of total sunlight. Normal leaves of Nicotiana and Quercus
transmitted 5-7% of visible, and 25-30% of total sunlight.
According to Seybold, a single chloroplast transmits from 30 to 60%
of visible light (depending on spectral distribution), absorbs 30 to 60% and
reflects about 10%. As mentioned in chapter 19 (Vol. I) an average leaf
contains the equivalent of from five to ten complete layers of chloroplasts;
these can easily account for the above-estimated absorption of 80% of the
incident white light. For a detailed discussion of the light absorption in
successive layer of chlorophyll molecules or plastides, see Seybold and
Weissweiler (1943).
In regularly patterned systems of colored and colorless materials, the absorption

sometimes depends upon the angle of incidence. WTien this is the case the absorption of
diffuse light may be different from that of collimated light. Seybold (1933'') made a
search for such an effect in leaves, but without sucêsSi The reason probably is that
leaves are such strong scatterers that collimated light is converted into practically com-
pletely diffuse light, long before it emerges from the leaf.
684 LIGHT ABSORPTION BY PIGMENTS JN VIVO CHAP. 22
3. Absorption by Nonplastid Pigments
When the total absorption of hght by a leaf, thallus or cell suspension

has been determined, the question arises as to what part of this absorption
is due to the chloroplast pigments. It has been assumed by many authors
—^from Reinke (1886) to Noddack and Eichhoff (1939) that a certain —
part of the absorption of white light in plants is due to the "colorless"
parts of the tissue, the cytoplasm, cell sap, nuclei, starch and cellulose.
Seybold arbitrarily ascribed one eighth of the total absorption to these
components, and seven eights to the chloroplast pigments. An absorption
curve of a white Pelargonium leaf, given by Seybold and Weissweiler (1942),
shows considerable absorption near the blue-violet end of the visible spec-
trum.
Of course, no really colorless substance can absorb visible light. But the
plant cells contain coloring materials associated with the cell walls or with
the cell sap rather than with the plastids, such as flavones, tannins, etc.
Some of these substances are only weakly colored —ûsually yellow; others,
although intensely colored, are present only in small quantities, as com-
pared to the plastid pigments. In some species, however, flavones and
anthocyanines are so abundant as to give the leaves a striking red color
(leaves of the purpurea variety, and many young leaves in the spring).
The color of these leaves advertises the fact that much of the light energy
they absorb goes to nonplastid pigments.
Figure 22.10 shows, as an example, the spectral transmission and re-
flection curves of three varieties of Corylus avellana, normal, aurea and
purpurea. Curves 1 and 2 illustrate the effect on light absorption of ex-
treme variations in the concentration of chlorophyll (c/. page 678), while
curve 3 shows the considerable increase in absorption, particularly in the
green, caused by the presence of anthocyanines in purpurea leaves. (Light
absorption in aurea and purpurea leaves will again be discussed in chap-
ters 28 and 30, in relation to the yield of photosynthesis.)
As far as green leaves and algae are concerned, the participation of non-
plastid pigments in light absorption remains a moot question. It probably
varies widely from species to species. For example, according to Thimann
(unpublished), leaves of Phaseolus vulgaris contain a large quantity of yel-
low, water-soluble pigments. The same is true of the needles of the coni-
fers (Burns 1942).
It was mentioned above (page 675) that "white" leaves may absorb
10 or 20% of incident white light (Seybold 1933S 1942, cf. fig. 22.12). This
absorption, too, is probably due to nonplastid pigments.
Most investigators who measured the yield of photosynthesis in rela-
tion to the amount of absorbed light silently assumed that the absorption
NONPLASTID PIGMENTS 685
by the nonplastid components was negligible; but this assumption is not,

or is not always, justified, particularly in work with blue-violet light. It is
therefore advisable to ascertain, before undertaking quantitative work on

photosynthesis, whether the plants chosen are free from, or at least poor
in, nonplastid pigments.
WAVE LENGTH, m^
400 500 600 700
30 '
' '
1
'
-x-"
(0) /
20
10
iS
C 30
o
u
_l
(xl
20
10
g
tn
lOOF
<
90
80
Q.
a:
o
en
OQ 70
<
60
50
40
686 LIGHT ABSORPTION BY PIGMENTS IiV VIVO CHAP. 22
cells —
bleached with bromine does not appear reliable, since bleaching by bromine may
not be restricted to plastid pigments.
It was suggested that the proportion of the total light absorption by leaves due to
plastid pigments can be determined by extracting the latter and comparing the absorp-
Timiriazev (1885) and Lazarev (1924,
tion of the extract with that of the original leaf.
1927) based this suggestion on the assumption that the absorption of white light by ex-
tracted pigments is about equal to the absorption by the same pigments in the leaf.
However, and band shifts can make the two magnitudes
light scattering, "sieve effect"
quite different. True, some of these factors enhance the absorption, while others
weaken it, but it would be an unusual coincidence if the net result were exactly nil.
Experimentally, Noddack and Eichhoff (1939) found that the increased absorption by
almost exactly compensates for the decreased absorption
live Chlorella cells in the far red
in the region 520-680 m/x (c/. fig. 22.21); but Seybold and Weissweiler (1942) could not
confirm this result, and found, to the contrary, that throughout the visible spectrum the
absorption by live cells is more complete than that by the extracts.
B. Spectral Properties of Plants*
1. Empirical Plant Spectra
from the preceding discussion that what is usually called a

It is clear
"leaf spectrum," or even a"spectrum of a cell suspension," is not the true
spectrum of the pigments contained in these materials (meaning by "true
spectrum" the plot of the absorption coefficient of the pigment mixture
16
EMPIRICAL PLANT SPECTRA 687
100
o
UJ
_l
li.
tlJ
a.
£
o
in
(/)
z
<
400 500 600 700

WAVE LENGTH, m,i
Fig. 22.12. Transmission (Jg), reflection {Rg) and absorption (A^,) of light by
a green leaf of Pelargonium zonale, and the corresponding constants for a white
leaf (index w) (after Se.ybold 1933). The figure indicates considerable absorp-
tion by "white" leaves, particularly in the blue- violet region. For other absorp-
tion curves of "white" leaves, see Seybold and Weissweiler (19430.
4.4
o
o
4.01—1
400 450 500 550 600 650
WAVE LENGTH, m^
Fig. 22.13. Transmission spectrum (log Ta/T) of leaves of Fatsia and Acuba
(after Meyer Absolute values of ordinates adjusted to give best agreement
1939).
with spectrum of extracted pigments.
against wave length). One may measure /, T and R {or S = T -{- R),
plot T/I, R/I, or A/I (= [/ - (T + R)]/I = [I - S]/I) against wave
length, X, and call the resulting curves "transmission spectra," "reflection
en
en
ir
350 400 450 500 550 600 650 700 750

WAVE LENGTH, m/x
Fig. 22.14. Transmission (T/I) of dark-green, light-green and yellow leaves of
Hibiscus rosa-sinensis, and of equivalent quantities of pigments in ether (after Loomis,
Carr and Randall 1941). With allowance for reflection, the leaves absorbed more
light than the extracted pigments in the green part of the spectrum, and less in the blue
and red.
•^"^^vÔOOSAmg./ml. ,
Chlorella
(Emerson
and Lewis)
(f)
Sphere
o
-1.4 -
400 500 600 700 800 400 500 600 700 800
WAVE LENGTH, m;i WAVE LENGTH, m/i
Leaf and chloroplast absorption spectra (on a log density basis and ad-
Fig. 22.16.
same height at 670 m/u.) (after Rabideau, French and Holt 1946). The
justed to the
Beckman curve of the chloroplastin solution (chloroplasts disintegrated by supersonics)
is an / - T plot (;'. e., it represents absorption plus scattering); the Ulbricht sphere
curve an / — T — R plot (pure absorption). An absorption curve of Chlorella (after

is
Emerson and Lewis) and a photosynthesis action curve (after Hoover et al.) are given
for comparison.
100
90
80
70
60
g
en 50
if)
z 40
<
a:
30
20
10
100
G, Fucus single cells according to Gaidukov
90 R, Phyllitis occordingto Relnke
£, Brown cells according to Engelmonn
80
500 600 700 •-

400 500 600 700
Fig. 22.19. The transmission spectra Fig. 22.20. Transmission T, reflec-
of brown algae (after Seybold 1934). tion R and absorption A of the red
Maximum of transmission is at 600 mî alga Delesseria (according to Seybold
instead of ^^550 mju as in green plants 1934). More recent curves for Delesseria
and algae (figs. 22.17-18) because of and Porphyra are in Seyboldgiven
the presence of fucoxanthol. For ab- (1943); and Gigartina, see
for Iridaea
sorption curves of Fucus and Laminaria, Van Norman, French and Macdowall
see Seybold and Weissweiler (1943')- (1948).
500 600 700 800

WAVE LENGTH, m/i
Fig. 22.21. Absorption spectra, (/ - S)/I = {I - T- R)/I, of Chlor-

suspension (after Noddack and Eichhoff 1939). About 1.1 X 10' cells
ella
and 1.6 X 10"^ g. chlorophyll a and 6 in 1 ml. A, 35 ml. pigment extract
in a vessel 3.90 cm. thick; B, cell suspension, same volume, same vessel.
spectra" or "absorption spectra," respectively; or one may use colorless

specimens as "blanks," determine To and iô (or So = To + ô), plot T/To,
R/Ro or .Sy,So against X and designate these curves as "transmission,"
"reflection" or "absorption" spectra, respectively —but, although each of
these plots is legitimate as representation of a certain property of the
specimen investigated, they are all different. (For example, fig. 22.11, p.
686, shows the transmission spectrum and the reflection spectrum of the
same leaf.) The true absorption spectrum on the other hand is an intrinsic
property of a molecular species (or, in the case of a mixture, the average of
intrinsic properties of several molecular species).
We will discuss the quantitative analysis of the empirical "leaf spectra"
1.4
L2
1.0
§0.6
_i
0.4
0.2
the last-named observer. Transmission spectra of suspensions of unicellu-

were studied by Noddack and Eichhoff (1939), Katz and Wassink
lar algae
(1939), Emerson and Lewis (1941), Wassink and Kersten (1946), Van
Norman, French and Macdowall (1948) and Tanada (1951). Several such
curves are reproduced in figures 22.21-22.24.
Albers and Knorr (1937) measured the absorption spectra of single
chloroplasts, in the narrow region 664-709 mix (cf. fig. 22.35). Vermeulen,
Wassink and Reman (1937), Katz and Wassink (1939), Wassink, Katz and
1 — r-T— —r— —r—r-

I 1
540 600 700 800 900 960

WAVE LENGTH, m/i.
Fig. 22.24. Transmission spectrum (To/T) of Oscillatoria (l)lue alga) (after Katz and
Wassink 1939). Curve 1, cell suspension; curve 2, chlorophyll extract.
Dorrestein (1939) and French (1937, 1940) observed the transmission spec-
tra of purple bacteria (figs. 22.25 to 22.28).
Egle (1937) and Loomis, Carr and Randall (1941) investigated the trans-
mission and reflection of leaves in the infrared. Figures 22.29 and 22.30
show that, from 800 to 1300 ni/x, T -\- R accounts for 85 or 90% of the inci-
dent light in the (comparatively thin) potato leaf, and for 75 or 85% in the
(0.6 mm. This region includes most of the
thick) leaf of Ficus elastica.
infrared radiation of the sun that reaches sea level. (About 75% of the
latter belong to the region 700 to 1500 m^.) Absorption bands at and
above 1.5 n, shown in figures 22.29 and 22.30, are due to water and carbon
400 500 600 700 800 900 1000

WAVE LENGTH, m^
Fig. 22.25. Transmission spectrum {To/T) of a Chromatium suspension

(after Vermeulen, Wassink and Reman 1937).
R, bonds of red pigment

spirillaxonfhin
G, bonds of green pigment

boctenoctilorophyll
800 900 1000 400 600 800

WAVE LENGTH, m>. WAVE LENGTH, m/i
Fig. 22.26. Absorption curve of ex- Fig. 22.27. Relative absorption curve
tracted pigment (broken line) compared of the pigments of Spirillum rubrum in
with transmission curve of pigment in living bacteria (measured photoelectric-
live cells of Streptococcus varians (strain ally using a scattering control of bleached
Cll) (after Frencli 1940). Absorption bacteria) (after French 1937).
bands of extracted pigment shifted to

let maxima coincide with those of the live
cells.
dioxide. Altogether, green plants absorb comparatively little light above

700 m/x —in the region where absorption would probably be useless for
photosynthesis, but could cause strong heating.
Cell suspension
640 700 800 840

WAVE LENGTH, m/x
Fig. 22.28. Absorption spectra of pigments of green sulfur bacteria in the red
and infrared region (after Katz and Wassink 1939).
600 1000 1400 1800 2200 2600

WAVE LENGTH, m/i
Fig. 22.29.Transmission and reflection of near infrared by potato leaf {Sol-

anum Note absorption bands
tuberosum) (after Loomis, Carr and Randall 1941).
due to water, > 1300 m^, but very low absorption (reflection transmission =
+
90-100%) in the 800 to 1300 mju region, which contains most of the near infrared
rays of sunlight.
Figure 22.31 illustrates the "whiteness" of leaves and conifer needles in

the near infrared, by a comparison of the reflection of infrared light by
leaves with that by a sheet of white paper. According to Mecke and Bald-
win (1937) this lack of absorption (rather than infrared fluorescence)
causes the striking brightness of vegetation on infrared landscape photo-
EMPIRICAX, PLANT SPECTRA 695
60
/
50
aJ 40
<
I-
u 30
o
a:
iij
°-
20
Fig. 22.31 A. Summer landscape photographed in infrared Hght (after Mecke and
Baldwin 1937).
graphs (fig. 22.31 A). Figure 22.32 shows the reflection spectra of three
leaves of the same species, but of different ages, as given by Shull (1929).
Table 22.11
Reflection Transmission and Absorption of Light by Leaves

OF Fraxinus excelsior (after Pokrovski 1925)
Wave length, m,. 480 500 550 600 620 650
Reflected, % 2.5 4.1 11.5 7.7 6.7 5.4

Transmitted, % 6.4 8.6 17.5 12.4 11.1 9.8
Absorbed, % .91.1 87.3 71.0 79.2 82.2 84.8
The figures of Pokrovski (1925), given in Table 22.11, illustrate the

fate of light of different wave lengths falling on a leaf of Fraxinus excelsior.
Figure 22.33 shows the reflection spectra of autumnal leaves of different colors
(averages for 20-80 different species), as given by Loomis, Carr and Randall (1941).
Spohn (1934) observed that the position of the absorption maximum of chlorophyll is
shifted in autumnal leaves, from 670 to about 660 niju, probably indicating the liberation
of chlorophyll from the pigment-protein-lipide complex present in photosynthetically
active cells.
CHLOROPHYLL BANDS IN PLANTS 697
Two characteristics strike the eye in all the above-reproduced curves;

the shift of band maxima toward longer waves (as compared with their
and the diffuse appearance of all bands, leading
position in solution spectra) ,
to considerable absorption (or apparent absorption) in those regions (green

and extreme red) where pigment solutions are almost completely trans-
parent. Of these two characteristics, the first one can be safely attributed
to changes in the intrinsic absorption curves of the pigments in the cell (cf.
page 698). The shapes of the bands, on the other hand, are affected
largely, but probably not exclusively, by scattering, "sieve effect," and
other phenomena of geometrical optics.
40 40
35- 35 -
30
Young
25
20
15
10
J_ _!_
Timiriazev (1872), in an early discussion of the effect of scattering on

the spectrum of leaves, suggested that scattering may not only broaden
the absorption bands, but also shift their maxima. However, the attribu-
tion of the "red shift" to scattering is not permissible, as shown, e. g., by
the return of the red band to the position it occupies in solution, after soak-
ing the leaves with ether, or immersingthem in boiling water (fig. 22.34)
(Willstatter and Stoll 1918, Seybold and Egle 1940, Seybold and Weiss-
weiler 1942). These treatments do not dissolve the pigments, and do not
make the tissues more homogeneous; they merely destroy the association
60
z
<
q:
Table 22.III
Absorption Maxima in the Spectra of Living Cells

Position of
Type Main red Other

of maximum, bands
Plants Observer spectrum" X, mtt (approx.)
Land Plants
Fatsia (fig. 22.13) Meyer (1939) T 677 486, 468, 440
Hibiscus (fig. 22.14) Loomis (1941) T 665 470
Pelargonium (fig. 22.12) Seybold (1932) A 670 580, 480, 440
Tilia (Fig. 22.32) Shull (1929) R 678

500-480
460-430
Eight spp. (figs. 22.15, 16). Ralideau, French A, R 670-680 660-690
and Holt (1946) 600-620
588-580
Fifty different spp Lubimenko (1927) T 663-690 Cf. below
Various spp Seybold (1942) T 678-684

R 680-684
Aquatic Plants
Potornageton (fig. 22.17) Seybold (1934^) 667 440, 420
Algae
Chlorella (fig. 21.29) Katz, Wassink
(1939) A 680
Chlorella (fig. 22.21) Noddack, Eichhoff
(1939) A 668
Chlorella (fig. 22.22) Emerson, Lewis
(1941) T 672 488, 473, 432
Chlorella Wassink, Kersten T 675,625(7)475, 430

(1946)
Chlorella Seybold (1942) A 680
Laminaria (fig. 22.19) Seybold (1933) T 669
Nitzschia Button, Manning

(1941) T 680
Nitzchia Wassink, Kersten T 675,630(7) 580,470,440

(1946)
Delesseria (Fig. 22.20) Seybold (1933) T 668,605 540-490-^
Iridaea Van Norman et al. T 675,620'' 550s 480

(1948)
Chroococcus (fig. 22.23) Emerson, Lewis
(1941) 683,625* 436
Chloroplasts
Protococcus, Spirogyra, Zyg-

nema (fig. 22.35) Albers, Knorr 681-684 698, 687, 676,
(1937) 679, 673, 675,
667, 669
« T = transmission spectrum, = reflection spectrum, A = absorption spectrum

R
(transmission +
reflection). * Phycocyanin. ' Phycoerythrin.
22.23 for Chroococcus, and 22.24 for Oscillatoria). The "red shift" is recog-
nizable in all of them, particularly in the last three figures, in which the
spectra of extracted pigments are compared with those of live cells. True.
Noddack's figure (fig. 22.21) indicates only a comparatively small shift
—from 660 to 668 mju— in live Chlorella cells, but Katz and Wassink (1939)
and Seybold and Weissweiler (1942) found, for the wave length of the red
band in Chlorella, the value generally given for leaves about 680 111^. —
Finally, it was stated on page 653 {cf. figs. 22.15, 22.16 and 21.29) that the
red absorption peak is situated at about 675 m/z also in aqueous chloro-
plastin extracts. The position of the red absorption peak in live cells is
thus determined, beyond doubt, by the state of the pigment (which is pre-
served, to a certain extent, in colloidal extracts), and not by geometrical-
optical conditions.
has often been suggested that the position of the red absorption
It
maximum of chlorophyll (and the number and position of secondary
maxima) varies in different species. Table 22. Ill gives a summary of ex-
perimental results. It shows the red peak at 675 mn ± 15 mn, with the
average position corresponding to a "red shift" of 15 mû, or 370 cm.-^
(compared to the position of the corresponding absorption band in ethereal
solution of chlorophyll a). The extreme limits of variation of Xâx. in
Table 22. Ill are 665 and 690 m^ — quite a wide range. However, because
of the diffuse character of the spectra, the position of the maximum often
cannot be determined precisely. It is therefore not certain whether any
of the tabulated variations in X,„«j are real. (It will be noticed that four
values are given for Chlorella, 680, 668, 672 and again 680 m/z!)
Lubimenko (1927), who made photographs of the absorption spectra of
a large number of leaves, insisted that they do exhibit real differences not —
only in the positions of the main peak, but also in the number end positions
in the secondary absorption maxima in the yellow, green and blue.
According to Lubimenko, the spectra of some species ("group 1") contain eight
bands in the visible region. An example is nettle {Urtica dioica), with the following
bands: I, 680-660; II, 650-645; III, 630-606; IV, 600-570; V, 550-540; VI,
512-480; VII, 450-430; and VIII, below 420 m^- The bands III, IV and V become
visible only when two leaves are used. Leaves of "group 2," which includes the largest
number of species, show seven bands (band VII of the above list is missing). Elodea
canadensis and Hedera helix belong to this group. "Group 3" (e. g., Prunus laurocerasus)
shows only six bands (bands I and II are fused). Finally, plants of "group 4" {e. g.,
the alga Ulva lactuca) have onW four bands: I, II, V, VI and VIII. The absence of
band III in these spectra is particularly remarkable. Table 22. IV shows the limits of
variation in the positions of the above-listed bands, as given by Lubimenko, and also
gives a tentative identification of these bands with the bands of chlorophylls a and b in
solution. According to Lubimenko, in passing from species to species, different bands
are displaced by different amounts, or even in different directions.
.
Table 22.IV
Absorption Bands in Leaves (after Lxbimenko 1927)
I II III V VI VII
Band (o) (fe) (a) IV (0,6) (a,6) (0,6) (6.0) VIII
Chi {a + h) in
ether", X . . . . 669- 648- 619- 599- 571- 547- 506- 467- 439- 415—
655 638 605 570 559 523 489 446 424
Leaves, X
from 700- 660- 630- 600- — 555- 510- 450- — 418-^
. . .
680 650 620 570 — 542 490 430 —

to 680- 648- 620- 595- — 545- 505- — — 430—
655 638 610 575 — 535 480 — —
° c/. Table 21.1.
Photometric curves prove that many of Lubimenko's visually recorded

"secondary bands" are only shoulders on the slopes of the main bands or
slight ripples on almost uniformly high absorption plateaus (c/., for ex-
ample, figs. 22.13 and 22.15). Apparent shifts in the positions of these
"maxima," or even the disappearance of some of them, could easily be
caused by changes in the ratios of chlorophylls a and h, as well as by varia-
tions in scattering. Lubimenko saw in these differences an evidence of
variability of chemical composition of "natural chlorophyll" (a name given
by him to a hypothetical complex formed by proteins with all the plastid
pigments)
Albers and Knorr (1937) found that the number and position of the ab-
sorption maxima and Zy-
in single chloroplasts of Protococcus, Spircgyra
gnema vary not only from species to species, but from specimen to also
specimen. In addition to the maxima at 681-683 m^ and 672-675 ui/jl,
which may be attributed to chlorophylls a and b, respectively, some chloro-

plasts showed secondary maxima at 667-669 mû, 678-679 m/x and rather —
unexpectedly — also in the far red, at 698 niju (c/. fig. 22.35).
Albers and Knorr considered these results as indicating variations in
the chemical nature of chlorophyll (e. g., oxidations or reductions) that they
thought might be associated with the participation of chlorophyll in photo-
synthesis (c/. Vol. I, chapter 19).
(b) Red Bund of Chlorophyll h
The main red absoiption Ijund of chlorophyll b is noticeable, according

to Lubimenko (1927), in some spectra of leaves (groups 1 and 2, cf. page
700) but not in others (groups 3 and 4). There is no doubt that this
l)and, too, is shifted toward the red from its position in solution (642.5
niju in but
ether), measure, because the
the extent of the shift is difficult to
band appears merely as a hump on the short-wave side of the main chloro-
phyll a band. Lubimenko's table (Table 22. IV) gives, for the band axis,
values from 643 to 655 ihm- This seems to indicate a somewhat lesser
shift than found with the a-component; Table 21. VI gives the impres-
is
sion that the same may be true also for the spectra of the two chlorophylls
in different solvents in vitro.
The clearest indication that chlorophyll b retains its identity in live
cells is provided by the fluorescence spectrum. According to Tables 24.1
and 24.11, the axis of the main fluorescence band of chlorophyll b in vivo
lies at about 656 m/x, i. e., only about 5 mn further toward the red than in
ether solution (while the axis of the corresponding a band is displaced at
least twice as much). This, too, indicates that the absorption band of
chlorophyll b in the living cell probably lies at 647-648 mju.
(c) Red and Infrared Barids of Bacteriochloi'ophyll
It was stated on page 642 that the bands of bacteriochlorophyll are more
susceptible to shifts under the influence of the solvent than are those of
chlorophyll. We two bands
are therefore not astonished to find that the
of bacteriochlorophyll, which are situated in methanol solution at about
605 and 770 m/x, respectively (c/. page 017, fig. 21.6), are replaced in live
bacteria by bands situated much further in the infrared at approximately —
800 and 850-870 m/x in figs. 21.30, 22.26 and 22.36, and at 825 and 875
m/x in fig. 22.25. Figures 21.30A and 22.26 {cf. also Table 22. V) indicate
a third absorption band close to or beyond 900 m/x, while figure 22.27 shows
only one, very strong infrared band beyond 900 m/x.
Wassink, Katz and Dorrestein (1939) found that the absorption spec-
tra of purple bacteria vary from strain to strain, as illustrated by Table
22.V and figure 22.36.
Alcoholic extracts from all organisms listed in Table 22. V showed only
one absorption maximum in the red (at 774 m/x, cf. fig. 21.30B) in place of
the two maxima of most Athiorhodaceae (a weak one at 800 and a sharp one
at 875 m/x), and three of some Athiorhodaceae and all Thiorhodaceae (at
approximately 800, 850 and 895 m/x). Analysis of the band shapes made
it probable that the spectra of all purple bacteria contain three infrared
bands, even if one of them may sometime be concealed by the other two.
Similar variations in the spectra of different species and strains of purple
bacteria were observed by French (1940).
The bands" with the "solution bands" of bac-
correlation of the "cell
teriochlorophyll (fig. 21.6) is not clear. Shall the cell band at 800 m/i be
considered as the displaced solution band at 605 m/i, and the cell band at
850-870 m/x as the displaced solution band at 770 m/i? This would mean
a shift by as much as 4000 cm.-^ for the "orange" and 1500 cm."'^ for the
"red" band. The first shift is so large that one is inclined to doubt the
I )
BACTERIOCHLOROPHYLL BANDS IN BACTERIA 703
correlation. An alternative is to consider both cell bands, that at 800 as

well as that at 850-870 m/x (and perhaps that at 900 mn, too) as related to
the one solution band at 770 m^t. This means "red shifts" varying be-
tween 500 and 2000 cm.~^, and implies that bacteriochlorophyll is present,
in purple bacteria, in at least three different pigment-bearing complexes.
2.0 _
_ strains strains;
( 1 Rhv. I
(2) Rhv. 2
0.5
ENERGY, e.v. ENERGY, e.v.

.6 1.5 1.4 1.3 1.6 1.5 14 1.3
""
—
'
1 H 1
1 r — I
T 1 r- — I
1
r— I
1
r-
740 800 900 960 740 800 900 960

55
704 LIGHT ABSORPTION V,Y PIGMENTS 7.V VIVO f'HAP. 22
Table 22.V
Absorption Maxima of Purplk Bacteria (after Wassink,
Katz and Dorrestein 1939)
m/i
Species and strain -~
III
Thwrhodaceae
Strain D (Roelefson) 895 854 803.5
Strains 4, 7,12 (v:ui \i<-l) 895 S50 .
1 ,
79f)
SoG"
Strains 9, b, 19 (van Niel-AIuller) 895 852.5 796
Strains 101, 201 Weak 865 1

803.5
Si mill, ;!01, 401 895 858 /
AOtiorltoddceae
865 — 802.5
Rhoduvibrio (2 strains)
864 — 803
Rhodohadllus palustn's (3 strains) 881
873
]
[
— 802
862. 5j
Phaeonwnas varians (Streptococcus varians) (3

strains) 885 1 r799
885 [ 850 . 799
892.5] (798.5
Rhodospir ilium rvhrum (2 strains) 875 \ 800

878 /
complexes in the individual species and strains. The complexes may be

formed by combination of the same pigment (bacteriochlorophyll) with
different proteins, each complex being perhaps specifically adapted to the
utilization of one reductant, such as hydrogen, sulfide, thiosulfate or an or-
ganic hydrogen donor (this hypothesis was suggested by Wassink, Katz and
Dorrestein). Other possibilities include several isomeric or tautomeric
forms of bacteriochlorophyll, or small differences in chemical composition,
or in the reduction level, of the pigment.
If this interpretation of the three cell bands is correct, the question
arises as to the reasons for the absence in live cells of a counteipart to the
605 mp solution band of bacteriochlorophyll. No answer can be given to
this question —
except that the matter requires renewed, and more syste-
matic, investigation. It was mentioned in chapter 21 (page 618) that the
role of the605 niM band is not quite clear even in solution spectra.
According to Katz and Wassink (1939), live green sulfur bacteria have
two absorption maxima (at 740 and 810 m^, respectively) instead of the
single band found in bacterioviridin extracts (at 668 m^, cf. fig. 22.28).
According to the statement on page 642 the width of the "red shift" is in
agreement with the hypothesis that bacterioviridin is a derivative of tetra-
hydroporphin (as assumed on page 445 in Vol. I).
—
CAKOTENOID BANDS IN PLANTS 705
{d) Blue-Violet Bands of Chlorophyll
The position, in live cells, of the second main band of chlorophyll or

bacteriochlorophyll — that situated in the blue-violet part
spectrum of the
has received much than that of the red band, the main reason
less stud}^
being that the presence of carotenoids and other yellow pigments tends to
make absorption in this region very heavy and diffuse. Table 22. VI con-
tains some values read from figures reproduced earlier in this chapter.
This table indicates a "red shift" by 5 or 10 mju. On the frequency scale,
this shift (250-500 cm.~0 is about equal to that of the main red band.
Table 22. VI. lii.rE-VioLKx Absorption Maxima in Living Plants
Organism ''max.' ™''
Fatsia {rf. fig. 22.13) 438

Chlorella {cf. fig. 22.22) 432
Chroococcus {rf. fig. 22.23) 436
Chlorophyll a in ether 427.5
(c) Protochlorophyll
French (1951) estimated, from the action spectrum of chlorophyll

formation, that the red absorption band of protochlorophyll lies, in vivo,
at 650 m/x. Because of its weakness, it has not yet been directly observed.
3. Absorption Bands of Accessory Pigments in Live Cells
Lubimenko (1927) could not find, in the spectra of green leaves, ab-
sorption maxima
identifiable with the absorption bands of the carotenoids
(which could easily be observed, at 510-480 mû, in the spectra of yellow
leaves or yellow parts of variegated leaves) He pointed out that the first
.
carotenoid maximum maybe masked by band VI of chlorophyll (cf. Table

22. IV) and that the second one, at 470-450 mix, should be visible between
the chloroph3'll bands VI and VII. Lubimenko concluded that the yellow
pigments do not exist "as such" in the green plastids. This conclusion, ob-
tained b}^ a purely ciualitative examination of the spectra, is untenable.
The spectra of leaf extracts, in which the chlorophylls and the carotenoids
certainly exist as separate entities, also do not alwaj's show the individual
maxima of all these pigments. In figure 22.47, for example, the absorption
curve of the extract from barley leaves shows only three maxima between
400 and 500 m^ — at 410, 428 and 450 niju — instead of the six maxima of
the separated green and mellow ])igmeiits. The two main carotenoid
. .
maxima (at 422 and 467 m^t) and one chlorophyll maximum (at 500 m^)
are lost by the superposition of the absorption curves.
Upon closer examination, carotenoid maxima can be identified at least
in some plant spectra. Peaks, which probably belong to the carotenoids,

are noticeable, for example, at 468 and 486 niju in the spectrum of the leaves
of Fatsia (Fig. 22.13, cf. also Table 22.III), and at 473 and 488 m/x in that
of Chlorella (Fig. 22.22). If one attributes both these peaks to luteol,
whose absorption bands in ethereal solution are situated at 442 and 472
m/i (cf. Table 21.1), it follows that the carotenoid bands in live cells are
shifted toward the red by as much as 25-30 m/x. (Av = 1250 cm.-^ or
three times the shift of the red and blue-violet absorption bands of chloro-
phyll.) Emerson and Lewis (1942) postulated, in their interpretation of
the Chroococcus spectrum (cf. page 723), a shift of the carotenoid bands
in vivo by 14 m/x from their position in ethanolic solution; according to
Table 21.1, this corresponds to a shift by 24 m/x relative to ethereal solu-
tion, ingood agreement with the preceding estimate (25-30 niju)
Menke (1940) extracted chlorophyll from chloroplast preparations
(made from spinach leaves; cf. Vol. I, page 369) and found that a brick-
;
red residue was left. A suspension of this residue in water showed absorp-
—
tion bands at 490 and 540 m/x much further toward the red than the caro-
tenoid bands have been observed in live green plants (470 and 490 m/x
were the positions quoted above for Fatsia leaves and Chlorella cells).
Moistening with ether led to a change of color, and a shift of the absorption
bands to their usual positions in carotenoid solutions (442 and 472 m/x)
The absorption spectrum of the brown alga Laminaria was found by
Menke to exhibit bands— probably due to fucoxanthol— at even longer
waves, namely, 499 and 545 m/x- Heating to 70° C. led to a shift of these
bands to below 510 m/x, and to a color change from brown to green.
The transmission curves of diatoms (Nitzschia dissipata) published by
Wassink and Kersten (1946), as well as the absorption curves of brown algae
given by Seybold (1934, 1943), clearly show an increased absorption (in
comparison to the green algae, such as Ulva or Chlorella) in the region 500-
580 m/x, but give no indication as to the position of the absorption peak
(or peaks) of the carotenoid responsible for this absorption. From the
comparison of the transmission curves of live diatoms (and of aqueous col-
loidal cell extracts, whose brownish color is similar to that of cell suspen-
sions) with the transmission curves of the (green) pigment exti-act in an or-
ganic solvent (methanol and petroleum ether), Wassink and Kersten esti-
mated that the fucoxanthol bands are shifted in vivo by about 20 m/x
(corresponding to about 700 cm.^^), toward the longer waves; but this esti-
mate is not at all reliable because of the absence of pronounced maxima.
These curves, and Karrer's absorption curves of fucoxanthol in solution,
make it appear uncertain whether the increased absorption of diatoms and
brown algae in the green (500-560 m/x) is due mainly (or exclusively) to a
.
PHYCOBILIN BANDS IN ALGAE 707
strong red shift of the fiicoxanthol band as a whole, or to a broadening of this

band toward the longer waves.
These observations give some information about the strength with which
the carotenoids are bound to the protein-pigment-lipide complex of the
chloroplasts. According to equation (21.4) and figure 21.23, the "red
shift" can be caused by association of the light-absorbing molecule with
other molecules (by adsorption, solution or complexing). The shift is ap-
proximately equal (on the energy scale) to the difference between the bind-
ing energies of the normal and the excited pigment molecule. In the case
of green leaves and algae, we found the chlorophyll bands to be shifted
in vivoby about 370 cm.-\ while the luteol bands were shifted by as much
as 1250 cm.-i (according to Meyer, and Emerson and Lewis), perhaps even
by 2220 and in some cases 2530 cm.-^ (according to Menke). The fuco-
xanthol bands in brown Laminaria are, according to Menke, shifted still
—
more widely by 2585 and 2815 cm.-^
All these shifts are relative to the band position in ether solution. A better idea of
the binding energies could be obtained by comparison with extrapolated positions of
the bands of isolated pigment molecules. Such an extrapolation was made for chloro-
phyll and bacteriochlorophyll on page 642, but it is not yet possible for the carotenoids.
The strong red shifts of the carotenoid bands— particularly those of

fucoxanthol—indicate clearly that these pigments form part, in chloro-
plasts, of some complex, and that the binding becomes particularly strong
when the carotenoid molecules are electronically excited. This fact may
be relevant for the transfer of electronic excitation energy from the caro-
tenoids (particularly fucoxanthol) to chlorophyll, a phenomenon that is
revealed by the occurrence of fucoxanthol —sensitized fluorescence of
chlorophyll in vivo (cf. chapter 24, page 814)— and that probably explains
also the participation of carotenoids in the sensitization of photosynthesis
(cf. chapter 30).
The absorption peaks of the carotenoids appear especially clear on
French's (1937) spectral transmission curves of purple bacteria {cf. fig.
22.27), because in this case the carotenoid bands fall between the two ab-
sorption bands of bacteriochlorophyll, at about 600 and 400 m^-
Differences in the carotenoid bands of brown and red varieties of Streptococcus

arians were described by French in a later paper (1941).
absorption bands of the phycobilins are clearly discernible in the

The
spectra of blue and red algae, e. g., in figure 22.20 at about 550 m/x (phyco-
erythrin) and in figure 22.23 in the neighborhood of 625 m/x (phycocyanin)
According to Emerson and Lewis (1942), the phycocyanin maximum is
shifted by about 6 m/x toward shorter waves in the aqueous cell extract
(figs. 22.23 and 22.48), while the absorption peaks of other pigments retain
the positions they had in living cells. This indicates that in the extract,
70S LIGHT ABSOHrTION BY PIGMENTS IM VIVO CHAP. 22
^v E~i ^^ ^-1 ^
\ ^^ E-( t-i
o o
bi e^ c>i c-
vj --, c/3 - \,
^7 o ^^^ 0000
K^ E~< K»| e~i e-H ^ E~(
be M bD be bC bC bO bC bC bC bC
<!
bB
o 00
bC bC
_o _o _o _o _o _o _o
H 10 10 (M O o O o
'O CO ^< CO
W
1-1
ITO CO CO CO o -}< CO iQ CO CO
O CO CI
Q 03
< lO
d 00 'r-
d o
CO 00 Oi O 1> .'^
o O -H --^
-O CO CI lo lO COTf<
o o o O O Co o o O O O o o
CO
00
St C4 CI
W
P5
CI CI CI
o
CO CO
C2 en
C5
CO
,-1 ^
CO
s 00 .OJ a;
o O
3
O CO '
'i
>.
o C5
s
CO .s -^
H o in C3 C2 !2 .rf
H
tn
SI CO 'l£ m CO ^
S
o
Ph CO
CO
o 3
'o
c
a CO
CO
o3
Ph O 3
O 73
CO O
« 42
o fl G
o g + s
bC bp
'a
o +
2 + CO <£
f3
'53
03
c
I
.3
o'S.
H CO o s
to bC S bO
iz; •as m o 00 o
H a a
Q 002 (H
o ^ p O C
h3
->i cr o .3 f^
ol-t o o.S P ^ <-^<SQk
El
CM (S o3
O (M CO -* 10
o
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O CO
CO
.<
o
Pi
o O
CO O
TRUE ABSORPTION SPECTRUM 709
the cyanobilin-proteid becomes dissociated from the pigment-protein-

Upide complex present in the Hve cell. It was stated in chapter 15 (Vol.
I, page 399) that, of all plastid pigments, only the phycobilin chromopro-
teids pass into true colloidal solution upon extraction with water.
To sum up, it is certain that all the pigments found in extracts from
plants retain their spectroscopic identity in live cells, despite their prob-
able close association in a common complex. The association causes,
however, considerable band shifts, and probably also changes the shape
of the bands, particularly those of accessory pigments, such as fucoxanthol.
4. True Absorption Spectrum of the Pigment Mixture in the Living Cell
In the two preceding sections, we tried to derive as much information as

possible from the positions of the absorption maxima in the empirical plant
spectra described and reproduced in section 1. It was mentioned re-
peatedly that the difference between the "plant spectra" and the spectra of
extracted pigments is not limited to band shifts, but includes also changes
in the height, width and shape of the individual bands. However, as
stated on page G97, the latter changes are, to a large extent, the product of
scattering and other geometrical-optical phenomena. In the present sec-
tion, we with a more detailed description of the appearance of
will deal first
the absorption bands in living plant cells, and then with the possibility of
deriving from the empirical plant spectra the true absorption curves of the
pigment mixtures contained in them.
General diffuseness was stated on page 697 to be the most striking char-
acteristic of plant spectra as compared to the absorption spectra of the
pigment extracts. The ratios of the "optical densities," log (I/S) or log
(To/T) in the absorption "peaks" and "valleys" can serve to illustrate this
statement.
Table 22.Vn shows that the ratio of the optical densities in the "green
minimum" and the "red maximum," which, according to Table 21. IB is
less than 0.01 in pure chlorophyll a, and about 0.05 in an ether extract
from barley leaves (which contains all the chloroplast pigments), is as high
as 0.3 in live algae and may reach 0.6 in green leaves.

The ratio (violet peak: red peak) also is changed: it declines from 1.75
in ethereal extract, to 1.6 in colloidal aqueous extracts, 1.4 to 1.5 in hve

algae, and 0.9 to 1.2 in green leaves.
It be noted that the ratios derived from true absorption spectra,
^^•ill
log (I/S), are not very different from those derived from the transmission
. —
spectra, log (Tq/T), obtained by comparison of colored with colorless tis-

sues (or, in the case of cell by comparison with pure water).
suspensions,
Similar data for a variety of green, yellow and red leaves can be found
in Seybold and Weissweiler's paper (1943).
The question can the leveling off of the absorption peaks and
arises:
the filling in of the absorption valleys be attributed entirely to geometrical-
optical effects (scattering and "sieve effect") or do they indicate a genuine
deformation of the absorption curves of the pigments (which could be
caused by close packing of pigment molecules in the chloroplasts, as well as
by their association with proteins and lipides) ?
The fact that the leveling off is much stronger in the spectra of leaves
than in those of algae indicates that geometrical-optical effects account for
a considerable part of the phenomenon. Tanada's (1951) observations
with glycerol (table 22.VII) support this interpretation. Clearly, scat-
tering and "sieve effect" must lead to an apparent decrease in the selec-
tivity of absorption, as far as plots of log {I/T) or of log (To/T) are con-
cerned, since, in these two representations, losses of the weakly absorbed
(green and extreme red) light by scattering obviously simulate absorption.
One could attempt to explain in this way the results under 4, 5, 7, and 8a-
8d inTable 22.VII, which were obtained by the use of blank cells with
pure water. On the other hand, the similarity of results of the experiments
listed under 6 and 8, in which the true absorption of Chlorella cell suspensions
was determined, with the results listed under 7, in which the transmission
of a similar suspension was measured, cannot be explained in this way.
In the case of leaf spectra too, it is not immediately obvious why diffuse
scattering should lead to transmission curves, log (To/T) (obtained by com-
parison of green with white specimens), characterized by high optical den-
sity in the regions of weak pigment absorption (green and far red)
If one would assume, for example (c/. Meyer 1939), that the weakening of the trans-
mitted beam by passage through a green leaf can be represented by the equation:
"('^"^+''^^
(22.9) T = I X 10
where K is a proportionality constant, equivalent to the product (concentration X thick-

ness of the absorbing layer) in Beer's law, and a a "scattering coefficient"; and assuming
also that the corresponding equation for the white leaf is:
(22.9A) To = I X 10~''>^
then the "transmission curve" would be given by the equation:
(22.10) log To/T = -Ka-^
In other words, the "transmission curve" would follow faithfully

except for a proportionality factar K— the true absorption curve of the
: :
pigments in the cell. Plotted on a semilogarithmic scale (log log [Tq/T] as

function of X), the curves with and without scattering would run parallel.
If this were the case, the ratios in table 22.VII derived from the transmission
curves, log (To/T),would be unaffected by scattering. Closer examination

shows why scattering can produce a distortion of the absorption curves
in the sense observed. This can be shown with the help of the simple
—
example discussed on page 673 that of repeated reflections in a plane-
parallel absorption cell. The equation usually applied for the calculation
of the absorption coefficient, a, is:
(22.11) a = (1/d) log i^To/T)
where d is the depth of the absorption cell, and T and Tq the fluxes trans-
mitted through the solution and the pure solvent, respectively. This
equation was sho\^Ti on page 674 to be a first approximation, neglecting the
difference in the reflectances of the two cells. As mentioned on page 674,
correct expressions for T and To can be obtained by summation of infinite
series. This summation leads to the following relationship
(22.12) T = To 10-"^ - ,rio-.«.)

(i
and thus to
„ = l[,o.^'-,o.(Lî;i5;=^)]
Since the term in parentheses is > 1, equation (22.13) shows that the value
of a, calculated in the usual way from (22.11), is too large and that the rela-
tive error increases with decreasing absorption. With r = 0.1 and To/T =
1.01, i. e., only 1%, the error in a is 2%. Such an error
an absorption of
can be neglected in most absorption measurements. What matters to us,
however, is the fact that the percentage error depends on the value of a
and therefore changes with wave length. In other words, it causes a distor-
tion of the transmission curve.
The character of this distortion —
increase of apparent a values for all
wave but particularly for those in which the true absorption coef-
lengths,
ficient is small —
remains the same in scattering media, where the effect
becomes much stronger than in plane-parallel cells filled with transparent
materials.
The loss of selectivity by scattering can be even more pronounced in
absorption spectra, log (//*S), than in transmission spectra, log (To/T).
Radiations that would be only weakly absorbed by straight passage through
a leaf (e. g., green or extreme red light) become more strongly absorbed
when path in the leaf is increased by scattering. On the other

their optical
hand, a certain proportion of radiations that would be almost completely
on
,
in a scattering medium, we will find that not only are the valleys less deep,
but also the peaks are less high than in a similar plot in a nonscattering
system.
This qualitative explanation of the effect of scattering on selective trans-
mission and absorption can be replaced by an exact analysis if the system
satisfies certain conditions —
namely, random distribution of scattering
centers and—in the simplest forms of the theory—equal probability of
scattering in all directions.
Equations for combined absorption and scattering in systems of this

t}T)e have been derived by several authors; we will mention here, as ex-
amples, the investigations by Wurmser (1941), Duntley (1942, 1943) and
Saunderson (1942). Figure 22.37 represents a nomograph constmcted by
Duntley (1942). In this diagram, the abscissae are the expressions:
{R' + P')/Q', and the ordinates the expressions: log {l/T) + log Q'
whose relation to the absorption coefficient (per unit path), a, and the
scattering coefficient (per unit path), a, is shown in the inserted formulae
{d being the depth of the layer measured). The constants P' and Q' are
characteristic of the asymmetry of the scattermg. If scattering is iso-
tropic, and the incident, transmitted and reflected fight are perfectly dif-
fuse, P' = and Q' = 1, and the abscissae in the figure are simply the
measured reflectances, while the ordinates are the measured optical densi-
ties (logarithms of inverse transmittance) The nomograph remains ap-
.
proximately correct also if the incident light is collimated, if only the re-
flected and transmitted fight are completely diffuse {cf. page 676). Under
these conditions, the absorption coefficient, a, and the scattering coefficient,
a, can both be read from the graph, if reflectance and transmittance have
been determined.
Another method of determination of the two coefficients was described
by Saunderson (1942). It requires two reflection measurements one —
with a thin layer and one with a layer of "infinite thickness" {i. e., having
practically negligible transmission).
Wurmser (1941) suggested that transmission measurements with two
layers of different optical density can be used to determine the coefficients
of absorption and scattering, and gave a sample nomograph for two specific
depths of the cell (0.035 and 0.1 cm.), which is reproduced in figure 22.38.
However, he did not point out that his two-constant formula is correct
only if both layers scatter sufficiently to ensure complete diffuseness of the
transmitted light.
Procedures of the above-described type can be appfied with a fair de-
gree of reliance to cell suspensions, and it is desirable that future investiga-

tions of spectra of such systems make use of them. The application of
Duntley's nomograph to leaves or thalli also is possible, but one has to re-
member that the statistical theory deals with random distributions of large
numbers and therefore does not take into account
of small colored particles,
the "sieve effect," which may
be caused by regular alignment of the com-
paratively large chloroplasts. The result of this effect is the admixture
0.8
0.7 -
0.6
0.5 h
> 0.4
en
o
0.3
0.2
0.1
O 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
log (Tq/T^)- loglTo/r,)
Fig. 22.38. Nomograph for calculation of absorption and scattering coefficients in

turbid media from two transmission measurements (after Wurmser 1941).
= "
(22.17) To/T cosh dVaia + a) + „ " , ^ sinh dV«(a + <r)
ity that a beam of light passes between all these chloroplasts, distributed
at random, is between 2 X IQ-^ and 5 X IQ-^. Consequently, if the dis-
tribution of the chloroplasts were random, the sieve effect would be
negli-
gible. However, the effect can become important if the alignment of

chloroplasts attains a high degree of regularity. The results of Schanderl
and Kaempfert (1933) (Table 22.1) point toward a measure of success
that nature has achieved in this regulation {cf. fig. 22.5). In very young
or thin tissues, the sieve effect is important even without an alignment
of
the chloroplasts. Meyer (1939) mentions that he was unable to measure

the absorption spectra of the seedlings of Tradescantia, and of oat, because
"chlorophyll in these seedlings was so granulated that they appeared in
transmitted light not green but checkered, consisting of white and dark
spots."
One way of viewing the sieve effect is to consider the mutual "shading" of molecules
in each colored particle, which prevents them from exercising their full absorbing capac-
light is markedly
ity; this interference obviously cannot become effective unless the
weakened by the passage through a single particle. This condition is satisfied in the
single chloro-
chloroplasts, since in the peaks of the absorption bands of chlorophyll a
plast absorbs more than 50% of incident light {cf. page 683).
Until quantitative theories have found actual systematic application to

if any,
cell suspensions, if not to leaves and thalli, the question— what,
changes in the true shape of the absorption bands can be deduced from the
spectra of live plants—will beg detailed answer. Several qualitative indices
that such changes do occur can be noted even now, but none of them is
entirely reliable. These indications are: enhanced absorption in the
far red and near infrared (to which we referred on page 654), decreased
absorption in the maximum of the red band, and the comparatively weak
absorption in the blue- violet region.
Increased absorption in the far redis exhibited not only by leaves {cf.,
for example, fig. 22.15) but by Chlorella

alsoand Chroococcus suspensions
(figs. 22.21, 22.22 and 22.23) and by aqueous protein-pigment suspensions
(fig. 21.28A). In the experiments of Smith (1941), this excess absorption
was observed to disappear upon the addition of a detergent, digitonin
(compare figure 21.28A with B); he therefore attributed it to scattering.
A difference that may be explained in the same way was noted by Rabi-
deau, French and Holt (1946) between the transmission and the absorption
spectra of chloroplastin dispersed by ultrasonic waves (cf. fig. 22.15).
However, as mentioned before, a strongly enhanced absorption in the
far red by Noddack and Eich-
live Chlorella cells is recognizable also in
hoff's figure which (unless the integrating apparatus failed to
(fig. 22.21),
function as intended) could not be affected by scattering in the way as-
sumed by Smith. It is true that, as explained on page 711, scattering, by
716 LIGHT ABSOKrTION BY PIGMENTS IN VIVO CHAr. 22
changing the average length of the hght path in the mediinn, could also
change the absorption spectrum (log I/S as function of wave length, which
is what fig. 22.21 represents). However, it appears impossible that the
light path in the Chlorella suspension could be so lengthened by scattering
as to replace the practically complete transparency of the pigment extract
at 800 mjLi, by an absorption of over 10%. If Noddack and Eichhoff's re-
sultscan be relied upon (which is not certain) we arc led to consider the
spread of the chlorophjdl absoriition in living cells into the far red and in-
frared, a genuine change in the absorption curve of the green pigment. It
may be noted (cf. fig. 22.48B) that the absorption curve of Chroococcus
(a Cyanophycea) calculated by superposition of the absorption curves of
,
all the extracted pigments, although it is very close to the transmissi(jn
curve of a living cell suspension (probably, because of the absence of chloro-

plasts and consequent reduction of scattering), nevertheless also shows
enhanced absorption in the far red.
Increased absorption in the green, which is so striking a feature of leaf
spectra, may or may not indicate a genuine change of the chlorophyll spec-
trum. The above-mentioned Chroococcus curve shows no similar effect.
In interpreting the absorption in this region, one has to consider, in addi-
tion to scattering, also the possible presence in living cells of protochloro-
phyll, pheophytin or other relatives of chlorophyll that have absorption
bands in the middle of the visible spectrum. The possible extensive spread
toward the longer waves of the absorption bands of the carotenoids also
has to be taken into account.
Decreased absorption by live cells in the maximum of the red band, shown
in figures 22.21 and 22.22, cannot be explained by a sieve effect (which is
negligible in cell suspensions); diffuse reflection, too, probably is insuf-
ficient to account for this effect. A certain flattening of the red band may
therefore also be characteristic of the true absorption curve of chlorophyll
in the living cell.
Table 22.VII shows that (as first noticed by Wurmser in 1921) the
ratio of the apparent absorptions in peaks of the blue-violet band and of the
red band is low in algae (about 1.5) and particularly in leaves (about 1.0),
as compared with pigment extracts (1.75). The blue-violet rays are scat-
tered more strongly than red ones; but this could not explain a decrease
in their absorption. (Such an effect would only be understandable if the
diffuse reflection of blue- violet light by leaves were stronger than that of red
light; but no such effect is revealed by figures 22.12 or 22.32.) The
comparatively weak absorption of blue-violet light by plants is made even
less understandable by the fact that yellow, water-soluble pigments present
in leaves must enhance the absorption in this region.
However, Seybold and Weissweiler (1943) opposed Noddack and Eich-
SPATIAL DISTRIBUTION OF PICMENTS 717
hoff's assertion (fig. 22.21)that live Chlordla cells absorb less light than the
pigment extract in the region 550-680 mn; they found the absorption
of the cells to be the same as that of extracts in the peaks of the bands, and
greater every^^here else.

The weaker absorption by living colls, compared with the pigment ex-
tract, in the blue and violet region was noticed by Emerson and Lewis
(1942) also in the spectrum of the blue-green alga Chroococcus (cf. fig.
22.48B), which is almost free of scattering effects.
C. Distribution of Absorbed Energy among Pigments*

The allotment of absorbed light energy to the several pigments is very
important for the interpietation of the quantum yield of photosynthesis
and, in particular, for the understanding of the role of the accessory pig-
ments —carotenoids and phycobihns.
1. Effects of Spatial Distribution of Pigments in the Cell
The step in the apportionment of absorbed energy is separation of

first
the absorption by the "photosjTithetic" pigments— chlorophylls, carote-

—
noids and phycobilins from that by pigments such as the flavones and
anthocyanines, which probably bear no relation to photosynthesis at all.
This question wâs discussed before (cf. page 685) figure 22.10 was given
;
as illustration of the extreme case of leaves of the "purpurea" variety, in
which a very considerable part of incident hght, particularly in the green,
is absorbed by the water-soluble red pigments.

by
The presence of pigments of this type complicates matters not only
adding new components to the composite absorption spectmm, but also
by raising the problem of "color filters": Generally the apportionment of

the absorbed light energy to different pigments, in the region of common
absorption, requires the knowledge not only of the true absorption curves
of the pigments in the state in which they are present in the living cells,
but also of their microscopic and submicroscopic distribution. In the
case of flavones and anthocjânines, it is definitely known that their dis-
tribution is different from that of chlorophyll— they are concentrated, not
in the chloroplasts, but in the cell walls and vacuoles, and thus form "color
before or between the chloroplasts {cf. the calculations of Noddack
filters,"
and Eichhoff 1939). This makes it particularly advisable to use, for quan-
titativestudy of photosjTithesis, plants containing as little nonplastid
pigments as possible.
Even if the flavones and anthocyanines are absent, and the object stud-
ied contains no separate carotenoid-bearing bodies, it is by no means certain
*Bibliography, page 738.
that the siibmicroscopic structure of the plastids (discussed in Vol. I,

chapter 14, and iUustrated by fig. 14.6) does not place some pigments in a
different position with respect to light absorption than others. We may
hope that studies with the electron microscope and investigations of the
optical properties of the plastids (birefringence, dichroism etc.) will reveal
more about the arrangement of molecules in these bodies. Pending these
developments, all estimates of the relative contributions of different pig-
ments to the absorption of light by plants must be based on the assump-
tion of an identical proportional composition of the pigment mixture in
every point of the cell or tissue.
2. Apportionment of Absorption in Uniform Mixture
If itcan be assumed that the pigment mixture in the cell or tissue under
—
investigation has uniform composition meaning that, wherever the mix-
ture is present, it has the same relative composition (but not that the same
absolute concentration of mixture is present everjovhere)— the contribu-
tion of the zth pigment to the total absorption by the mixture at a given
wave length, Ai, is proportional to the product CjOJi, where Ct is the concen-
tration and «< the absorption coefficient of this component.
(22.18) Ai =A (CiaifSiCiai)
Whatever the length and shape of the path of the light beam in the
medium, equation (22.18) applies to absorption in every infinitely small

element of this path therefore, it applies also to the integral light absorp-
;
tion, independently of scattering or other geometrical-optical phenomena.

The relative concentrations, Cf, can be determined, e. g., by extraction
and photometric estimation; the correct value of the total absorption, A
(i. e., the value corrected for reflection and scattering), can be determined,
for each wave length, by the methods discussed in part A. The applica-
tion of equation (22.18) therefore hinges primarily on the knowledge of the
true absorption coefficients of all pigments in the state in which they are
present in the cell, and here the difficulty comes in.
In part B, we referred to statistical theories whose application may per-

mit the determination of the average absorption coefficients of the pigment
mixture, from measurements of transmission and reflection (or two reflec-
tion measurements, or two transmission measurements with different opti-
cal densities). In figures 22.37 and 22.38 we gave examples of nomographs
that could be used for this purpose, provided both the transmitted and re-
flected light fluxes are perfectly diffuse; and we suggested that these (or
other similar theories) be used in the future in the optical study of cell sus-
pensions, leaves and thalli.
APPORTIONMENT OF ENERGY TO CHLOROPHYLLS 719
Even after the average absorption curve of the pigment mixture has been
determined, this will not give us the desired knowledge of the absorption
curves of the individual pigments; but it will be a step in the right direc-
tion: Some sections of this average absorption curve will be due to a single
pigment or a small group of related pigments (e. g., the part above 550 mM
in green plants, to chlorophylls a and h, and in brown algae, to chlorophylls
a and c). The changes in shapes of the absorption bands, found in these
regions, may be considered, by analogy, as valid also for the bands of the
same pigments in the regions of composite absorption. (However, the dif-
ferent polarizabilities of a molecule in different electronic states, and the

possibility of resonance effects between molecules with overlapping absorp-
tion bands call for caution in the use of such analogies.)
By constructing true absorption curves of cells or plastids with var>ang
contents of the individual pigments, one can hope to assemble material
whose analysis will permit derivation of the absorption curves of the indi-
vidual components. (Here, too, caution will be needed because resonance
phenomena may destroy simple additivity of absorption coefficients.)
In the light of these considerations, all attempts undertaken so far to
apportion the light energy absorbed by plants, among individual pigments,
are but first crude approximations. In some of these studies, the analysis
was made entirely on the basis of comparison of the spectra of extracts
with those of the solutions of separated pigments. In others, a certain

improvement was achieved by assuming that all bands were shifted in vivo
by the same amount, without change of shape. In a third group of inves-
tigations, the analysis was further improved by assuming individual values
for the shifts of the bands of different pigments; but still no attempt was
made to take into consideration the possible changes in shapes of the bands.
3. Absorption by Chlorophylls a, b, c and d
All absorption of light by chlorophyllous plants or plant organs at

wave lengths longer than 550 m/x can be attributed to the chlorophylls,
except in red and blue algae, where phycobilins may absorb light up to 650
or 700 mfji {cf. figs. 21.39 and 21.40), and purple bacteria, which contain
carotenoids with absorption maxima at 550-570 m/x {cj. fig. 22.27 and Table
21.VIII).
No serious attempts have been made to apportion the absorption be-
tween chlorophylls a and h. A crude idea of this distribution in an ex-
tract from a green alga is given by INIontfort's figure (fig. 22.39). (It will
be noted that the abscissae in this and other figures of Montfort and Sey-
bold are absolute values of absorption in each separate solution, not per-
centages of the total absorption by the mixture, and thus do not add to
100%.) This figure is too crude to reveal an increased participation of

chlorophyll b in the region of its band maximum in the red; but shows
450 500 550 600 650 700 750

WAVE LENGTH, m/x
Fig. 22.39. Absorption of pigment extracts from Ulva tacluca (green alga)
(after Montfort 1940).
9?
o
_J
_l
>-
X
a.
o
a:
o
_i
X
o
>
m
z
g
\-
a.
tr.
o
CD
m
<
100 100
500 600 500 600 700
WAVE LENGTH, m/i WAVE LENGTH, m>i
Fig. 22. -40. Light absorption by Fig. 22.41. l'roi)ortions of light absorbed
cliloi'ofucin (chlorophyll c), in per hy cliloroi)hylls a and d in a methanol ex-
cent of total absorption, by a meth- tract of Erythrophyllum delesserioides (after
anol extract from diat(jms (after Manning and Strain 1943). Absorption by
Strain and Manning 1942). red and yellow pigments is not considered
in the calculation.
clearly such an effect in the blue, lêtween 450 and 530 mî, where the ab-
sorption by the 6-component is up to five times stronger than that by chloro-
phyll a.
ABSORPTION BY CAROTENOIDS IN GREEN PLANTS 721
jMiich more precise measurements were made b}'- Strain and Manning
(1942) with the chlorophyll a -\- mixture extracted from diatoms, and bj-
c
Manning and Strain (19-43) with the chlorophyll a -i- d mixture from red
algae. Figiu-es 22.40 and 22.41 show that chlorophyll c (chlorofucin)
contributes about 90% of total chlorophyll absorption at 570 mix; while
chlorophyll d account.s for 60% of the total chlorophyll absorption at 470
and 90% at 710 m/x.
These figures arc for methanol extracts. The relative roles of the
chlorophyll components in the absorption of light in vivo are uncertain.
In the case of chloroph}-!! h, not even the position of its red absorption peak
in vivo is kno^vn with any certainty (c/. page 701). It was suggested (cf.
page 612) that alternation of the absorption maxima of chlorophylls a and

b in tlie red, orange and 3'ellow, best shown by figures 21.1 A and B, may be
nature's means to ensure most effective light absorption throughout this
region; but all these bands are so broadened in leaf absorption spectra
that it is doubtful whether the absorption by the natural mixture a -\- b
is, at any wave length above 550 mn, markedly different from what would
prevail if either of the two components were alone present in equivalent

concentrations. The situation is different below 550 mî. The region of
prevalent absorption by the 5-component, which we noted in extract at
450-530 mix (fig. 22.39), in all probability exists also in hve green cells.
Because of the "red shift" it probabh^ extends in vivo from about 460 to
about 540 m/x. In this region, the presence of chloroph^dl b may be of
considerable importance from the point of view of enhanced light absorp-
tion by green leaves and algae.
4. Absorption by Carotenoids in Green Plants
The distribution of light between the chlorophylls and the yellow caro-
tenoids of green plants in the region below 550 m/x has been much discussed.
The first estimate was made by Warburg and Negelein (1923). In their
calculations of the quantum yield of photosj^nthesis (cf. chapter 29), they
decided, from extract spectra, that the carotenoids of ChlorcUa accoimt for
30% of the total absorbed light at 436 m/x.
Figure 22.42 shows the results of the first more detailed estimate by
Seybold (1936), based on spectroscopic measurements with an extract
from leaves of Phaseolus vulgaris. Figure 22.39 represented similar data
for the multicellular green alga Ulva lacluca; this graph shows the absorp-
tion by carotene and the carotenols separately (as well as that by the chloro-
phylls a -\- b, a and b). The two figures show the absorption by the caro-
tenoids becoming noticeable below 530 m/x (in solvents of low polarizabil-
ity) and below about 570 m/x in carbon disulfide. The largest percentage
participation of the carotenoids in total absorption Is indicated between 450

and 520 m/x in methanol, and up to 550 m/x in carbon disulfide.
A considerably more precise analysis was made by Emerson and Lewis
(1941, 1942, 1943). They measured the transmission of a Chlorella sus-
400 500 600 700

WAVE LENGTH, m/i
Fig. 22.42. Absorption by all pigments {a + b -\- c + x) from 100 cm.*

of leaves of Phaseolus vulgaris in methanol, compared with absorption by
an extract of the chlorophylls a + 6 in methanol, and by extracts of the caro-
tenoids c + X in methanol (1) and carbon disulfide (2) (Seybold 1936).
1.2 /s —
1.0
p 0.8
b 0.6
o
o
_)
0.4
0.2
—
ABSORPTION BY CAROTENOIDS IN BROWN ALGAE 723
and accounts for about 50% of total absorption between 500 and 450 m^;
below 450 mû, the absorption by the chlorophylls again becomes predom-
inant.
Figure 22.44 is an attempt to interpret the conditions in living cells.
It shows the transmission spectrum, log (To/T), of the cell suspension
it would be better if it were the absorption spectrum, log I/S. Also shown
isa composite absorption spectrum of the pigments obtained by shifting
the chlorophyll bands, derived from the preceding figure, above 550 mû,
toward the red by 10 m^, and below 580 m^, by G m^ (it would be simpler
to make the plot on the frequency scale and use a uniform shift!); the
carotenoid bands, derived from the preceding figure, were shifted by 14 m/x.
(The values used for chlorophyll may be a little low; cf. page 706.)
Two cell curve are at points where cell suspension was
breaks in intact
stirred and filtersmonochromator were changed. The dotted curve
in
shows fraction of total absorbed light absorbed by carotenoids, based on

the curve for extracts after introduction of wave length shifts.
The difference between the composite absorption curve for total pig-
—
ments and the much more diffuse —
transmission curve of the cell sus-
pensions can be due (as discussed in part B) partly to scattering and partly
to intrinsic changes in band shapes. A theoretical treatment, as de-
scribed on pages 711-714, could eliminate the scattering effect and reveal
more clearly the intrinsic alterations of the pigment spectra; but no at-
tempt was made to take scattering into account, and the proportion of
light absorbed by the carotenoids was calculated simply by comparison of
the absorption curve of the combined extracts in figure 22.44 with those of
the individual extracts in figure 22.43 (after appropriate "red shifts" of
the latter). The results, indicated by the dotted line, show 40% absorp-
tion by the carotenoids at 520 m^, a maximum of 75% at 500 mju, a second-
ary maximum of about 55% at 460 m/x and a decline to about 25% below
440 niM-According to these results, carotenoids contribute significantly
to the total absorption of plastid pigments in Chlorclla from 530 m^t down-
ward.
5. Absorption by Carotenoids in Brown Algae
Brown algae (including diatoms) contain no chlorophyll b, and should

thus, according to page 720, transmit more freely than green plants in the
region 460-540 mix. Instead, as their color shows, they absorb consider-
able —
amounts of green light (510-580 mju) much more than do the green
cells. This must be ascribed to the presence of a specific carotenoid,
fucoxanthol. If figure 21.35A is correct, and the absorption spectrum
of fucoxanthol in solution does not extend toward the red any further than
—
724 LIGHT ABSORPTION BY PIGMENTS IN VI VU CHAP. 22
that of luteol i. e., not much beyond 510 —

niju the brown color can be
attributed to fucoxanthol only by assuming a very wide shift of absorption
bands (or their strong broadening toward longer waves). According to

page 706, Menke had in fact found indications that the absorption peaks of
fucoxanthol in live Laminaria cells are situated as far toward the red as at
499 and 545 mji (instead of 457 and 492 mî in ethanol solution, according
to Table 21. IX). Attempts to analyze the absorption by brown algae,
described below, luive not taken into account the possibility of such a wide
shift —
not to speak of any changes in the width of the bands.
On page 657 we referred to a more recent measurement of the extinc-
tion curve of fucoxanthol in hexane by Karrer and Wiirgler, which showed
a considerably broadened band, extending to or even beyond 530 van (fig.
21.36).
100
^~N
80
5 60
Q.
<
20
;
this absorption to 00% in the region 500-550, and by 30% in the

by 40
region 450-500 mn. In bro^vn algae and diatoms, the absorption increase
due to the carotenoids is much larger, from 160% to 400%. This is a
consequence of the absence of chlorophyll b and the presence of fucoxanthol
these two pigments substitute for each other, despite their difference in
color.
Table 22.VIII
Ratio of Absorption by Individual Pigments in Methanol or Benzene and by
THE Total Pigment in Methanol" (after Montfort 1940)
550-500 m^ 500-450 iiim
X. X.
(including f including
pl> F.) C. b
Organisms Clil. F.) Clil. /3
Diatoms 0.34 0.40^ 3 0.20 0.82 0.24 5

Phaeophijceae
Laminaria digitata
taken from 12 m.
depth 0.38 0.69= 2.6 0.32 0.93 0.33 3.1
Fucus vesiculatus
from the surface . 0.24 0.80 4.2 0.22 0.91 0.30 4.5
(0.33)'= (0.47)"=
Chlorophyceae
Ulvalactuca 0.73'' 0.34 1.37 0.75'' 0.56 0.25 1.33
(0.19)^
Leaves
Phaseolus vulgaris^ .
— — 1.6 — — 1-3
The figures in the table do not add to unity because they do not represent the
"
proportions in which the energy absorbed by the pigment mixture is divided between the
individual pigments, but rather the ratios (.absorption by the separated pigments):
absorption by the mixture). Chi. = chlorophyll (in methanol), X. = carotenols (in
(,
methanol), F. = fucoxanthol (in methanol), C. = carotene (in benzene).

* /3 =
factor by which total absorption is increased by the presence of carotenoids.
" F. alone.
Chi. a
''
+
b.
« Chi. a alone.
/ According to page 684, the leaves of this species contain a considerable quantity
of water soluble yellow pigments.
A
considerably more detailed analysis of the absorption by diatom pig-
ments was made by Button and Manning (1941); the results are repro-
duced in figure 22.46. They are based on measurements with acetonic pig-
ment extracts from Nitzschia In constructing the figure,
closterium.
Button and Manning postulated a shift of all absorption bands, those of
chlorophyll as well as those of the carotenoids, by 20 m/z. (They based
this assumption on the observation that, in the spectrum of this diatom, the
maximum of the red chlorophyll band was recognizable at 680 m^; the
maximum of the blue-violet band, although not distinct in the cell spectrum
72G LIGHT ABSORPTION BY PIGMENTS IN VIVO CHAP. 22
itself,could be recognized in the spectrum of a bile salt extract and ap-

peared to be shifted by about the same amount— 15 m/x.) We know, how-
ever, that the blue-violet band of chlorophyll is shifted by approximately
the same amount as the red band on the frequency scale (and not on the
wave length scale), and the bands of the carotenoids—particularly those of
— — —r— — — — —r—
1 [—1 I 1 I I I r-
T— — —r— — — — —
I I I 1 I I
I I I—— I
I I I I I
Chlorophyll o
Total carotenoids
Fucoxanthol
Caretenoids other
than fucoxanthol
400 500 600 700

WAVE LENGTH, m/i
Fig. 22.46. Distriliution of alisorption in acetone extracts from Nitzschia

closterium (after Button and Manning 1941). Lowest curve, chlorophyll a.
Middle curve, chlorophyll a + carotenoids other than fucoxanthol. Top
curve, all pigments.
fucoxanthol— probably are shifted much further than those of chlorophyll.

Thus, figure 22.46 must contain considerable errors. The absorption by
fucoxanthol above 500 m/x probably is much stronger than shown, with
the consequent increase in general absorption in this region. (Figure 22.46
represents a green rather than a brown mixture!)
A still more detailed analysis of the spectrum of a diatom, Navicula

minima, was undertaken by Tanada (1951). By extracting the pigments
stepwise with aqueous methanol of different concentrations, he obtained
evidence of the following "red shifts" in the blue- violet region (compared
to methanolic solution) : chlorophyllc, 20 m^t; fucox-
a, 8 m^t, chlorophjdl
anthol, 40 m/x; other caroteaoids, 20 m/x. A
comparison of the cell spec-
trum with the sum of so adjusted pigment spectra can be found in fig.
30.9B.
How wary one must be in drawing conclusions based on a uniform shift of the bands
of several pigments is illustrated by from Strain (1938): It shows the
figure 22.47, taken
absorption curves of barley leaf extracts in ethanol and in ether, and the corresponding
curves for the separated yellow and green pigments, in the same solvent. In ether, up
to 90% of the total absorption in the region 470-500 m^ is due to the yellow pigments,
and less than 10% is absorbed by chlorophyll; in alcohol, on the other hand, the green
400 440 480 520 560 600 640 680 400 440 480 520 560 600 640 680
WAVE LENGTH, mî WAVE LENGTH, m,i
Fig. 22.47. Effect of solvent on absorption spectra of barley leaf pigments (after
Strain 1938). a referred to 1 g. fresh leaves in 1 1. solution. I, all ether-soluble pig-
ments; II, chlorophyll a and b; III, carotenoids.
pigments account about one half of the total absorption in the same region. The
for
from ether to ethanol causes a stronger
difference is due to the fact that the transition
shift of the bands of the more polar pigment — —
chlorophyll than of those of the less
polar carotenoids (11 and 5 mî versus 1 and 4 m/x, respectively).
6. Absorption by Carotenoids and Phycobilins in Blue-Green Algae
Emerson and Lewis (1943) analyzed absorption by the pigments in

the blue-green alga Chroococcus, in the manner described on page 722 for
Chlorella. Figure 22.48A illustrates the absorption in alcoholic extract
containing chlorophyll and the carotenoids, and in aqueous extract con-
taining the phycocyanin-protein complex. The separation of the ab-
sorption regions is much neater than in green plants: above 650 m/x,
0.9 1 1
1
\
0.9 0.6
I
—
\
\
« Alcohol-soluble pigments -" Intact cells
0.8 •— Chlorophyll 0.8 -« Combined extrocts 0.5

—• Carotenoids -« Water extract
0.7 —a Phycocyanin
X Phycocyanin (Svedberg)
0.6
^ 0.5
if
g 0.4
0.3
0.2
0.1
400 440 480 520 560 600 €40 680 400 440 480 520 560 600 640 680 720
WAVE LENGTH, m/x WAVE LENGTH, m/i
A B
Fig. 22.48. Analysis of spectrum of Chroococcus (blue alga.) (after Emerson and
Lewis 1943). (A) Absorption spectra of extracted pigments in ethanol, and of phyco-
cyanin in water. (B) Absorption spectra of intact cells (1.26 mm.' cells/ml., layer 1.4
cm. thick), combined extracts (calculated from A by shifting bands as described in text)
and of water extract (containing all pigments). Water extract curve has changed shape,
indicating alteration of the phycocyanin-protein complex by extraction.
absorption is due mainly to chlorophyll; between 530 and 640 m^c, to

phycocyanin; and between 460 and 510 m/x, to the carotenoids (because
of the absence of chlorophyll b) Below 440 m^, chlorophyll again becomes
.
the main absorber.

Figure 22.48B is the attempt to reconstruct conditions in the cell. The
chlorophyll and carotenoid bands were shifted, as in the treatment of
Chlorella, the first ones by 10 m/x in the red, and 6 m/x in the blue and violet,
and the second ones by 14 m/x. The phycocyanin band was shifted by
6 m/x compared to its position in aqueous extract. In the case of chloro-
phyll and the carotenoids, the extraction was assumed to be complete; in
that of phycocyanin, the fact that the 620 m/x absorption peak in the ex-
CAROTENOIDS AND PHYCOBILINS IN BLUE-GREEN ALGAE 729
tract was considerably lower than in the cell spectrum was taken as an indi-
cation of extraction losses {cf. Vol. I, page 418, about difficulties of quanti-
tative extraction of phycobilins). Therefore, in the construction of the
''combined extract" curve in figure 22.48B, the phycocyanin curve of
figure 24.48A was not only shifted, but also increased in height by a factor
sufficient to make the phycocyanin peak of this curve coincide with that
of the cell curve.
Emerson and T.ewis pointed out that the agreement between the "cal-
culated" absorption curve and the empirical transmission curve (fig. 22.48)
is much better than in a corresponding construction for Chlorella. They
saw the explanation of this fact in the absence of chloroplasts in Cyano-
phyceae,and consequent reduction of scattering. The remaining differ-
100
80
a.
(T
O
CO
< 20
carotenoids. Above 700 m/x, the absorption by purple bacteria is due en-
tirely to bacteriochlorophyll, as shown in figure 22.26 by comparison of the
cell spectrum with the spectrum of the extracted green pigment.
Appendix. Natural Light Fields*

The importance of the various pigments in the light economy of plants
cannot be fully understood on the basis of curves discussed in the preceding
section, because it also depends on the spectral composition of the light
available to the species or individual plant under natural conditions.
>-
o
on
UJ
UJ
>
UJ
q:
NATURAL LIGHT FIELDS 731
in the depths of the sea and the periodicity adaptation shows itself in the
;
distinction between long-day plants of the arctic zone and short-day plants
of thetemperate and tropical zones.
The spectral composition and intensities of light fields in different habi-
tats of plants have been measured and discussed by several authors, for
example, Wiesner (1907), Ursprung (1918), Seybold (1934, 1936) and Egle
(1937). The factors that determine the total intensity of the available
light (in the photosynthetically important region 400-700 m/x) are the
height of the sun over the horizon, the clearness or haziness of the air,
cloudiness and the position of the plant in direct sunlight or in the shade.
<
Q.
UJ
>
I-
<
_i
LJ
q:
20 30 40
HEIGHT OF THE SUN, degrees
Fig. 22.51. Total irradiation and relative intensity of sunlight in relation to height of
sun ov or the horizon (after Seybold 1936). Inner scale at left, cal./(cm.- miu.).
Reflection by the surrounding surfaces may also be of importance, particu-

larly for the evergreens (reflection by snow!) and shore plants (reflection
by water!) (cf. Egle 1937). Figure 22.50 shows the intensity distribution
of the combined light of the sun and the sky for different heights of the
Sim over the horizon. The curves show the transition from the red light
of the setting sim (ma.ximum intensity at 680 m/x) to the yellow light of the
sun in the zenith (maximum intensity at 520 m/i). Figure 22.51 shows the
increase in the total energy of the light, from 0.1 cal./cm.^ min., at 10°
elevation, to 0.6 cal./cm.^ min., at an elevation of 50°. (The figures are
for a surface normal to the direction of the incident light; for a horizontal
surface the variations are much wider.) When the sun is obscured by light
clouds, the total light intensity decreases to 10 or 20% of its full value (i. e.,
to 0.1-0.2 cal./cm.- min. at midday). When the sky is entirely overcast.

;
LIGHT ABSORPTION BY PIGMENTS IN VIVO CHAP. 22

732
or even less,
the light intensity can drop to as low as 0.02 cal./cm.'- min.,
At the same time, the spectral composi-
i. e., to only 1% of full sunshine.
tion changes in the way characterized by figure 22.52, takenfrom a paper

by Taylor and Kerr (1941); the light intensity becomes almost uniform
throughout the visible spectrum, and the common designation of such
days as "gray" proves to be correct.
400 500 600 700

400 440 480 520 560 600 640 680
Average energy distribution
Fig. 22.52. Fig. 22.53. Energy distribu-
curves for daylight (afterTaylor and Kerr tion in the shade (after Seybold
1941). (A) Zenith sky, color temperature 1936). (G) Edge of wood (black
13,700° K.; (B) north sky on 45° plane, square = 2.5 X 10 "^ cal. per
color temperature 10,000° K.; (C) totally (cm.2 min.); (1), (2), (3) three
overcast sky, color temperature 6,500° K. shade habitats of Oxalis (blaf;k

(D) sun plus sky on horizontal plane, color square = 0.025 X 10 "^ cal. per
temperature 6000 ° K. (E) direct sunlight,
;
(cm.^ min.).
color temperature 5335° K.
When the plant is in the shadow house or mountain, and re-

of a rock,
ceives light mainly from the blue sky ("blue shade"), the spectral composi-
tion of its light field is entirely different from that to which it is exposed in
direct sunlight, as shown by figure 22.52. The intensity of radiation from
a clear blue sky is of the order of 20% of that of full sunlight (i. e., about
0.1 cal./cm.2 jj^jn ) at sea level. It decreases mth increasing altitude,
as the scattering air layer above becomes thinner and the color of the sky
a deeper blue.
The plants that live in the shadow of other plants, e. g., the floor vege-
tation in the forest, receive their light filtered through the chlorophyll layers
of the overhanging foliage. They Hve in the "green shade." Figure 22.53,
taken from Seybold (1936), shows the spectral compositions of the light
field in the midst of a forest, compared with that at the edge. Character-
istic is the minimum at 650 m/x, clearly corresponding to the absorption
maximum of chlorophyll. A large part of radiations reaching the floor of
a forest are either infrared or deep red, scarcely visible to the eye and use-
less for photosynthesis. The total intensity of the light field under the
trees (400-700 mû) is less than 10% of that of full sunlight above the forest
and can drop to as low as 1% in a dense pine forest (Seybold).

An even stronger alteration in the intensity and spectral composition
of the light field occurs when sun rays pass through thick layers of water.
Table 22. IX gives some data on the decrease in total intensity with depth.
The main cause for this drop in light intensity is the absorption by water
Table 22.IX
Decrease in Light Intensity with Depth
50 100 200 ft.
Atlantic Ocean
Brightness" 1076 114 37 4.4
1 2 5 10 20 m.
Titisee Lake
Intensity-* 100 57 32 21 13 10
Bodensee Lake
Intensity*. 100 54 30 15 9 6
« Beebe and HoUister (1930), Hulburt (1932).

'•Seybold (1936).
itself. higher bands (overtones) of the vibrational spectrum of

Some
water lie in the visible spectrum (the fundamental frequencies are in the
near infrared). They decrease in intensity from red to blue; in the violet
and near ultraviolet, however, the absorption increases again, probably
due to weak electronic bands. Figure 22.54 shows the extinction curve
of water in the region 360-800 mû, according to the measurements of
Aschkinass (1895) and Sawyer (1931) (c/. Dorsey 1940). A water layer
10 m. thick reduces the light intensity at 640 m/x by the factor of 10; a
layer 1 m. thick does the same at 760 mju 100 m. are required for a similar
;
reduction at 440 m^. Because of this absorption in red, yellow and violet,
thick layers of pure water are bluish green in color.

The absorption by natural waters in the blue and in the violet is usually
much larger than that by pure water, due partly to the presence of certain
inorganic ions (e. g., iron) and partly to that of organic matter (e. g., the
chlorophyll of the phytoplankton). This increased absorption at the
short-wave end of the spectrum gives natural waters a pure green or even
a yellowish-green tinge, as compared with the bluish-green color of water

as such. Figure 22.55 shows the spectral composition of light in different
depths of the Mediterranean on a clear day according to Seybold (1934).
100
360 440 520 600 680 720 400 500 600 700
WAVE LENGTH, m^ WAVE LENGTH, m/i
Fig. 22.54. Absorption curve of pure Fig. 22.55. Energy distribution under
water (a = specific absorption coefficient). 1-50 m. water (after Seybold 1934).
S, Sawyer (1931). A, Aschkinass (1895).
Table 22.X
Spectral Composition of Light under Water"
Italics indicate position of maximum of transmission.
A. transmission by a five meter thick layer*

IXlfl
Water 700 650 600 550 500 450 400 350
Pure water (blue)
B. SPECTRAL COMPOSITION OF LIGHT IN DIFFERENT DEPTHS''

Per cent
Depth
Similar sets of curves, showing a less rapid decline in intensity at the

red end of the spectrum, were given by Johnson and Kullenberg (1946) and
Levring (1947) for sea water at the West Coast of Sweden. Levring at-
tributed the absorption in violet and blue —which often converts the blue-
—
green color of pure water into a yellow-green to the presence of suspended
particles. Some additional data can be found in Table 22.X.
Algae are found to a depth of 120 m. These deep water species live in a
light field that contains almost exclusively green radiation (practically no
red light is available below 10-20 m.). The total intensity of light avail-
able to them is only a few per cent of the light enjoyed by the species living
close to the surface of the sea.
400 500 600 700 500 600 700

WAVE LENGTH, m/i WAVE LENGTH, mM
Fig. 22.56. Light absorption by algae in different depths (in meters) in per
cent of light incident on the surface (after Seybold 1934).
We have discussed in chapter 15 (Vol. I) the ways in which plants adapt

themselves to the chromatic composition of the light fields. There is no
doubt that the brown algae, containing fucoxanthol, are capable of absorb-
ing more light in the middle of the visible spectrum than the green plants,
and that the presence of phycobilins in the Rhodophyceae and Ctjanophyceae
increases their absorbing capacity in this spectral region even more strongly.
Table 22.VIII showed what these changes in the pigment system mean
for the absorption capacity of the plants in different spectral regions.
However, this table referred to illumination with lightwhose intensity is
constant throughout the spectrum. same changes
The consequences of the
for the absorption of natural light were discussed by Seybold (1934), Mont-
fort (1934) and Levring (1947), and we must refer to their papers for de-
tailed results. We merely mention, as an example, that, according to Sey-
bold, a Rhodophycea will absorb up to 95% of the light available in a 50 ra.
depth, where a Chlorophycea will be able to absorb only 30^0% (cf. fig.
22.56).
Light Absorption by Pigments in the Living Cells
A. Light Absorption by Plants
1856 Bohm, J. A., Sitzber. Akad. Wiss. Wien, Math, naturw. Klasse, 22, 479.
1861 Sachs, J., ibid., 43, 265.
1880 Stahl, E., Botan. Z., 38, 297, 321, 345, 361, 377, 393, 409, 868.
1883 Reinke, J., Ber. deut. botan. Ges., 1, 395.
1885 Timiriazev, C, Cornpt. rend., 100, 851.
1886 Reinke, J., ibid., 44, 161, 177, 193, 209, 225, 241.
1888 Detlefson, E., Arbeit, des botanischen Instituts, Wilrzburg, 3, 534.

1901 Linsbauer, L., Botan. Zentr., Beihefte, 10, 53.
1905 Brown, H., and Escombe, F., Proc. Roy. Soc. London, B76, 29.
1908 Senn, G., Die Gestalts- und Lageveranderungen der Chromatophore, Engel-
mann, Leipzig, 1908.
1909 Senn, G., Ber. deut. botan. Ges., 27, ( 12.)
Stahl, E., Zur Biologie des Chlorophylls. Fischer, Jena, 1909.
1911 Weigert, F., Die chemischen Wirkungen des Lichts. Enke, Stuttgart, 1911.
1912 Coblentz, W. W., Bull. U. S. Bureau Stand., 9, 283.
1914 Purevich, K. (Purjewitsch), Jahrb. wiss. Botan., 53, 210.
1917 Senn, G., Verhandl. naturforsch. Ges. Basel, 28, 104.
1918 Willstatter, R., and Stoll, A., Untersuchungen uber die Assimilation der
Kohlensdure. Springer, BerHn, 1918, p. 122 ff.
TJrsprung, A., Ber. deut. botan. Ges., 36, 122.

1919 Senn, G., Z. Botan., 11, 81.
1921 Wurmser, R., Arch. phys. bioL, 1, 33.
1922 Liese, W., Beitr. allgeni. Botan., 2, 323.
1924 Lazarev, P. P., /. Applied Phys. USSR, 1, 142.

Benecke, W., and Jost, L., Pflanzenphysiologie, Vol. 1, Jena, 1924.
1925 Pokrovski, G. I., Biochem. Z., 165, 420.

Warburg, 0., ibid., 166, 386.
1927 Lazarev, P. P., ibid., 182, 131.
1929 Shull, C. A., Botan. Gaz., 87, 583.

Briggs, G. E., Proc. Roy. Soc. London, B105, 1.
1932 Seybold, A., Planta, 16, 195.

Seybold, A., ibid., 18, 479.
1933 Schanderl, H., and Kaempfert, W., ibid., 18, 700.

Voerkel, S. H., ibid., 21, 156.
1934 Seybold, A., Jahrb. wiss. Botan., t9, 593.

1935 Mestre, H., Cold Spring Harbor Symposia Quant. Biol., 3, 191.
1937 Mecke, R., and Baldwin, W. C. G., Naturwissenschaften, 25, 305.

Egle, K., Planta, 26, 546.
1939 Noddack, W., and Eichhoff, H. J., Z. physik. Chem., A185, 241.
1941 Loomis, W., Carr, and Randall, H. M., Gibson Island A. A. A. S. Sym-
posium on Photosynthesis (unpublished).
1942 Burns, G. R., Am. J. Botany, 29, 381.
Seybold, A., and Weissweiler, A., Botan. Archiv, 43, 252.
1943 Seybold, A., and Weissweiler, A., Botan. Arch., 44, 102,456.
1946 Rabideau, G. S., French, C. S., and Holt, A. S., Am. J. Botany, 33, 769.
1947 Looniis, W., A.A.A.S. Symposium on Photosynthesis, Chicago (unpub-
lished).
1948 Kok, B., Enzymologia, 13, 1.
1949 Loomis, W., Photosynthesis in Plants, Iowa State College Press, Ames,
1949, p. 5.
B. Spectral Properties of Plants
1870 Hagenbach, A., Ann. Phys. (Poggendorf), 141, 245.

1871 Gerland, E., ibid., 143, 585.
1872 Timiriazev, C, Ber. deut. chem. Ges., 5, 329.

1884 Engelmann, T. W., Botan. Z., 42, 81, 97.
1886 Reinke, J., ibid., 44, 161, 177, 193, 209, 225, 241.
1887 Engelmann, T. W., ibid., 45, 393, 409, 425, 441, 457.
1904 Gaidukov, N., Ber. deut. botan. Ges., 22, 23.
Gaidukov, N., Hedwigia, 43, 96.
1907 Ivanovski, D. (Iwanowski), Ber. deut. botan. Ges., 25, 416.
1913 Ivanovski, D. (Iwanowski), Biochem. Z., 48, 328.
1918 Ursprung, A., Ber. deut. botan. Ges., 36, 86, 111.
Willstatter, R., and Stoll A., Untersuchungen ilber die Assimilation der
1921 Wurmser, R., Arch. phys. biol., 1, 33.
1925 Pokrovski, G. I., Biochem. Z., 165, 420.
Baas-Becking, L. G. M., and Ross, P. A., /. Gen. Physiol, 9, 111.
1926 Wurmser, R., /. phys. Radium, 7, 33.
1927 Lubimenko, V. N., Rev. gen. botan., 39, 547.

1929 ShuU, C. A., Botan. Gaz., 87, 583.
1932 Seybold, A., Planta, 16, 195.
1933 Schanderl, H., and Kaempfert, W., ibid., 18, 700.
Seybold, A., Planta, 21, 251.

1934 Seybold, A., Jahrb. tuiss. Botan., 79, 593.
Spohn, U., P/anfa, 23, 241.
1936 Seybold, A., Jahrb. loiss. Botan., 82, 741.
1937 Albers, V. M., and Knorr, H. V., Plant Physiol, 12, 833.
Vermeulen, D., Wassink, E. C, and Reman, G. H., Enzymologia, 4, 254,
.
Egle, K., Planta, 26, 546.

Mecke, R., and Baldwin, W. C. G., Naturwissenschaften, 25, 305.
1938 Strain,H. H., Leaf Xanthophylls, Carnegie Inst. Wash. Pub. No. 490.
1939 Katz, E., and Wassink, E. C, Enzymologia, 7, 108.
Wassink, E. C, Katz, E., and Dorrestein, R., ibid., 7, 113.
Noddack, W., and Eichhoff, H. J., Z. physik. Chem., A185, 241.
1940 French, C. S., J. Gen. Physiol., 23, 483.
Menke, W., Naturwissenschaften, 28, 31.
Seybold, A., and Egle, K., Botan. Arch., 41, 578.
Zscheile, F. P., and Comar, C. L., Botan. Gaz., 102, 463.
Emerson, R., and Lewis, C. M., Gibson Island A. A. A. S. Symposium
on Photosynthesis (unpublished)
Button, H. J., and Manning, W. M., Am. J. Botany, 28, 516.
Loomis, W., Carr, and Randall, H. M., Gibson Island A. A. A. S. Sym-
posium on Photosynthesis (unpublished).
Wurmser, R., Rev. brasil biol, 1, 325.
1942 Emerson, R., and Lewis, C. M., J. Gen Physiol, 25, 579.
Duntley, S. C, /. Optical Soc. Am., 32, 61.
Saunderson, J. L., J. Opt. Soc. Am., 32, 727.
Seybold, A., and Weissweiler, A., Botan. Arch., 43, 252.
1943 Seybold, A., and Weissweiler, A., ibid., 44, 102.
Seybold, A., and Weissweiler, A., ibid., 44, 456.
Duntley, S. C, J. Opt Soc. Am., 33, 252.
1946 Rabideau, G. S., French, C. S., and Holt, A. S., ibid., 33, 769.
Wassink, E. C, and Kersten, J. A. H., Enzymologia, 12, 3.
1947 Iljina, A. A., Zhurnal fys. Khimii, 21, 145.
1948 Van Norman, R. W., French, C. S.. and Macdovvall, F. D. H., Plaid
Physiol, 23, 455.
1949 Loomis. W.. Photosynthesis in Plants. Iowa State College Press. Ames,
1949, p. 5.
1951 Tanada, T., Am. J. Botany, 38, 276.
French, C. S., and Koski, V. M., Proc. Soc. Exptl Biol, (in press).
C. Distribution of Absorbed Energy among Pigments
1923 Warburg, 0., and Negelein, E., Z. physik. Chem., 106, 191.
1936 Seybold, A., Jarhb. iviss. Botan., 82, 741.
1938 Strain,H. H., Leaf Xanthophylls. Carnegie Inst. Wash. Publ. No. 490.
1939 Noddack, W., and Eichhoff, H. .J., Z. physik. Chem., A185, 241.
1940 Montfort, C, ibid., A186, 57.
1941 Button, H. J., and Manning, W. M., Am. J. Botany, 28, 516.
Emerson, R., and Lewis, C. M., Gibson Island A. A. A. S. Symposium
on Photosynthesis (unpublished).
1942 Strain, H. H., and Manning, W. M., /. Biol Chem., 144, 625.
Emerson, R., and Lewis, C. M., /. Ge7i. Physiol., 25, 579.
1943 Emerson, R., and Lewis, C. M., Am. J. Botany, 30, 165.
Manning, W. M., and Strain, H. H. J. Biol. Chem., 151, 1.
1951 Tanada, T., .4//;. J. Botany, 38, 276.
Appendix. Natural Light Fields
1895 Aschkinass, E., Ann. Phys. (Poggcndorf) 55, , 40L

1907 Wiesner, Der Lichtgenuss der Pflanzen. Engelmann, Leipzig.
J.,
1908 Schmidt, W., Sitzber. Akad. Wiss. Wien Math, naturw. Klasse, Aht., lid,
117, 237.
1918 L^rsprung, A., Ber. dent, botan. Ges., 36, 111.
1922 Knudsen, M., Conseil permanent intern, exploration mer, Publ. No. 76.
1928 Hulburt, E. 0., /. Opt. Sac. Am., 17, 15.
1929 Shelford, V. E., in E. Abderhalden, Handbuch der biologischen Arbeits-
methoden. Part 2, 9, p. 1495.
1930 Beebe, W., and Hollister, G., Bull. N. Y. Zool. Soc, 33, 249.
1931 Sawyer, W. R., Contrib. Canad. Biol. Fisher., 7, 75.
1932 Hulburt, E. O., /. Optical Soc. Am., 22, 408.
Atkins,W. R. G., /. conseil permanent intern, exploration mer, 7, 171.
1934 Seybold, A., Jahrb. iviss. Botan., 79, 593.

Montfort, C., ibid, 79, 493.
1936 Seybold, A., ibid., 82, 741.
1937 Egle, K., Planta, 26, 546.

1938 Manning, W. M., Juday, C, and Wolf, M., /. Am. Chem. Soc, 60, 274.
1940 Dorsey, N. E., Properties of Ordinary Water Substance. Reinhold, New
York, 1940.
1941 Taylor, A. H., and Kerr, G. A., J. Optical Soc. Am., 31, 3.
1946 Johnson, N. G., and Kullenberg, B., Svensk. hydr.-biol. Komm. Skr., Ill,
1, No. 1.
1947 Le\Ting, T., Goteborgs Kungl. Vetenskaps. Vitterhets Samh. Handl. (VHB),
5, No. 6.
Chapter 23
FLUORESCENCE OF PIGMENTS IN VITRO
Fluorescence phenomena have two aspects. In the first place, the fluor-
escence spectrum offers a welcome addition to the absorption spectrum in
the study of the term system and molecular structure of a chemical com-
pound. In the second place, the yield and duration of fluorescence gives
significant information as to the fate of the excitation energy and thus
provide clues to the mechanism of photochemical reactions of the light-
absorbing compound. In the case of chlorophyll, we are particularly in-
terested in the second, photochemical aspect of the fluorescence phenomena.
Because of the division of this treatise into a chemicaland a physical
part, the photochemistry of chlorophyll already was dealt with in the first
volume (chapters 18 and 19), while fluorescence could first be discussed
in the present, second vohune. Certain conclusions derived from fluores-
cence studies had to be anticipated in Volume I some of them will have
;
to be repeated and amplified here.

Some new facts and considerations have been added to this field since
the appearance of Volume I their fluorescence aspects are discussed in
;
the present chapter, and their photochemical aspects in chapter 35.
A. Fluorescence of Chlorophyll in Vitro*
1. Fluorescence Spectra of Chlorophyll and Its Derivatives in Solution
The was discovered by Brewster more than

fluorescence of chlorophyll
a hundred years ago (1834). was first studied spectroscopically by
It
Stokes in 1852, and a by-product of this study was the discovery that the
leaf pigment consists of two green and two yellow components. Dher^
(1914) and Wilschke (1914) contributed the first photographs of the
fluorescence spectrum. However, because the fluorescence bands of
chlorophyll are situated in the far red and infrared, and the sensitivity of
red- and infrared-sensitized plates varies strongly with wave length, the
photographic method is not very suitable for the quantitative study of
chlorophyll fluorescence.
740
FLUORESCENCE SPECTRUM OF CHLOROPHYLL 741
Figure 23.1 A shows the appearance of the fluorescence spectrum of

chlorophyll on a panchromatic plate, and figure 23. IB, on an infrared-
Wave length, m/i
Reference spectrum (He + Hg)

'
Absorption
Chloroptiyll a
Fluorescence
Reference spectrunn (He + Hg)

'
Absorption
Fluorescence
Chlorophyll a
with self-
absorption
Exciting light ( \ < 470 m/i)
Calibration spectrum (Nernst lamp)
Reference spectrum (He + Hg)
oo o oo
inoui
r-- 1^ lO lO
742 FLUORESCENCE OF PIGMENTS IN VITRO CHAP. 23
(1943) by means of a photoelectric spectrophotometer. The maximum of
the first emission band Hes, in this figure, at 664.5 mn for chlorophyll a and
CVJ>0 00
FLUORESCENCE SPECTRUM OF CHLOROPHYLL 743
first fluorescence band is followed bj^ a second one, of lower intensity, situ-
ated in the far red, which is visible in figure 23. IB as well as in figure 23.2;
and by a third, weak one, situated in the near infrared, which is not re-
corded in these figures.
In an earlier investigation of Zscheile (1935), an additional band was observed in the

fluorescence spectrum of chlorophyll b at 672.8 m^; but Dhere and Biermacher (1936)
and Biermacher (1936) ascribed it to contamination with chlorophyll a, and this ex-
planation was accepted by Zscheile and Harris (1943).
According to Biermacher (1936), the fluorescence spectrum provides the most sensi-*
five test for the purity of chlorophyll b. A purification method based on this test was
described in chapter 21.
100
—^— Chlorophyll o
>- Chlorophyll b
cn
z
UJ
o
z
LD
o
(/)
Ijj
q:
o
iij
>
UJ
620 660 700 740 780

WAVE LENGTH, m/i
Fig. 23.2. Fluorescence spectra of chlorophylls a and b in ether. Photo-

metric curves corrected for self-absorption (after Zscheile and Harris 1943).
Table 23. lA shows the positions of the main fluorescence bands of the
two chlorophylls in ethyl ether, as found by several investigators, and
Table 23. IB, the positions of the same bands in various solvents.
A spectrophotometric curve of the fluorescence of a benzine extract
from Brassica (containing both chlorophyll components) can be found in
a paper by Vermeulen, Wassink and Reman (1937).
In Table 23.1, some values represent band maxima, Xj^^x., as determined
by photoelectric photometry, and others band axes, X, i. e., the arithmetic
means of the extension limits of the bands on spectrum photographs.
These limits depend strongly on the spectral sensitivity curves of the
photographic plates used, and on the length of the exposure. Biermacher
(1936) insisted, however, that, as long as only the peaks of the bands are
photographed, e. g., by using suitably short exposures, the axes coincide
Table 23.1
Fluorescence Bands of the Chlorophylls a and b
A. BAND POSITION IN ETHYL ETHER

FLUORESCENCE SPECTRUM OE CHLOROPHYLL 745
Table 23.1 {Continued)
C. BAND SHIFTS IN DIFFERENT SOLVENTS"

Biermacher (1936)
Zscbeile and Harris (1943)
Chi. o Chi. 6
Chi. a,
Solvent AX„ m/i Solvent AX, m^
Methanol 10 Pentane 4.5 4.5
2-Ethylhexanol 10 Hexane 4.5 4.5
2-Methylpropanol 9.5 Pyridine 4.5 3.5
Carbon tetrachloride 4 3
Benzene
Isopropyl ether
9
9 Methanol 3.5 —
Acetone 8.5 Aniline 3 5
1-Butanol 8.5 Benzene 2.5 4.5
Methyl oleate 8 Dioxane 2.5 5
Carbon tetrachloride 8 Ethanol 2.5 6.5
Olive oil 7.5 Cyclohexanol 2 4
Dioxane 7 Chloroform 2 2.5
Cyclohexane 5.5 Carbon disulfide 2 6
Ethyl ether 4.5 Acetone 1.5 8.5
Ethyl ether 0.5 5
Paraffin (liq.) 0.5
" Photographic.
* Photoelectric photometer.
" Concerning subsidiary maxima, see page 748.
<*
Biermacher (1936) found no fluorescence at all in methylene iodide (as well as in
nitrobenzene).
^ This figure is quoted by Seybold and Egle (1940) from Stern.
^ After Stewart, Knorr and Albers (1942).

" AX = X(fluorescence) —
X( absorption).
with the true band maxima. He therefore denied that the difference be-
tween the X vahies measured by him and the Xâx. vahies found by earher
investigators covild have been due to the dechne in the sensitivity of his
photographic plates in the far red; he suggested instead that this differ-
ence was caused by the faihire of other observers to avoid "self-absorption,"
i. e., reabsorption of fluorescent light befoi-e its escape from the chlorophyll
solution. (Because of the position of the fluorescence band of chlorophyll

closeon the red side of the absorption peak, self-absorption must cause an
apparent shift of the fluorescence band maximum toward longer waves.)
The correctness of this interpretation was acknowledged by Zscheile and
Harris (1943), who made a spectrophotometric redetermination of the
fluorescence bands, varying the chlorophyll concentration systematically,
and using a capillary vessel to reduce self-absorption. The extent of the
self-absorption effect is illustrated by figure 23.3. It shows how large a
part of the fluorescence band is overlapped by the (shaded) absorption
band, and how, in consequence of this overlapping, the position of the
maximum of the fluorescence band can be displaced, by self-absorption,
by as much as 12 mju. The position of the second fluorescence band (at 720
m^) found to be practically unaffected by reabsorption; this is natural,
is
band leads to a vibrating state of the chlorophyll molecule and

since this
consequently does not occur in absorption at least not with a marked in- —
tensity.
Table 23. IC compares the most reliable figures in Table 23. IB those of —
Biermacher, and Zscheile and Harris —with the wave lengths of the ab-
sorption bands as listed in Table 21. IV. This comparison shows that the
fluorescence maximum remains on the long-wave side of the absorption
maximum in all solvents, but that the distance between the two maxima
sometimes falls to as little as 1 m/x.
^^— Fluorescence absorbed by minimum layer

of chlorophyll solution
Fluorescence obsorbed by V4mm. loyer

Fluorescence absorbed by 3 V* mm. layer

Fluorescence obsorbed by 10mm. layer

>////^^. Absorption (from fig. 21.1)
620 660 700 740 780

WAVE LENGTH, m/i
Fig. 23.3. Effect of self-absorption on fluorescence spectrum of ciilorophj'll a in
ether (after Zscheile and Harris 1943).
It is difficult to say whether any of the variations in the shift, AX, indi-
cated by Table 23. IC, are significant; whether the conspicu-
in particular,
ous difference in the order of solvents found for the two chlorophylls, a and
&, is real.
Seybold and Egle (1940) suggested that AX is abnormally large (^^15 myu) in chloro-
phyll solutions in Hpides such as lecithin. (This assumption was necessary for their
"two-phase theory" of the state of chlorophyll in vivo; cf. Vol. I, page 393). This sug-
gestion is not plausible in itself, and therefore cannot be accepted without confirmation
by reliable measurements.
As to the reason for the "red shift" of the fluorescence bands compared
with the absorption bands, the explanation must lie in the loss of vibra-
tional quanta in the interval between excitation and re-emission, or after
re-emission. Quite generally, the molecule has a somewhat different nu-
clear configuration in the excited and in the normal state. Therefore, ac-
cording to the so-called Franck-Condon principle, electronic excitation is
accompanied by the excitation of a certain amount of vibrations. A large

part if not all these vibrational quanta are dissipated before re-emission of
FLUORESCENCE AND ABSORPTION BANDS 747
light.After the emission, the molecule finds itself for a second time in a
deformed state, and, for a second time in the fluorescence cycle, some
energy converted to vibrational energy. The magnitude of AX indicates
is
that the vibrational quanta concerned must be of the order of 100 cm.-^,
much smaller than the quanta (1000-1400 cm.-^) postulated on page 630 to
account for the sequence of the visible absorption bands of chlorophyll.
Another explanation of the red shift could be derived from the hy-
pothesis (c/. page 631) that the main red absorption band, Xo -^ Yo {cf. fig.
21.20), conceals a weak band, Xo ^
Ao, which belongs to the yellow-orange
band system. If this is true, and if the red fluorescence band is the pure
Fo -^ Xo band, the somewhat different position of its maximum is under-
standable. This explanation is less likely because the displacement of the
fluorescence band toward the red is a general phenomenon, while the over-
lapping of the bands Xo -^ Fo and Xo ^ Ao, if it exists at all, can be only an
accidental occurrence.
The influence of the solvent on the position of the fluorescence band must
be attributed to the same cause as its influence on the absorption spectrum,
i. e., to the difference in the solvation energy of the pigment in the ground
state and in the excited state. Table 23.IC shows that within a homolo-
gous group of solvents an approximate parallelism exists between the posi-
tion of the fluorescence band and the refractive index of the medium. This
regularity already was noted and discussed in chapter 21, when we dealt
with the absorption spectra of chlorophyll in different media.
A new light on the effect of solvents on the fluorescence of chlorophyll
was thrown by the observations of Livingston, Watson and McArdle
(1949), which will be described further below. These experiments indicate
that the solvent effect is twofold: In the first place, the presence of at
least a small amount of solvent molecules of a certain type (Avater, alcohols,
amines) appears to be needed to bring out the fluorescence (presumably,

by converting chlorophyll from a nonfluorescent into a fluorescent tauto-
meric form). Alter the fluorescence had been "activated" in this Avay, its
spectrum and intensity are independent of the specific nature of the "ac-
tivator," and determined only by the nature of the bulk solvent. In other
words, whether the fluorescence of chlorophyll a in benzene is "activated"
by methanol, or piperidine, or water, its spectrum and intensity are char-
acteristic of benzene as medium. Of course, when larger quantities of the
"activator" are added, the spectrum must sooner or later approach that
characteristic of the chlorophyll solution in the pure activator; but these
transitions have not yet been studied.
Like the two main chlorophylls, a and b, chlorophyll c {chlorofucin) also
has a red fluorescence band; its axis lies at 631.5 mju in ether (Dhere and
Fontaine 1931), and at 635 m/x in ethanol (Wilschke 1914).
Chlorophyll d, too, was described by Manning and Strain (1943) as

exhibiting a deep red fluorescence. Its spectrum shows a first maximum at
693 mju (in ethereal sokition) and indications of a diffuse second maximum
at 750 mM-
No quantitative data appears to be available on the fluorescence spec-
trum of allomerized chlorophylls.
Proiochlorophyll has a fluorescence band at 626.5 mju, in ether (Dh6r6
1930). The fluorescence bands of pheophytin a lie and 730.5 mju
at 676
in ether (Dhere and Raffy 1935) and at 677.5, 717, 750.5 and 804 mju in
dioxane (Stern and Wenderlein 1936).
A photometric curve of the fluorescence spectrum of bacteriochlorophyU
in solution was obtained by Vermeulen, Wassink and Reman (1937). It
showed two bands, a weaker one at 695 mju, and a stronger one at 810 m/j
(fig. 23.4). The relationship of these two bands is not clear (c/. p. 751).
The fluorescence of these and many other porphin derivatives was re-
viewed by Dhere (1937, 1939).
J3
O
650 700 750 800 850

WAVE LENGTH, m/i
Fig. 23.4. Fluorescence spectrum of bacteriochlorophyU in solution

(after Vermeulen, Wassink and Reman 1937).
counterpart of the strong blue-violet absorption band appears in chloro-

No
phyll fluorescence, even if white, violet or ultraviolet light is used for excita-
tion (c/. Dhere and Raffy 1935, Prins 1934, and Vermeulen, Wassink and
Reman 1937) . The same is true of the weaker absorption bands in the mid-
dle of the visible spectrum. True, Prins (1934) said that the red band oc-
curs in fluorescence without the yellow and orange bands only if fluores-
cence is excited by red light (660-680 m/x); and Ivnorr and Albers (1933,
1935) observed "subsidiary" fluorescence bands on the short-wave side of
the main one—at 633 and 637 mju in chlorophyll a and b, respectively (c/.
Table 23.1) but Zscheile and co-workers (1935, 1943), who excited fluores-
;
cence with white light, and Vermeulen, Wassink and Reman (1937), who
used ultraviolet and blue exciting light, obtained fluorescence curves with-
out any indication of such additional bands (cf. fig. 23.2).
Zscheile and Harris (1943) foimd that the fluorescence spectmm of
chlorophyll was exactly the same whether excited by the mercury lines
FLUORESCENCE OF CHLOROPHYLL DERIVATIVES 749
365, 404.7, 435.8 or 546 m/x, or by white light filtered through a red, orange
yellow, violet, blue or green filter.
Whether chlorophyll capable of emittmg a weak, but long-lasting

is
cj. page 753) is

infrared fluorescence (originating in a metastable state,
uncertain. Calvin and Dorough (1947) have described such a "phosphores-
cence," but Livingston and co-workers (1948) could not confirm their ob-
servations (c/. page 795).
Figure 23. IB shows, beside the fluorescence spectra of the two chlorophylls, also
those of the two pheophorbides, and of two porphyrins. The pheophorbides {i.e.,
chlorophyllides in which hydrogen has been substituted for magnesium) fluoresce not
less strongly than the chlorophylls or chlorophyllides themselves; but certain other sub-
stitutions in the same position in the molecule {e. g., copper instead of magnesium) cause
complete disappearance of fluorescence.
Stern and Molvig (1935, 1936^), Stern and Dezelic (1936) and Stern
(1938) have investigated the fluorescence of numerous porphyrins and
chlorins. They found that, similariy to chlorophyll, all of them fluoresce
with red light, evenwhen excited by violet or ultraviolet radiation. The
main fluorescence band always lies close to the first absorption band in the
—
red whether this band is the weakest of the whole absorption spectrum
(as in some porphyrins) or the strongest one (as in chlorins and phorbins).
Stern (1938) found that tetrapyrrole compounds without the closed por-
phin ring system (e. g., the bile pigments), as well as compounds in which
the conjugation in the porphin ring is interrupted, do not obey this rule,
and do not show sharp fluorescence bands at all. He therefore considered
a sharp red fluorescence band as an important characteristic of the all-
round conjugated porphin ring system.

According to the term systems given in figures 21.9 and 21.25, the ap-
pearance of the red band the exclusion of all the other
in fluorescence, to
bands, means that all excitation energy in excess of that corresponding to

the lowest, nonvibrating, excited electronic state is dissipated before
fluorescence can occur —probably first by internal distribution of this
energy among vibrations within the pigment molecule, a process known
as "internal conversion," and then by gradual transfer of vibrational quanta
to the medium. The dissipation is interrupted at the lowest excited level,
whether this is level .4 (in porphyrins), Y (in chlorins and phorbins) or
Z (in bacteriochlorophyll), long enough to allow a significant proportion
of the excitation energy to escape as fluorescence.
It was suggested by Franck and Herzfeld (1937) that the capacity of
chlorophyll to convert rapidly quanta of larger size into smaller red (quanta
may be important for the function of this pigment in photosynthesis, be-

cause it prevents the occurrence of undesirable photochemical reactions
that could be sensitized by the larger quanta. This surmise may or may
not be correct, but since the same property is shared by all porphyrins and
chlorms, it cannot explain the special suitability of chlorophyll as photo-
catalyst in photosynthesis.
750 FLUORESCENCE OF PIGMENTS /iV VITRO CHAP. 23
Table 23.11
A. Fluorescence Bands of Porphin in Dioxane
Band no.
INTERPRETATION OF FLUORESCENCE SPECTRUM 751
the sake of uniformity, all figures in Table 23.IIB are based on the data of
Dhere and Raffy in Table 23.IA.)
In bacieriochlorophyll the distance between the two fluorescence bands
(shown in fig. 24.4) is much larger than in chlorophyll— 2400 cm.-^; and
the short-wave (red) band is weaker than the long-wave (infrared) band.
This points to two different electronic transitions, rather than two vibra-
tional bands in a common band system. The two fluorescence bands may
even belong to two different molecular species. Only the stronger of them
—that at 810 myu- appears to be correlated with a known absorption band
of bacteriochlorophyll, that at 770 m/x (c/. fig. 21.7). (This correlation im-
plies that AX, the displacement of the fluorescence band relative to the
absorption band, is of the order of 40 mn, as against < 15 mn in ordinary
chlorophyll.)
This be the place to mention the luminescence that occurs when a chlorophyl
may
is heated to 125° C.
solution in tetralin This phenomenon was first described by Rothe-
mund (1938) and investigated spectroscopically by Stewart, Knorr and Albers (1942).
The maximum of the luminescence band was found at 677.5 m^. After the tetralin
solution was heated for five minutes, chlorophyll showed a change— its
fluorescence
at 688.5 to 671.0 and was reduced to one
band was shifted from its original position niyu
third its original intensity; the absorption spectrum also had undergone a transforma-
tion, especially in the blue-violet region. The origin of this luminescence is as yet un-
known, and its interpretation as chemiluminescence, suggested by the investigators,
although plausible, requires confirmation.
2. Yield of Fluorescence and Life-Time of the Excited States

of Chlorophyll
The fluorescence yield can be defined either as the proportion of ab-

sorbed energy re-emitted in the form of radiation or, more significantly,
as the proportion of re-emitted photons. The two figures coincide only
in
the case of resonance fluorescence; usually (particularly in condensed sys-
tems) the emitted light is of a lower frequency than the absorbed light
("Stokes' rule"), and the "energy yield," e/, is therefore smaller than the
"quantum yield," (p.
The relation €f < f holds main fluorescence bands of all
true for the
derivatives of porphin and chlorin, and the difference becomes particularly
large if blue, violet or ultraviolet light is used for excitation. It was stated
above (page 748) that only red emitted in the fluorescence of these
light is
compounds this means that the absorbed ultraviolet, violet or blue energy
;
ciuanta are transformed into much smaller red quanta, while up to 50%
of
the absorbed light energy is dissipated. Since the "theoretical" life-
time of the excited state B (upper state of the blue-violet band system) is
of the order of 5 X 10 "« sec.
{of. page 634), the absence of even 0.01%
fluorescence in this system shows that the electronic energy of the state B
is dissipated m less than 5 X IQ-^^ sec, i. e., after less than one hundred
molecular vibrations.
It appears that all porphyrins, chlorins and phorbins, when brought

into an electronic state with an energy higher than that of their lowest ex-
cited state, rapidly lose this excess energy, and revert into state A (por-
phyrins), Y (chlorins and phorbins) or Z (bacteriochlorophyll and its
derivatives). Differences in the yield of red fluorescence among various

compounds of these three classes must then be due to variations in the
longevity of the lowest excited states {A, Y or Z). In ''nonfluorescent"
compounds (e. g., copper pheophorbide, wdth <p <0.01%) the energy of the
lowest excited state must be dissipated within 10 ~^^ sec. ; while in "strongly
fluorescent" compounds, such as chlorophyll itself, this state must persist
for 10~^ or 10~^ sec. to allow at least several per cent of the excitation
energy to be re-emitted as fluorescence.
Prins (1934) mentioned that the quantum yield of the fluorescence of
chlorophyll a in ethanol is smaller if by blue than if excited by red
excited
light —an observation that seemed to indicate that the conversion of
chlorophyll molecules in state B into those in state Y is less than 100% ef-
ficient,perhaps due to competition on the part of a photochemical reac-
tion with the solvent (cf. page 756). However, this result needed confirma-
tion; Button, Mannmg and Duggar (1943), who worked with acetonic
solutions of chlorophylls a and b, found identical yields of fluorescence when
using excitation by violet light (436 m/x) or yellow light (578 mju). More
recently, measurements of the relative yield of the fluorescence of chloro-
phyll, excited by the Hg lines 435.8 and 577-579 m;u, and by narrow bands
centered at 645 and 681 m/x (isolated by means of Farrand's interference
filters) were made by Livingston and co-workers (1949). Table 23.IIC
shows their results.
Table 23.IIC
Effect of Wavelength of Exciting Light on Relative Quantum Yield
OP Chlorophyll Fluorescence
YIELD OF CHLOROPHYLL FLUORESCENCE 753
(c/. Livingston mentioned that a similar decline of fluorescence

chapt. 30).
in the long-wave region was noted by Solomin with other dyestuffs. vSuch
a drop would be understandable if the long-wave wing of the red absorption
band were covering another band, leading to a different excited electronic

state. If, is due to transitions originating in
however, the long-wave wing
vibrational levels of the normal state and leading to the same excited state
as that reached in the peak of the band (and this is what one would think
offhand), then the lower quantum yield of fluorescence and sensitization is

peculiar and awaits interpretation.
Transition to
fluorescent stote
Fluorescence
10%
Scheme 23.1. The metastable state of chlorophyll {T may mean "triplet"
or'' tautomeric").
In contrast to the observations in the far red the confirmation by Liv-

ingston of Prins's observation— that the fluorescence yield of chlorophyll
in solution is considerably smaller when fluorescence is excited by blue-
violet than when it is excited by red light— has no parallel in the wave-
length dependence of the quantum yield of photosynthesis in live cells
chapter 30,), and (what seems to be even more remarkable) in the
{cf.
quantum yield of chlorophyll-sensitized autoxidations in the same

medium (alcohol) chapter 18, page 513). This discrepancy con-
(cf.
firms the supposition that photochemical sensitization by chlorophyll does

not compete directly with fluorescence, but is brought about by transfor-
754 FLUORESCENCE OF PIGMENTS 7A" VITRO CHAP. 23
mat ion of chlorophyll into a long-lived active (tautomeric, isomeric or

metastable electronic) state. This transformation may perhaps occur not
only from state A, but also directly from state B (fig. 21.9 and scheme
23.1). Molecules excited to the higher state B, have the choice of going
directly to T, or first to A and thence to T. Consequently, the total prob-
ability of conversion to a metastable state is higher for molecules excited
by blue-violet, than for those excited by red light. In the numerical ex-
ample indicated in scheme 23.1, this probability is 95% for molecules in
state B and 90% for molecules in state A —
leaving for fluorescence, 5%
in the first case and 10% in the second.
More measurements of the yield of fluorescence of chlorophyll under
different conditions are greatly needed for better understanding of the
photochemistry of this compound. At present, our knowledge of the

absolute yield of chlorophyll fluorescence in solution is limited to a single
estimate by Prins (1934), who found it to be of the order of 10% (in a 10"^
M solution of chlorophyll a in ethanol). He did not take into account
self-absorption (c/. fig. 23.3), and, not knowing the exact experimental ar-
rangement, impossible to estimate its influence on the yield.
it is
Perrin (1929) calculated 3 X 10"^ sec. for the hfe-time of chlorophyll

fluorescence, from polarization measurements in four solvents of different
viscosity.
If we accept the Prins estimate as substantially correct, it follows that,
even in so-called "strongly fluorescent" chlorophyll solutions, 90% of the
excited pigment molecules lose their excitation energy before they have an
opportunity to fluoresce. The actual mean life-time of the excited state Y
under these conditions must be of the order of 0.1 X 8 X 10-^ = 8 X 10"^
sec. (cf. equation 22.3). Not all of the excitation energy needs to be lost
during this period. The fate of the residual excitation energy is not defi-
nitely established, but in the next section we will discuss indications (men-
tioned in Volume I, page 483) that the chlorophyll molecules in state A,
which fail to emit fluorescence, are converted into a long-lived active form
(level T in scheme 23.1), which may represent a metastable triplet electronic
state, or a tautomer, or an oxidized or reduced molecular species.
The fluorescence yield of allomerized chlorophyll a in methanol is, ac-
cording to Livingston (1949) about one-half, and that of allomerized chloro-

phyll h about twice, that of the intact pigment. The change is the same
when the "allomerization" of the pigments is caused by iodine, or acceler-
ated by catalysts such as LaCls.
This confirms that all these reagents lead to the formation of the same
product —which, according to Fischer (Volume I, page 461), is chlorophyll
oxidized at the carbon atom C(10).

FACTORS LIMITING THE YIELD 755
3. Factors Limiting the Yield of Fluorescence
Whatprocess (or processes) cause the abbreviation of the natural Ufe-

time of the excited state A of dissolved chlorophyll molecules and prevent
a 100% yield of fluorescence? If a single molecule in vacuum absorbs a
light quantum, the probability of fluorescence is 100%, since fluorescence
is the only way in which the molecule can get rid of excess energy. A tem-
porary distribution of this energy between the internal degrees of freedom
or even its transformation into chemical energy, by isomerization or dis-
sociation, can delay the emission of fluorescence, but cannot prevent it.
Sooner or later, the original composition and configuration of the molecule
will be restored, excess energy will again be converted into the original form
of electronic excitation and emission of fluorescence will take place.

In condensed phases, on the other hand, the yield of fluorescence is usu-
ally, if not always, less than unity. This weakening of fluorescence by
the medium has several causes. The presence of a large number of foreign
molecules can make both the above-mentioned "fluorescence-delaying"
—
processes the dissipation of the electronic excitation energy within the
absorbing molecule ("internal conversion") and the transformation of
excitation energy into chemical energy—partly or completely irreversible,
and thus reduce the probability of fluorescence. The proximity of foreign
molecules adds new possibilities of chemical utilization of light energy,
since the excited molecule can now react with other molecules encountered
during the excitation period. \ fourth possibility of energy loss in condensed
systems — in addition to (1) chemical reaction
internal conversion, (2)
within the excited molecule and (3) chemical reaction with molecules of
—
the medium is (4) bulk transfer of electronic energy from the excited
molecule to a foreign molecule close by. Finally (5), if the fluorescent
molecules are present in sufficient concentration, "self-quenching," i. e.,
energy dissipation by interaction of excited with nonexcited pigment mole-
cules, may become significant.
It is important to keep in mind that the interaction of a fluorescent
molecule with a given medium can be complex. For example, the presence
of the medium may make internal energy dissipation irreversible, but also
slower. Furthermore, the general eff"ect of the solvent surrounding the
fluorescent molecule may be one of quenching, but the specific effect of as-
sociation of certain groups in the medium with certain groups in the fluores-
cent molecule may be one of "protection", i. e., stimulation of fluorescence,
and so on.
We will now consider in more detail the five ways in which fluorescence
can be affected by the mutual closeness of molecules in condensed systems.
(a) Internal Conversion (Physical Dissipation of Excitation Energy)
In complex molecules, electronic excitation states are likely to be con-

verted, before fluorescence has had time to occur, into strongly vibrating
states. The reason for this is that the nonvibrating (or weakly vibrating)
excited electronic state may have not only the same energy, but also the
same nuclear configuration as strongly vibrating, electronically nonexcited
states. (This the many-dimensional equivalent of the "crossmg of po-
is
tential curvês" of diatomic molecules.) It was stated above that, in an
isolated molecule, internal conversion is reversible, and therefore can only

delay but not prevent fluorescence. In condensed systems, on the other
hand, the vibrational quanta of the "converted" molecule can be lost, by
collisions, to the molecules of the medium. The loss of one or a few vibra-
tional quanta may be sufficient to make return into the original, electroni-
cally excited state impossible, and thus prevent fluorescence. The re-
maining vibrational quanta can then be lost, at leisure, one by one, to the
surrounding molecules.
(b) Isomerization or Dissociation ("Monomolecular'' Chemical Quenching)
Theexcitation energy can be used, within the absorbing molecule

(either directly, or after "internal conversion" into vibrational energy), for
a chemical change, e. g., isomerization, or dissociation. Here again, the
reversible character that the process has in vacuum may be lost in the
presence of foreign molecules. The photochemically formed isomer or
tautomer may, for example, lose one or several vibrational quanta by col-
lisions, and thus become incapable of reconversion into the original, elec-
tronically excited form. Similarly, the recombination energy of the disso-
ciation fragments may be lost to foreign molecules serving as "third bodies,"
so that the original molecule will be formed directly in the nonexcited
ground state. (In this case, the dissociation remains chemically reversible,
but recombination is not the exact reversal of photochemical dissociation,
smce it occurs without chemiluminescence.)
(c) Reaction with Foreign Molecules {" Bimolecular" Chemical Quenching)
The presence of foreign molecules opens the possibility of "bimolecular"

chemical reactions of electronically excited molecules. The reaction part-
ners may be the molecules of the solvent, or molecules of an accidental im-
purity (e. g., dissolved oxygen) or specially provided "quenchers." In this
case again, even if the photochemical reaction is reversed afterward, the
reversal is without chemiluminescence; in other words, the
likely to occur
light energy used for the photochemical forward reaction will not be avail-
able for re-emission in the back reaction.
: :
As to the specific nature of the quenching reactions, two types appear

most hkely: Oxidation-reductions and complex formation. The first
type can be represented by equation (23.1 A or B)

(23.1 A) Chi* +Q > oChl + rQ
(23.1B) Chi* +Q > rChl + oQ
(where o stands for oxidized, r for reduced, Q for quencher and Chi* for
excited chlorophyll). The second tj^pe is described by equation (23.2)
(23.2) Chi* +Q > Chl*Q * ChlQ > Chi +Q
the quenching effect being due to accelerated internal conversion of ex-
citation energy into vibrational energy in the complex Chl*Q.
If Q is the solvent, only reactions (23.1 A and B) can be classified as
"chemical quenching," while reaction (23.2) becomes identical with "phys-
ical"energy dissipation in the solvated pigment molecule.
Photochemical reactions with foreign molecules interfere with fluores-
cence only if they take place directly, i. e., by encounters of electronicallj''
excited molecules with the quencher. on the other hand, reactions
If,
of this kind are preceded by monomolecular steps such as isomerization

(or dissociation) of the excited molecule, their effect on fluorescence may
become negligible (since the molecules that take part in the photochemical
reaction are the ones lost for fluorescence anyhow; (c/. second scheme on
p. 483, Vol. I). We will use this concept below; cf. page 788 in interpreting
the nonquenching of chlorophyll fluorescence by certain compounds whose
autoxidation is sensitized by this pigment.
Sometimes the isomeric molecule, formed after light absorption, has a
certain chance of reverting into the original electronicalh' excited state
(e. g., with the help of thermal energy) . Similarly, photochemical dissocia-
tion products may have a certain chance of forming an electronically ex-
cited molecule by recombination. If this exact reversal of the primarj^
photochemical process occurs after a period that is long compared to the
duration of ordinary fluorescence (10~'^ sec), we obsei-i'e the emission of
"delayed fluorescence" or "phosphorescence." Chemical reactions of the
metastable, isomeric photoproduct (or of the dissociation products) will
cause quenching of this delayed emission. This is the mechanism of the
strong quenching of phosphorescence of many dyestuffs by oxygen {cf.
page 789).
{d) Bulk Transfer of Electronic Energy
Transfer of electronic excitation energy "in bulk" to another type of

molecules can lead to the quenching of the fluorescence of the originally
excited molecular species, either by substitution of a secondary (stronger
or weaker) "sensitized" fluorescence of the quencher, or (if the quencher

is nonfluorescent) by complete conversion of the excitation energy into
heat.
Three types (or rather three Hmiting cases) of electronic energy trans-
fer mechanisms are known. The first, which is the only one possible when
the distance between the excited molecule and the quencher is >10~^ cm.
(for visible light quanta), is trivial —
the emission of a light quantum by the
primarily excited molecule and its reabsorption by a molecule of the
ciuencher, a process similar to the "self-absorption" of fluorescence (page
745). The second mechanism is energy transfer by kinetic collisions
second kind"), or "encounters," to use a term
(so-called "colhsions of the
more appropriate for molecules in solution. It is associated with the
mutual disturbance of the electronic structures of the two molecules in
contact, and requires approach to within the kinetic collision diameter
(10~^ to 10"'' cm.). In this case, the energy exchange is not contingent
on "resonance" between the electronic excitation states of the two part-
ners, since a considerable fraction of electronic energy can be converted into
vibrational or kinetic energy in the collision. A third and perhaps most
interesting possibility is the "resonance transfer" of electronic excitation
energy between two practically undisturbed molecules, which can occur
when these molecules are within a distance smaller than the wave length
of the exchanged quantum (^^10~-' cm. for visible light), and does not
require an actual "contact" between them. The probability of this kind
of transfer depends decisively on resonance between the energy-exchanging
molecules(i. e., on the mutual overlapping of the fluorescence band of the
donor and the absorption band of the acceptor). The phenomenon was
first discussed by Kallman and London in application to sensitized fluores-
cence in gases. Similar considerations were afterward applied to solutions
by J. Perrin (1926, 1927), who used classical electrodynamics, and by F.
Perrin (1929, 1932), who first attempted a quantum-mechanical treatment.
F. Perrin used this energy transfer mechanism to interpret so-called
"concentration depolarization" of fluorescence in solution (decrease in the
degree of polarization with increasing concentration). Subsequently,
several other phenomena in fluorescence and photochemistry have been
ascribed to energy exchanges of this type, and improved theoretical treat-
ments were evolved by Vavilov and co-workers (1942, 1943, 1944), Forster
(1946, 1947, 1948) and Arnold and Oppenheimer (1950). Because of
the importance of the resonance transfer concept for the photochemical
mechanism of photosynthesis (in particular, for the possible participation
of phycobilins and carotenoids in it), these papers will be discussed in
greater detail in chapters 30 and 32. Here, we are concerned only with
the possibility of quenching (or excitation) of chlorophyll fluorescence being
due, insome cases, to resonance transfer of- excitation energy not requiring
molecular contact. Examples will be found on p. 778 (quenching of dye
fluorescence by other dyes), p. 790 (chlorophyll q fluorescence sensitized
by b) and chapters 24 and 32 (energy transfer between pigments in
in
vivo). Self-quenching, too, may be caused by resonance (p. 797).
(e) Self-Quenching
Experience shows that quenching by molecules identical with the ex-

cited one often is particularly strong. This is revealed by rapid decrease
in the yield of fluorescence of many substances with increasing concentra-
tion of the fluorescent pigment. This strong self -quenching probably is
due to very between
close resonance the fluorescent molecule and the
quencher. However, resonance transfer of electronic energy does not in
itself explain self-quenching, because, from the point of view of the yield
of fluorescence, it should be irrelevant whether the excitation energy

stays with the originally excited molecule or is transferred to another
molecule of the same kind. Nevertheless, self-quenching can result from
resonance, if some additional phenomena are taken into account. Effective
energy dissipation can result either from kinetic encounters of excited and
normal pigment molecules ("kinetic self-quenching"), or from their close
average proximity ("static self-quenching"). In the first case, we can pos-
tulate transient formation of dimeric molecules during the encounter.
Resonance between the two structures, D*D ^ DD*, creates an attraction
force, leading to a more intimate contact of the two electronic systems
than that established in an encounter of two nonresonating molecules.
This can bring about accelerated conversion of electronic into vibrational
energy; the molecules which met as D* -|- D will then separate as D + D.
Resonance transfer of excitation energy over distances wider than a
collision diameter also can explain self-quenching, if one makes certain
auxiliary hypotheses. Forster (1947, 1948) suggested, for example, that
even dyestuff solutions which reveal no equilibrium dimerization (i. e.,
show no effect of concentration on the absorption spectrum; cf. below),
contain a small proportion of nonfluorescent, dimeric molecules. If the
resonance exchange of excitation energy is so fast that this energy visits.
during its life time, a considerable number (say > 100) pigment molecules,
the presence of even a single dimer in this series of "hosts" may suffice to
"trap" the excitation energy, and dissipate it into heat. (Of course, for
this mechanism to be effective, the absorption band of the dimer and the
fluorescence band of the monomer must overlap sufficiently to permit reso-
nance exchange.)
Franck and Livingston (1949) suggested another possibility —that
energy traps are provided by monomers which are in the state of excep-
tionally strong thermal agitation. (This hypothesis explains also the de-
cline in the intensity of fluorescence, usually observed with rising tempera-
ture.) Accelerated dissipation of electronic energy in "hot" molecules is
plausible, since in order to convert electronic energy into vibrational energy,
a configuration of the nuclei must be reached in which the electronic sys-
tem has the same energy in the excited and in the normal state ("crossing
point of two potential curves" in the diatomic model). This configuration
usually can be achieved only by combination of electronic excitation with
vibrations of appropriate kind; the excited molecule must wait until an
accidental fluctuation of thermal agitation supplies the critical degree of
freedom with the amount of vibrational energy required to make internal
conversion possible. The higher the temperature, the shorter will be this
waiting period, and the greater the probability of internal conversion oc-
curring during the electronic excitation period, and competing successfully
with fluorescence.
One may ask: how can resonance migration of excitation energy assist
this mechanism of dissipation? Does it make any difference whether the
excitation stays with one molecule and awaits there the thermal fluctua-
tion that will permit it to be dissipated, or whether it visits a thousand
molecules during the same total life-time, spending a correspondingly short
time with each of them? We said elsewhere in this book that a man cannot
change his life expectancy by sleeping every night in a different bed!
Whether this analogy applies here or not depends on the relative duration
of a thermal fluctuation and electronic excitation. If the fluctuation is
short-lived, in comparison not only with the total duration of electronic

excitation, but also with the time during which the excitation remains
with a single host molecule, then migration can have no effect the chance —
of being hit by lighting is the same whether one spends the thunderstorm
under a single tree or shifts every minute from one to another (identical)
tree. If, however, the state of abnormal thermal agitation lasts long com-
pared to electronic excitation of a single molecule, then resonance exchange

will increase the chances of the two meeting in one molecule. (If one house
in a hundred in a town is quarantined for smallpox, then a visitor who

comes to town will be much safer if he stays the whole time in the first
house he has entered than if he visits a hundred houses, spending a corre-
spondingly short time in each of them.)
In mathematical form, the probability of two independent events, one
lasting T sec. (electronic excitation) and another t sec. (thermal fluctuation)
overlapping each other in a single molecule, is changed, by subdividing T
into n periods of T/n sec. each, by the factor:
(nt/T) + 1
:
which is significantly different from 1 if f is not < T/n, i. e., if the duration
of the thermal fluctuation much shorter than that of electronic ex-
is not
citation of a single molecule. The former can be postulated to last for a
period of a few molecular vibrations, thus t ^ 10 -^^ sec. The total period
of electronic excitation is T ^ 10 -« sec. (for example, in alcoholic chloro-
phyll solution, the natural life-time of excitation is X 10-^ sec; the ^5
actual life-time must be ten times shorter, as indicated by a fluorescence
about 10%). Under these conditions, for t to be not much shorter
yield of
than T/n, the number n must be higher than 10^ i.e., excitation energy
must be exchanged more than ten thousand times before its dissipation
(staying < 10-^^ gee. at each molecule visited). The role of thermally
excited ("hot") monomeric dyestuff molecules in the concentration quench-
ing of fluorescence thus is predicated on this minimum length of energy
exchange chains, and on the possibility of internal conversion occurring
during the extremely short sojourn of the electronic energy in the hot mole-
cule. It may be suggested that conversion requires (at least) a period of a
single molecular vibration (^10-^^ sec). This would restrict quenching
by hot molecules (in the case of chlorophyll) to exchange chains not shorter
than 10^ and not longer than 10^ molecules.
In addition to the various physical mechanisms of self-quenching which
were considered so far, two chemical mechanisms also are feasible, analogous
to the two chemical mechanisms of quenching by foreign substances, dis-
cussed in section (c). They are: an oxidation-reduction reaction between
the excited and a normal molecule (photodismutation)
(23.3) Chi* + Chi > oChl + rChl
and formation of nonfluorescent dimers by two normal molecules:
(23.4) Chi + Chi ,

Chl2
Dimerization of dyestuff molecules is favored by the fact that marked

resonance attraction must occur not only between an excited and a normal
molecule (as discussed above), but also between two molecules in the non-
excited state. (This application of London's theory of intermolecular
forces to pigment molecules was suggested by Rabinowitch and Epstein
1941.) The tendency of dyestuff molecules to dimerize (or polymerize)
in solution may be attributed to such resonance phenomena. In many
cases— perhaps the majority of those observed so far—self-quenching of
fluorescence appears to be due to "permanent" dimerizations (or polymeri-
zations) rather than to reaction (23.3) or dimerization after excitation.
The dechne in yield of fluorescence with increasing concentration is in this
case a direct measure of the degree of association. Like the unstable dimers
formed in light, the stable dimeric molecules formed in the dark can be
nonfluorescent either because of rapid internal conversion of excitation

energy, or because of the occurrence of internal oxidation-reductions (dis-
proportionations). It was already mentioned above that combination
of dimerization with resonance exchange of energy can lead to strong
quenching even when the number of dimeric molecules is very low.
Whether self-quenching is due to pre-existing dimers (or polymers) or
to encounters between excited and nonexcited monomers (in which dimers
are formed) can often be deduced from observations of the absorption
spectra: In the first case the absorption spectrum of the dyestuff must
change with concentration (as observed, e. g., by Rabinowitch and Epstein
with thionine and methylene blue) in the second case. Beer's law must
;
be obeyed {i. e., the absorption spectrum of the dye must be independent
of concentration). Quenching by a chain of energy exchange reactions,
with a dimer as occasional link in the chain, suggested by Forster, also does
not require marked deviations from Beer's law, since the number of dimers
present can be very small.
Which of the several possible quenching or self-quenching processes
actually limits the yield of fluorescence in a given solution is not easy to
say. If the pigment is and its fluorescence is unchanged

stable in light,
after long illumination, "physical" quenching is the likely mechanism.
True, even when chemical quenching does occur, the dyestuff may be
photostable, if the quenching reaction is reversible; and the yield of
fluorescence may remain unchanged with time, even when quenching ini-
tiates an irreversible sensitized chemical reaction, if the products of this
reaction do not quench fluorescence stronger (or weaker) than the originally
present molecular species. Usually, however, chemical quenching is not
entirely reversible, but causes a more or less rapid chemical change of the
fluorescent pigment; and sensitized chemical reactions often do lead to
the formation of products whose presence changes the intensity of fluores-
cence. When this is the case, the fluorescence yield must change with time
—either because the original fluorescent compound converted into a new
is
one, with different properties, or because the fluorescence yield of the

original species is changed by the accumulation of the products of the
sensitized reaction. Therefore, whenever the yield (or the spectrum) of
fluorescence changes with time, the indication is strong that chemical fac-
tors account for at least part of the quenching (for examples, see page 764).
A systematic study of the effects of solvent, concentration, admixtures,
temperature and other factors on the yield of fluorescence is needed to
elucidate the quenching problem, which was discussed above on the basis
of general possibilities more than on the basis of actual observations with
chlorophyll. Studies of this kind would be' of particular interest for under-
standing the mechanism of sensitized photochemical reactions, such as
—
INFLUENCE OF SOLVENT ON YIELD 763
photosynthesis. Since this summary was written, a number of pertinent
data have been collected by Livingston and co-workers. A discussion of

these will be found below.
If we consider the few presently available data on the intensity of
chlorophyll fluorescence in different media, we acquire the impression that

physical energy dissipation and chemical quenching must both play a part
in these systems; but much remains to be done before their relative roles
will become clear.
The possibility that the yield of chlorophyll fluorescence in solution

may be limited by photochemical dissociation of chlorophyll {e. g., into
"monodehydrochlorophyll" and a hydrogen atom) was suggested by
Franck and Wood (1936). This hypothesis was discussed in chapter 18
(Vol. page 484), and it was pomted out that a quantum of red light
I,
(with an energy of about 40 kcal/emstein) is unlikely to disrupt a carbon-

hydrogen bond (whose standard energy is about 100 kcal/mole), even if
some energy might be gained by the solvation of the hydrogen atom. Thus,
if light absorption does cause a reversible photochemical
change of chloro-
phyll, it is more likely to be either tautomerization, or reaction with the sol-
vent. Dissociation becomes more likely when excitation occurs in the blue-
violet band, with quanta of about 60 kcal./einstein; it was mentioned be-
fore that this is one possible explanation of the lower yield on fluorescence
in this region.
not unlikely that energy dissipation by internal conversion is the
It is
basic factor limiting the yield of fluorescence of chlorophyll in condensed
systems; it is also possible that—as suggested by Franck and Livingston
(1941)— tautomerization occurs as a more or less regular intermediary
stage in this dissipation (c/. Vol. I, p. 490). Chemical interactions with
solvent or admixtures, as well as self -quenching, are then to be considered
as contributmg factors, which further depress the yield of fluorescence
under certain conditions. (According to Lewis and Kasha cf. pp. 790-2
—formation of a metastable triplet state could play the role ascribed above
to tautomerization.)
4. Influence of Solvent on Yield of Chlorophyll Fluorescence
Chlorophyll fluoresces in all (or most) organic solvents (as well as in

wax and paraffin), but with different intensity. No precise measurements
of the fluorescence intensity in different solvents are available, but Franck
and Levi (1934), Albers and Ivnorr (1935), Ilnorr and Albers (1935),
Knorr (1941) and Zscheile and Harris (1943) gave some preliminary results;
all these investigators agree that wide differences occur both in the initial
intensity of fluorescence in different solvents, and in its change with time
(cf. Table 23.III).
Table 23.III
Changes of Fluorescence Intensity of Chlorophyll with Time in Different
Solvents" (after Zscheile and Harris 1943)
.
INFLUENCE OF SOLVENT ON YIELD 765
Obsei-vations of Albers and Ivnorr (1934, 1935) and Kjiorr (1941) con-
cerning the changes in the fluorescence spectra of chlorophylls a and b
with time, in different solvents (ether, acetone, benzene and methanol), and
under different atmospheres (air, oxygen, carbon dioxide and nitrogen)
revealed a bewildering variety of shifts in positions, shapes and intensities
of the fluorescence bands, which do not lend themselves to easy mterpreta-
tion, but indicate complex chemical changes. Apparently, both the sol-
vent and the dissolved gases participated in chemical reactions with ex-
cited chlorophyll molecules. In some systems, these reactions led to a
complete disappearance of fluorescence after less than one hour of illumin-
ation. One reason for the complexity of the results of Knorr and Albers
may have been the use of unfiltered light from a powerful mercury arc.
Strong ultraviolet irradiation may have caused chlorophyll to react with
substances that would not have affected it in visible (particularly red)
light.
One strange observation of Ivnorr and Albers is that the fluorescence of

chlorophyll a (but not that of chlorophyll h) in acetone (but not in other
solvents) is best preserved under an atmosphere of oxygen (where it dis-
appears only after twelve hours of illumination, whereas, in nitrogen or

carbon dioxide, it vanishes completely in less than one hour). As de-
scribed in chapter 18 (Vol. I, page 491), this may mean that the quenching
of fluorescence is caused, in acetone, by a reduction of the pigment (and
oxidation of the solvent) and that oxygen restores the reduced pigment to
its original fluorescent form (c/. chapter 36)
We have considered so far only those changes in the intensity of fluores-
cence which could follow from the interaction of the chlorophyll molecule
in the excited state with the medium. It wâs mentioned, however, in the
introduction that another type of fluorescence effects is possible one in —
which the state of the chlorophyll molecule is altered already in the dark,
prior to excitation. This alteration must manifest itself in a change of the
absorption spectrum. One possibility of this type is that chlorophyll may
dimerize (or polymerize) in some solvents, and remain monomeric in others.
Dimerization is known to cause the disappearance of fluorescence of many
dyestuffs (such as methylene Ijlue) in aciueous sohition. In the case of
chlorophyll, no similar effect has as yel Ix'on discoN^crod— unless owv con-
siders the nonfluorescence of colloidal solutions and solid chlorophyll as
the result of "quenching by polymerization." Another type of associa-
tion, howêver, —
appears to be important in this case association of chloro-
phyll molecules with hydroxyl groups or amine groups present in solvent
molecules; in contrast to thequenching effect of dimerization, association
of this type seems to be necessary to bring out the fluorescence of chloro-
phyll.
This conclusion follows from some remarkable observations described

by Livingston, Watson and McArdle (1949). Contrary to what was gener-
ally assumed before, they found that chlorophyll solutions in nonpolar
all (or only very weakly), but that traces
organic solvents do not fluoresce at
of polar admixtures, such as water or methanol, are sufficient to "activate"
their fluorescence.
In this study, Livingston and co-workers used a mercury arc for excita-
tion (mainly the lines 436 and 405 m/x) The fluorescent light was filtered
.
through a deep-red filter, so that only the second fluorescent band of chloro-
phyll a (720 m/i) was measured. This eliminated self-absorption. (It was
also stated that the position of the second band is less strongly affected by
the solvent than that of the first band, which can be shifted by as much
as 7.6 mû, cf. table 23. IC; but this seems strange, since one would not
UJ
o
z
UJ
o
(/)
UJ
cr
o
_I
U.
u.
o
z
UJ
0.2 0.4 0.6 0.8 1.0
SQUARE ROOT OF MOLE FRACTION OF ALCOHOL
Fig. 23.5. Intensity of fluorescence of chlorophyll a in the system octanol-benzene

(after Livingston 1948).
anticipate substantial differences in solvent effects on two emission bands

originating, presumably, in the same excited state and leading to two ad-
joining vibrational levels of the same lower state.)
Chlorophyll solutions in dry hydrocarbons were obtained by evaporat-
ing in vacuum a solution of chlorophyll a or h, dissolving the residue in dry
hydrocarbon, evaporating again, and repeating this operation until all
water (which may have been present in chlorophyll from its preparation)
had been removed. (Disappearance of fluorescence was used as criterion
of dryness.) Various polar "activators" were then added, dissolved in the
same dry hydrocarbon.
In purest dry benzene, the intensity of fluorescence (F) was <3% of that
ordinarily observed in the same solvent (Fo) even this weak fluorescence
;
ACTIVATION OF FLUORESCENCE 767
could perhaps be due to residual moisture (or other polar admixtures).

Addition of 0.01% water (6 X 10"^ mole/1. HoO) brought F back to the
usual level, Fo-
Other solutions that proved nonfluorescent were those in n-heptane,
iso-octane, styrene, chlorobenzene, carbon tetrachloride and diphenyl
ether. Solutions in methanol, ethanol, octanol, dimethyl ether and diethjd
ether, on the other hand, remained fluorescent even after drying. It was
IQ-'^ 2x10"'*
MOLALITY OF ACTIVATOR
Fig. 23.6. Intensity of fluorescence of chlorophyll a in a hydrocarbon as function of
the concentration of an activator (after Livingston 1948).
Curve number 1 2 3 4 5
Solvent n-Heptane Benzene Benzene Isooctane Benzene
Activator Phenylhydrazine Benzyl alcohol Cetyl alcohol Methanol Piperidine
Chlorophyll ... a a or 6 a a a
considered possible, however, that the fluorescence in the last two solvents
was due to residual impurities.
Figure 23.5 shows the intensity of fluorescence as function of composi-
tion in a mixture of benzene and octanol. The fluorescence is completely
activated by 0.0016 mole alcohol in a mole of hydrocarbon, corresponding
to a concentration of about lO"^ mole/1. Similar relationships were found
in mixtures of —
benzene wuth other polar solvents alcohols and amines.
Figure 23.6 shows the initial parts of five activation cui-ves. Table 23.IIIA
gives, under [Ac]i/j, the molar concentrations of the activators needed to
raise the fluorescence intensity to V2 ^0; they range from 6.8 X 10 "^
mole/1, for dimothylamine in benzene, down to only 6.5 X 10~^ mole/1,

for piperidine in benzene. Water is about half as effective as piperidine.
Although amines generally are the strongest activators, diphenylamine
and diphenylhydrazine are without effect. It is noteworthy that phenyl-
hydrazine acts as activator in low concentration, and as quencher in high
concentrations.
The maximum which chlorophyll in a given
intensity of fluorescence to
nonfluorescent solution can be raised by activators is independent of the
specific activator used, at least in the first approximation.
Activation seems to be completely reversible; in other words, fluores-
cence disappears again if the activator is distilled away, and reappears
upon its renewed addition.
Table 23.IIIA
Efficiency of Activation of Fluorescence of a 5 X 10^*^ Mole per Liter Solution
OF Chlorophyll a
(after Livingston, Watson and McArdle, 1949)
Solvent
:
nonactivated one, independent of the nature of the activator, and bears

is
no relation to the absorptionspectrum of chlorophyll in pure activator.

The fluorescence of activated solutions is quenched by rising tempera-
ture (15-70° C), particularly strongly in the region of partial activation.
The maximum intensity, Fo, is a linear function of temperature over a
comparatively wide range (a similar relationship was found also by Zscheile
and Harris, 1943). In pai'tly activated solutions, the temperature curve
is not only steeper than in fully activated ones, but also shows with some —
activators at least —a definite curvature.
The concentration of the strongest known activators, required to achieve
complete activation, is of the same order of magnitude as that of chloro-
~^
phyll which in the experiments of Livingston et al. was 5 X 10
itself,
mole/1. This shows that the effect cannot be due to kinetic encounters
between chlorophyll and activator (which are too rare at such low concen-
trations) nor can it be ascribed to a change in properties (such as dielec-
;
tric constant) of the solvent as a whole. Rather, the effect must be caused
by the association of chlorophyll molecules with the molecules of the activa-
tor. The change in absorption spectrum supports this assumption. If the
nonassociated form is totally nonfluorescent, the intensity of fluorescence
can be used to calculate the proportion of chlorophyll molecules in the as-
—
sociated form assuming that the absorption coefficient of associated
chlorophyll is the same as that of the nonassociated pigment. Figvire
21. 26. A shows that this is not quite true for chlorophyll a at 436 ni/x; but
Livingston neglected this difference. Assuming a one-to-one complex
[ChlAc ] the equilibrium constant of association
can be calculated from the half-activating concentration [Ac]i/,:
K[Chl]o[Ac]o
(23.4B) [ChlAc] =
1 + K[Ac]o
^^^•*^^
Fo
" 1 + A'[Ac]o
(23.4D) A' = ^
Ac],/,
chapter 27, eq. 27.12). This is a simplified solution, based on the as-
{cf.
sumption [Ac] ^^ [Ac]o; in other words, it assumes that the amount of

activator boundcomplex is small compared to the total amount
in the
added to obtain activation. In Table 23.IIIA, the concentration [AcChl]
at half-activation is 2.5 X 10 ~^ mole/1, for piperidine; since [Ac Jo =
6.5 X 10-^ [Ac]i/, = 4 X 10"" molc/1. In other words, the simplified
equations are not quite applicable to piperidine (and four other systems
at the bottom of Table 23.IIA. In this case the approximate equation

(23.4D) should be replaced by the exact equation:
(23.4E) K = [Ac]y, -0.5[Chl]o
Livingston used, however, the simplified equation for all the systems
studied by him. Furthermore, he calculated the constants K, not from
the value of [Acl^/^, but from the average slope of the activation curve.
He obtained in this way the K-values given in Table 23.IIIA, which differ
somewhat (although not in the order of magnitude) from values that could
be calculated by means of equation (23.4E). (For example, the latter
would give, for piperidine, K = 2.2 X 10^ instead of 1.6 X 10^).
Deviations from straight line in the plot of log [(Fo/F) — 1] against
[Ac]o were noted by Livingston and co-workers at low concentrations of
the activator, and were ascribed by them to the presence of an "adventi-
tious" activator, Ac' (probably water). They held the latter responsible
for the weak fluorescence still noticeable even at [Ac]o = {e.g.,F ^ 0.03Fo
in purest benzene). using the same assumptions as before («chiAc =
By
achi for excitation light and [Ac']o »
[ChlAc']), and assuming the adventi-
tious activator to be water, Livingston and co-workers obtained theoretical
curves fitting well the experimental results.
From the equilibrium constant K and its change with temperature,
values of AF, AH and AS were calculated for three systems shown in
Table 23.IIIB.
Table 23.IIIB
Thermodynamic Constants for the Association of Chlorophyll a
WITH "Activators" in Benzene
Activator
and amines to the formation of hydrogen bonds with the amino or

alcohols
hydroxy group, shown in formula 23.1.
I I
I
^c^\^ ^c^'^c^ -c<\^
H H I
Chelated Enol Form Keto Form Stabilized Keto Form

(noiifluorescent, stable (unstabilized) (fluorescent, stable in the
in pure hydrocarbons) presence of amine or alcohol)
Formula 23.1. Tautomerization and fluorescence of chlorophyll (after Livingston,

Watson and McArdle, 1949).
The fluorescence of ethereal chlorophyll solutions not covered by this

is
hypothesis, and further experiments are needed to show Avhether this

fluorescence would persistupon more stringent purification. As an alterna-
tive explanation of the phenomenon of activation, Li\nngston considered
the possibility that chlorophyll forms nonfluorescent dimers in pure hydro-
carbons, and that these dimers are dissociated into fluorescent monomers
by association with polar molecules. If this were the case, however, one
would expect the extent of activation to depend on concentration of chloro-
phyll; while a few — —
admittedly preliminary experiments showed no dif-
ference in the values of F/Fo between partially activated chlorophyll solu-
tions in benzene containing 4.8 X lO'S 1.5 X 10"* and 2.3 X 10"^ mole/1,
chlorophyll, respectively.
Observations bearing obvious relation to those of Livingston and co-
workers, have l)een made also by Evstigneev, Gavrilova and Krasnovsky
(1949^. In studying the effect of oxygen on the absorption spectrum and
fluorescence of chlorophyll, they first found this effect to depend on the
solvent. In toluene, heptane and carbon tetrachloride, oxygen increased
the absorption (page 648) and activated the fluorescence, while in pyridine,
ethanol, ethyl acetate, acetone and (commercial) benzene, it had no effect
on absorption,and quenched fluorescence. Later (1949^) the same
investigators found that these effects were due not to oxygen, but to water
vapor. In moist toluene, oxygen quenched fluorescence in the same way
as in ethanol or other polar solvents.
Evstigneev and co-workers also discussed two conceivable mechanisms
of the action of polar solvents. One was the same as Livingston's alterna-
tive hypothesis —solvent on dimerization. The other differed from
effect
Livingston's preferred hypothesis: it assumed attachment of polar mole-
cules to the free co-ordination places at the central magnesium atom. This
772 FLUORKSCENCK OF PIGMENTS IN VITRO CHAF. 23
last hypothesis is supported by the observation that polar molecules do

not affect the absoi-ption spectrum and fluorescence of pheophytin; even
more convincing is the fact that a similar difference in behavior occurs
between phthalocyanine and its magnesium complex, although these
compounds contain no cyclopentanone structure, so that Livingston's
interpretation cannot be applied to them.
5. Influence of Concentration on Yield of Fluorescence. Self-

Quenching. Fluorescence of Chlorophyll in Colloids and Adsorbates
The self-quenching of fluorescence in chlorophyll solutions was studied

by Weiss and Weil-Malherbe (1944). They found that with a constant
intensity of illumination, the intensity of fluorescence of an ethyl chloro-
phyllide solution in ethanol increased with increased concentration, be-
tween 1 X 10^^ mole/1., due to increased absorption. The
10"^ and 1 X
latter became practically complete above 1 X
10^^ mole/1.; instead of be-
coming constant from there on, the fluorescence intensity declined rapidly,
dropping at 1 X 10 -^ mole/1, to one sixth of its maximum value. Six

points were measured between [Chi] = 2 X 10"* and 1 X
10 --^ mole/1.,
and found to lie on a hyperbola:

(23.5) F = C/{l+k[Ch\])
where F is fluorescence intensity and k and C are constants. The constant

k was evaluated graphically and fc = 2 X 10'^ sec.-^ was found. Making
the— unjustified— assumption that the intensity of fluorescence is limited
exclusively by self-quenching, one can easily show (c/. equations on page
546, Vol. I) that the constant k has the meaning:
(23.6) k = ke/kf
where h is the bimolecular rate constant of self-quenching:

K
(23.7) Chi + Chi* > Chl2 ( > 2 Chi)
and kf the monomolecular rate constant of fluorescence
(23.8) Chi* > Chi + hu
The reciprocal, l/kj, is the average life-time of the excited molecule

when limited only by fluorescence. According to page 634, k/ ^ 10^
sec.-i; with fc = 2 X 10-^ sec.-\ this gives, according to (23.6):
(23.9) A-c ^2 X 1012 sec. -1
This calculation is based on the assumption that reaction (23.7) is the

only one that limits the yield of fluorescence. However, equation (23.5)
would remain correct, i. e., self-quenching would follow a hyperbolic curve,
: :
CONCEJSTKATION QUENCHING 773
also if fluorescence were competing, not only with the bimolecular reaction
(23.7), but also one (or several) mononiolecular (or pseudomonomolec-
A\-ith
ular) transformations of the excited molecule, such as energy dissipation by

internal conversion, or tautomerization, or a reaction with the solvent.
In this case, the constant k in (23.5), instead of meaning (23.6), would have
the meaning (23.10):
(23.10) k = kc/ikf + kd)
where mononiolecular rate constant of the competing energy-

ka is the
dissipating process (or processes), (corresponding to the sum -{ kt, fc,-
internal conversion plus tautomerization, in equation 19.5 in Vol. I, page

546).
Furthermore, in this case, the constant C would not be equal to Fo (the
fluorescence intensity with quantum yield 1), as assumed by Weiss and
Weil-Malherbe, but would have the meaning:
(23.11) C = FoA7/(A7 + kd)
With the assumption C = Fo, the data of Weiss and Weil-Malherbe indi-
cate a yield of fluorescence (<p = F/Fo = F/C) of 4% at 1 X 10"^ mole/1,
and 21% at 2 X 10"* mole/1, (and, of course, 100% at infinite dilution);
but since no check was made by an experimental determination of Fo, the
true value of Fo may have been > C, i. e., the quantum yield of fluorescence
may have been correspondingly lower (not reaching 100% at infinite dilu-
tion).
Comparison of (23.10) with (23.6) shows that by correcting the deriva-
tions of Weiss and Weil-Malherbe for the possibility of internal conversion
or other mononiolecular deactivation processes, one would obtain, from
their value of A", a value of k^ even larger than the one given in (23.9)
(23.12) Ac > 2 X 10'2
The makes one

extraordinarily large value of this bimolecular rate constant
skeptical about the correctness of Weiss and Weil-Malherbe's experiments,
or, at least, of their interpretation. Assuming that Chi* is deactivated by
—
the first encounter with Chi which is not implausible the above value —
of kc gives for the average interval between two encounters (at [Chi] = 1
niole/1.)
(23.13) i = 5 X 10-3 sec.
This is at least one whole order of magnitude less than can be estimated
from the formula for the frequency of collisions in gases, and two or three
orders of magnitude less than the estimate based on the rate of diffusion
of dyestuff molecules in solution.
The rates of those reactions in solution that occur by the first (or one of the first)
molecular collision are determined by the rate of diffusion (which brings the molecules
.
together in an "encounter") rather than by the frequency of collisions. The distinction

between and collisions, and its importance for reaction
eiicounters (or "co-ordinations")
kinetics of condensed systems was discussed by Rabinowitch (1937).
The unusual magnitude of the constant (23.12) is illustrated by the

fact that for nine other dyes, investigated by the same authors, the self-
quenching constants were at least 2000 times smaller (and thus of the
order of magnitude to be expected from diffusion constants)
It seemed thus that the conclusions of Weiss and Weil-Malherbe were
in need of confirmation. Their results might have been vitiated by self-
absorption (which the authors dismissed as unimportant "because there
was no evidence of surface fluorescence"). In their set-up, the fluores-
cent beam was in line with the (ultraviolet) excitmg beam an arrangement —
that favors self-absorption. Less mutual overlapping of the fluorescence
band and absorption band may explain why self-absorption did not affect
equally strongly the results obtained with the other nine dyes.
That the observation of Weiss and Weil-Malherbe was due to self-
absorption was later confirmed by Livingston and co-workers (1948), who

showed experimentally that, if care is taken to avoid self-absorption, no
concentration quenching of chlorophyll fluorescence can be noted up to
1.5 X 10~^ mole/1. Weiss (1948) agreed with this, but stated that con-
centration quenching does occur at still higher concentrations. Livingston
and co-workers (1948), using a cell 1 mm. thick, extended their measure-
ments up to 0.10 mole/1, of chlorophyll in butyl ether and found in fact a
strong concentration quenching above 2 X 10~^ mole/1, (fig. 23.7); at
7 X 10 ~^ mole/1. the fluorescence yield was only 7% of that in dilute solu-
,
tion. The insert in fig. 23.7 shows the sigmoid shape of the curve.
The absorption spectrum of chlorophyll a in butyl ether, measured at
2 X 10 ~^ mole/1, (a concentration at which self-quenching reduces the
fluorescence to about 40% of the maximum), appeared not to differ sig-
nificantly from the spectrum of dilute solution.
A further pertinent observation was that the partly quenched fluores-
cence of a concentrated chlorophyll solution was more strongly depressed
by an increase in temperature than the fluorescence of a dilute solution.
—
These observations the unchanged absorption spectrum, the sigmoid
quenching curve and the enhancing effect of temperature on concentration
quenching —can all be fitted into the picture of quenching as the result of
dissipation of excitation energy in a "weak link" in the energy exchange
chain, such as a dimeric molecule (Forster) or a "hot" molecule of the
monomer (Franck and Livingston). The concentration at which the
quenching becomes noticeable (2 X 10"^ mole/1.) is of the same order of
magnitude as that calculated by FSrster for the onset of the energy ex-
change (of. chapter 32).
CONCENTRATION QUENCHING 775
Numerous observations are known showing that the close packing of

chlorophyll molecules in solid chlorophyll, or in chlorophjdl monolayers on
water, causes a complete disappearance of fluorescence. Chlorophjdl
colloids too are, as a iiile, nonfiuorescent (Willstatter and St oil 1918;
Stern 1920, 1921; Albers 1935; Meyer 1939). Meyer described certain
fluorescent solutions of chlorophyll in ethanol as "colloidal," but no rea-
sons for this description were given (cf. Smith 1941). Meyer's aqueous
chlorophyll colloids, in which the density of the particles was similar to
that in the chloroplast grana, were nonfiuorescent. Chlorophyll is non-
0.80
0.60
0.40
10.20
OOI 002 0.03 0.04 0.05 O06 0.07
Fig, 23.7. Concentration quenching of fluorescence of chlorophyll a in butyl ether

(after Livingston and Ke, 1949). Ordinate, F/Fq. Abscissa, [Chi] in mole/1.
fluorescent also in the adsorbed state, e. g., in starch columns used for
chromatographic separation (Sej-^bold and Egle 1940).
If we attribute the nonfluorescence of solid, colloidal and adsoibed
chlorophyll to self-quenching, and consider a consequence of close pack-
it
ing, the restoration of fluorescence by certain "protective" substances can

be attributed either to simple dilution of the pigment, or to effective inter-
mption of the interaction between neighboring pigment molecules. Among
the compounds reportedly capable of protecting the fluorescence of chloro-
phyll, we find first of all lipides and lipophilic solvents. Stern (1920.
1921) found, for example, that chlorophyll-liiiide emulsions in water are

fluorescent, while pure aqueous chlorophyll sols are not. The fluorescence
of chlorophyll colloids in the presence of lecithin was confirmed by Bakker
(1934). This may be either a true case merely a dilution
of protection, or
effect, since the concentration of chlorophyll molecules in the lipide drops

may be lower than in the particles of the hydrosol.
Seybold and E^lc (1940) found that chlorophyll on filter i)aper is
fluorescent only if the solution from which it was adsorbed contained lipides,
waxes or other lipophilic organic materials. (They even used the ab-
sence of a fluorescent rim on a filter paper strip dipped into a chlorophyll
solution as a control of the purity of both solution and paper.) Nonfluores-
cent chlorophyll adsorbates on starch "light up" if they are wetted by or-
ganic solvents, or if ether vapor is blown at them.
Wakkie(1935) prepared a series of colloidal chlorophyll solutions (b}'
diluting alcoholic solutions with increasing amounts of water), with and
without a lipide (sodium oleate) If no oleate was added, the shift of the
.
absorption band from 660 to 672 m/x (completed within a narrow concen-
tration range, and considered indicative of the transitionfrom a monomole-
cularly dispersed to an aggregated state) was accompanied by complete
disappearance of fluorescence. In the presence of oleate, the absorption
band began to shift at the same dilution, but the displacement ceased at
670 instead of 672 m/x; and, at the same time, fluorescence reappeared.
By salting out, fluorescent, birefringent chlorophyll oleate "coacervates"
could be precipitated from these fluorescent colloidal solutions.
From these and similar observations, it appears that lipophilic mole-
from self-quenching even
cules can protect the fluorescence of chlorophyll
without diluting the pigment, and without disrupting the chlorophjdl-
protein or chlorophyll-cellulose bond. One can visualize the protecting
molecules as enveloping the lipophilic parts of the adsorbed pigment mole-
cules (e. g., in the case of chlorophyll, the phytol "tails"), and thus inter-
rupting their mutual interaction. The "wrapping up" of flexible parts of
the molecule may stiffen the latter and interfere with the internal conver-
sion of electronic into vibrational energ3^ This stabilization effect may
become manifest in a single pigment molecule, as well as within a complex
of several such molecules. In the light of the above-described, more recent
experiments by Livingston, one has also to consider the possibihty that
tautomeric transformations from a nonfluorescent into a fluorescent form
of chlorophyll could be responsible (or coresponsible) for effects of this type.
It does not seem that the association of chlorophyll with proteins can
in itself protect fluorescence. True, natural "chloroplastin" preparations
apparently are fluorescent. Although Smith (1938) called aqueous chloro-
phyll-protein extracts from spinach leaves "nonfluorescent," Noack
QUENCHING BY ADMIXTURES 777
(1927), StoU and Wiedemann (1938) and Fishman and Moyer (1942)
found that they fluoresce weakly, and that fluorescence is preserved also
in precipitates prepared from such extracts by salting out. Wassink,
Katz and Dorrestem (1942) observed that the yield of fluorescence was
about the same (~0.1%) in live purple bacteria and in aqueous, colloidal
bacteriochlorophyll-protein suspensions prepared from them. However,
chloroplastin fluorescence probably must be attributed to the presence of
as much as 30% hpophilic material. Artificial complexes containing only

chlorophyll and proteins do not fluoresce. Accordmg to Noack, adsorb-
ates of chlorophyll on globin sometimes fluoresce faintly; but Seybold and
—
Egle (1940) suggested probably with justification —that this must be as-
cribed to the presence of impurities of a lipide nature.

The bearing of these results on the problem of the state of chlorophyll
in the IW'mg cell was discussed in chapter 14 (Vol. I). It was argued there
that the nonfluorescence of pure chlorophyll adsorbates on proteins does

not prove Seybold's hypothesis that the chlorophyll fluorescence in vivo
is caused by a small fraction of chlorophyll dissolved in a lipide phase;
more probably, all chlorophyll in the plants is weakly fluorescent {despite
its high density and irrespective of its association with proteins) because of
its simultaneous association with protective substances such as fats or
phospholipides (Hubert, 1935). Livingston's experiments, (p. 766), in-

dicate association with "activating" groups (such as OH, NH, or SH) as
another possible explanation of fluorescence.
6. Quenching and Activation of Chlorophyll Fluorescence by Admixtures
While the limitation of chlorophyll fluorescence in pure solutions may

be caused equally well by physical dissipation or by photochemical reac-
tions (tautomerization or reaction with the solvent), strong quenching by
small amounts (<10~^ mole/1.) of foreign substances must be attributed
to chemical interactions, since the rate of physical energy dissipation is not
be affected by the comparatively rare encounters of excited dyestuff
likely to
molecules with the molecules of the "quencher," or by changes in the aver-
age properties of the solvent caused by the presence of the latter. (An
exception may be the case of resonance —to be discussed below.)
As mentioned on page 757, the two most likely mechanisms of chemical
quenching are oxidation-reduction (equations 23.1), and complex formation
(equation 23.2). The second one is particularly probable when the
([uencher is another dyestuff with overlapjiing bands, so that self-quenching
conditions are closely ai)proximated. In this case, permanent association
of the ciucncher with the fluorescent molecule also becomes a likely possi-
bility (like the permanent dimerization discussed on page 761). Study

of the effect of the quencher on the absorption spectmm of the fluorescent
material, and of the dependence of quenching on the concentration of the
quencher, may help to distinguish between "quenching by complex forma-
tion" and "quenching by kinetic encounters"; but, at present, very few
such data are available for chlorophyll solutions. Finally, in analogy to
the above-discussed mechanisms of self-quenching (page 759), still
another possibility of quenching should exist when the quencher is "in

resonance" with the fluorescent molecule: quenching without complex
formation and without kinetic encounters, through transfer of excitation
energy over distances considerably larger than the collision diameters.
If the molecules of the quencher are nonfiuorescent, they will serve as
"traps" in the same way as was postulated by Forster for the dimers.
Vavilov and co-workers (Pekerman 1947, Vavilov et at. 1949^'^) found
convincing examples of this type in the quencing of dye fluorescence by
resonating nonfluorescent dyes. However, strong quenchers in Table
23.IIIC (p. 782) certainly do not owe their effectiveness to a resonance
transfer mechanism. They are all oxidants and this points to chemical
interaction rather than physical energy transfer. In the second place,
they have no absorption bands in the red, and are thus not in resonance
with excited chlorophyll molecules. Their lowest excited states must be
considerably higher than the fluorescent state A of chlorophyll.
Among the substances whose quenching effect on the fluorescence of
chlorophyll has been investigated in some detail are the reaction partners
in chlorophyll-sensitized autoxidations —molecular oxygen and oxidation
substrates such as benzidine, allyl thiourea etc. Because of the importance
of these results for the analysis of the mechanism of sensitized autoxidation,
they have already been anticipated in part in Volume I (c/. chapter 18,
pages 483 and 518, and chapter 19, page 546).
The quenching action of oxygen on the fluorescence of different dyestuffs
(including chlorophyll) was first investigated by Kautsky and co-workers.
Kautsky and Hirsch (1931) had discovered that the fluorescence of certain
dyestuffs adsorbed on silica gel is considerably weakened by oxygen at pres-
sures of several hundred millimeters, and that their afterglow (phosphores-
cence) iscompletely destroyed even by very much lower pressures of this
gas. Later, a similar ciuenching was observed in fluorescent dyestuff
solutions, including chlorophyll solutions in acetone. According to Kaut-
sky, Hirsch and Flesch (1935), the quenching of chlorophyll fluorescence
is proportional to the partial pressure of oxygen, and shows no "saturation
effect" even under a partial pressure of one atmosphere.
Franck and Levi (1934) and Weil-Malherbe and Weiss (1942) found
that the fluorescence of chlorophyll or ethyl chlorophillide solutions in
:
QUENCHING BY OXYGEN 779
ethanol reduced under one atmosphere oxygen by 30-35% (compared

is
with its This indicates that 50%

intensity in the absence of oxygen).
quenching must require about two atmospheres oxygen, corresponding to a
concentration of about IQ-^ mole/1, (c/. table 23.IIIC). Life-time of the
fluorescent state of chlorophyll in ethanol solution was estimated on page
634 as 8 10-» sec. The average time that an excited chlorophyll mole-
X
cule has available before its first encounter with a molecule of a solute
whose concentration is of the order of 10 -^ mole/1, also is of the order of
10"^ sec. (No exact formulae for the calculation of encounter intervals in
solutions are available, but appears likely that these intervals are some-
it
— —
what perhaps 10 or 100 times longer than the collision intervals in gases
of the same concentration.) It thus seems that excited chlorophyll mole-
cules in the fluorescent state A undergo a quenching reaction by the very
first, or one of the first, encounters with an oxygen molecule.
The nature of this interaction is not known, but it is most likely to be
the autoxidation of chlorophyll. A different hypothesis was suggested by
Kautsky and maintained by him despite much criticism. According to this
hypothesis, the interaction a hulk transfer of electronic excitation energy
is
from Chi* to O2. This concept originated in certain obsei-vations made by

Kautsky and de Bruijn and by Kautsky, de Bmijn, Neuwirth and
(1931)
Baumeister (1933) study of the autoxidation of leuco malachite
in the
green, adsorbed on silica gel. They found that this reaction can be sensi-
tized, in an atmosphere of oxygen of very low pressure (10"^ mm.), by the
dyestuff trypaflavine, adsorhed on separate particles of the gel. Kautsky ex-
plained the "transmission" of the sensitizing action across the air gaps sepa-
rating the dyestuff from the acceptor by the assumption that excited dye-
stuff molecules transfer their energy to oxygen molecules
(23.14) D* + O2 > D+ O2*
This process he also made responsible for the quenching of fluorescence.

Energy transfer was supposed by him to convert ordinary oxygen into a
metastable active form, which Kautsky identified as the state ^S, known
from spectroscopic data to be situated 37.3 kcal/mole above the ground
state ^n. After Gaffron remarked that infrared excitation of bacterio-
chlorophyll provides <37 kcal, Kautsky (1937) suggested that the state
Â (23 kcal/mole) could sei-ve the same purpose. Both states are meta-
stable because their multiplicity (singlet) is different from that of the
ground state (triplet). The same principle underlies Lewis and Kasha's
more recent theory of metastable triplet states in molecules with singlet
ground states (c/. p. 730).
An alternative chemical explanation of the mechanism of quenching
: —
7S0 FLUORESCENCE OF PlfiMENTS IN VITRO CHAP. 23
by oxj^gen was suggested by Weiss (1935). He postulated an autoxidation

(dehydrogenation) of the dye:
(23.15) D* + O2 > HO2 + oD

(o signifies oxidized). The radical HO2 can diffuse across air gaps and
cause the oxidation effects ascribed by Kautsky to metastable oxygen
molecules.
A reaction of the type (23.15) could be responsible not only for the
quenching of the fluorescence of adsorbed dyes, but also for that of the
fluorescence of dyestuffs dissolved in organic solvents. Since the quantum
jaeld of irreversible photoxidation of chlorophyll in pure organic solvents
is very low (Vol. I, p. 496), reaction (23.15), if it is responsible for quenching,
must be practically completely reversible (at least as far as the chemical
composition of chlorophyll is concerned). The restoration of oxidized
chlorophyll may be brought about either by direct reversal of (23.15),
or — if the HO2 radicals are partly consumed by dismutation or side reac-
tions —by interaction with the solvent. In the latter case, the net result
is sensitized autoxidation of the solvent S
(23.16a) Chl*+ O2 > oChl + HO.

(23.16b) oChl + S >oS + Chi
(23.16c) HO. > ^ H2O + f O2

(23.16) S + i O2 >oS + m.O
For the solvent, one may substitute an oxidizable substrate e. g.'
benzidine, or potassium iodide —thus obtaining a mechanism of chloro"

phyll-sensitized autoxidation of such substrates. (This mechanism wa^
discussed in Volume I, equations 18.33, and chapter 19, cf.
chapter 18, cf.
scheme 19.11; there, tautomerization was added as a preliminary step.)

An alternative interpretation of ser^sitized autoxidation, also discussed
in Volume I, chapter 18 (cf. equations 18.40), envisages a primary reaction
between excited chlorophyll inolecules and the oxidation substrate (rather
than oxygen). Whenever this mechanism operates, oxidation substrates
should quench the' fluorescence of chlorophyll more effectively than does
oxygen. Franck and Levi (1934) measured the quenching of chlorophyll
fluorescence by benzidine and potassium iodide. Their quenching curves
are not labeled and therefore do not permit reading off the half-quenching
concentration, but the authors state that, under the conditions of Noack
experiments on the chlorophyll-sensitized autoxidation of benzidine (cf.
page 528), quenching by benzidine must have been many times more ef-
ficient than that by oxygen. If this is true, then the mechanism of this
reaction must be different from that of chlorophyll-sensitized autoxidation
of substrates such as allylthiourea (cf. below).
niYRICAL AND OHEMK'AL QUENCHING 781
Li^•ingston and co-workers (Livingston 1948, Livingston and Ke, 1949)

made the first systematic investigation of changes in the intensity of
chlorophyll fluorescence, produced by small admixtures. They used
various organic compounds, certain salts and several gases.
As long as the fluorescence remains the only property measured, all
the observed changes can be described as quenching (if fluorescence be-

comes weaker) or stimulation (if fluorescence becomes stronger —as it
actually does upon amine to a chlorophyll

addition of traces of an alcohol or
solution in dry hydrocarbon, or upon the addition of iodine to an alcoholic
solution of chlorophyll h) It would be best,
. however, to restrict the terms
"(luenching" and "stimulation" to cases in which the admixture does not
affect the composition or state of the light-absorbing molecules in the
dark, but acts only on molecules which have been excited by the absorption
of hght. We may refer to these phenomena as "true quenching" (or
"true stimulation"— if the latter does exist at all, which is doubtful).
True quenching can be due to physical or chemical processes. In the first
case (physical quenching), kinetic encounters of light-excited, fluorescent
molecules with the molecules of the quencher, or mutual proximity of these
molecules, lead to accelerated conversion of electronic excitation energy into
vibrational energy and, ultimately, into heat. The accelerated dissipa-
tion can occur within the excited molecule itself (because its configuration
or charge distribution change under the influence of the quencher), or in a
complex formed by the excited molecule and the quencher, or even within
—
the quencher molecule alone which in this case, must first take over the
electronic excitation energy "in bulk" and then dissipate it, by internal
conversion to vibrational energy. (It is also possible for this energy to be
re-emitted by the quencher as sensitized fluorescence.) In the second

case (chemical quenching), either the excited molecule or the molecule of
the quencher (or both) are changed chemically in the process of quenching.
In this case, a kinetic encounter of the two molecules is needed. If the
photochemical reaction responsible for quenching is completely reversible
by a dark reaction, the net result is the same as in physical quenching—
conversion of light energy into heat. Otherwise, a net photochemical
change remains; and only if this change does not involve the fluorescent
speciesis a steady yield of fluorescence observable in the presence of the
quencher.
Contrasted to true quenching can be the changes in the yield of fluores-
cence which are caused by alterations in the composition or structure of
the light-absorbing molecules produced by the addition of the admixture.
(AH cases probably belong to this class.) These processes
of "stimulation"
can be distinguished from true quenching by the fact that the absorption
spectrum of the solution also is changed by the presence of the quencher
"
(or stimulant). The absorption changes may be major or minor, depending

on the character of the interaction (complexing, tautomerization, dissocia-
tion, oxidation, reduction, etc.), but should never be entirely absent.
Furthermore, chemical changes in the dark will often (albeit not always)
proceed at a measurable rate, thus causing the yield of fluorescence to
depend on the length of time between the preparation of the mixture and
the illumination. Finally, the dependence of quenching on the concentra-
tion of the quencher should be different in the case when the absorbing
molecule and the quencher combine (or react) in the dark than when they
interact only after the absorption of light.
Table 23.IIIC
Quenching Data for Chlorophyll a in Methanol, Ethanol and
Acetone (after Livingston and Ke, 1949)
Quencher Solvent X (excit.), mî [0] 1/2 *' ''
mole/L
Chloranil Me.CO 645

Quinone Me2C0
MeOH
Methyl red MeOH
Trinitrotoluene MeOH
MeOH
m-Dinitrobenzene MeOH
MeOH
Duroquinone MeOH
/3-Nitroso-a-naphthol MeOH
/3-Nitrostyrene MeOH
Nitric oxide EtOH
/3-Nitro-i3-methylstyrene MeOH
Oxygen EtOH
Nitrobenzene MeOH
/3-Nitro-)3,7-hexene MeOH
o-Aminophenol MeOH
Phenylhydrazine EtoO
MeOH
Dimethylaniline MeOH
2-Phenyl-3-nitrobicyclo-
[l,2,2]-heptene-5 MeOH
2,6-Diaminopyridine MeOH
"Half-quenching concentration. * Cf. equation 23.16C, p. 785.
These general considerations should be kept in mind in the evaluation

of the results of quenching experiments. They make it particularly im-
portant that measurements of the intensity of fluorescence be combined
with measurements of the absorption spectrum (and if possible, also of the
fluorescence spectrum) of the light-absorbing species.
QUENCHING EFFICIENCY '83
The experiments of Li\'ingston and Ke (1949) dealt largely with what

appears to be true chemical quenching: changes due to reversible reactions
of excited chlorophyll molecules with certain organic and a few inorganic
molecules. In some cases, at least, this interpretation was confirmed by
observations of the constancy of the absorption spectrum, and by the in-
stantaneous character of the change.
Table 23.IIID
NONQUENCHERS OF CHLOROPHYLL FLUORESCENCE IN METHANOL, EtHANOL OR AcETONE"
(after Livingston and Ke, 1949)
Reagent Solvent [Q], m./I. Fo/F
Nitropropane MeOH .09 1 .00

Nitropropane MeOH 0.2 1.02
Butvl nitrate MeOH 0.9 1 .02
Butvl nitrite MeoCO 0. 138 101
Phenvlhvdroxvlamine MeOH 0.021 1 .02
Phenvlhydroxylamine MeOH 07 . 1 . 05
Aniline Et.OH 0.16 1 .02
Hydrazine MeoCO 1 .05 1.0
Urethan MejCO .07 1.0
Thiourea Me.CO 0.02 1 .0
2-Aminopvridine MeOH .08 1 .00
Phenvlurea MeOH 0.05 1.01
Urea^ MeOH 0.19 1.00
Guanidine carbonate MeOH (Satd.) 1.0
Phenol MeOH 0.09 1.0
Hvdroqninone* MejCO 0.03 1.0
Phenolphthalein MeOH .04 1.0
Dimethvlo;lvoxime MeOH 07. 1.0
/e/7-Hexvlmercaptan MeOH 0.11 1 00.
Benzaldehvde MeOH 0.38 1.0

Benzoic acid Me.CO 0.08 1.0
Camphor MeOH 0.15 1.0
Boric acid MeOH (Satd.) 1 .00
Sodium methoxide MeOH 0.05 1.0
Sodium cyanide MeOH 0. 15 1 .01
Sodium oxide MeOH 0.11 1.0
Nitrous oxide EtOH (605 mm.) 1.0
Carbon dioxide EtOH (576 mm.) 1.0
Carbon monoxide MeOH (640 mm.) 1 .00
Potassium thiocyanate MeOH 05 1 . 02
Potassium thiocyanate MejCO . 004 . 92
" Other nonquenchers: a^^corbic acid; alkali iodide (Evstigneev and Krasnovsky,
1948): allylthiourea(r/. naffe789).
^ The measurements of Evstigneev and Krasnovsky (1948) indicate that hydroquin-
one at much higher concentrations has some quenching action, but is less efficient than
2-diaminopyridine, the weakest quencher in Table 23.IIIC.
Li\nngston and Ke used a 1.2 X 10~^ molar solution of chlorophyll a

in methanol, ethanol, ether or acetone, which they excited by the mercury
line 435.8 mju, or bj^ a Tables 23.IIIC and
red band centered at 645 m/z.
D show that quenching was similar for both types of excitation. The
quenchers are listed in the first table in order of declining efficiency; it is
clear that oxidants (quinones, diazo dyes, nitro and nitroso compounds, and
oxygen) are strong quenchers, while redudants (such as amines) are at best
only weak quenchers. Among compounds listed in Table 23. HID, only
phenylhydroxyamine showed any measurable quenching at all, at concen-
trations up to 0.1 mole/1. See also footnote b to Table 23. HID.
1.00
j
:
QUENCHING BY COLLISIONS AND BY RESONANCE 785
However, deviations from the linear relation required bj^ equation (23.16B)
do occur, particularly at the higher values of [Q] (fig. 23.8). Livingston
and Ke obtained a better approximation by using an empirically generalized
equation
Fo
(^^•^^^^ ^ - 1 + kdQ] +k2lQr-
The broken lines in figure 23.8 show how this equation can be made to fit
the data. The values of /ci are given in Table 23.IIIC. Livingston com-
pared the empirical two-constant equation (23.1()C) with a theoretical
equation of ^\nvilov and Frank:
(23.1GD) F
elQ]p + iAT/v)[Q\
Here, the first exponential term in the denominator represents "static

ciuenching," i. quenching determined by average distance between
e.,
quencher and fluorescent molecule (p = effective radius of energy ex-

change). The second term accounts for "kinetic quenching" {A = const.,
ij = viscosity of the medium). Quenching by resonance exchange of
energy is a possible mechanism of static quenching; chemical reaction
by the first, or one of the first, kinetic encounters (the frequency of which
isdetermined by the rate of diffusion, and thus indirectly by viscosity),
can be suggested as one mechanism of "kinetic" quenching.
Developing the exponential in (23.16D) and retaining only the first
term, one can obtain an equation of the type (23.16C). Livingston and Ke
calculated from their empirical constants (h and fc2) the "action ^radii", p
for different quenchers, and obtained values between 23 and 7 which A—
they considered as plausible in view of Forster's calculations of the range
of energy exchange (chap. 32). However, Forster's calculations were for
the case of resonance between the two molecules taking part in the energy
exchange, while the molecules listed in Table 23.niC have no absorption
bands which could resonate with the red fluorescence band of chlorophyll.
Therefore, no exchange of excitation energy over distances wider than a
—
molecular collision diameter appears possible unless one resorts to theo-
retically feasible, but experimentally as yet unsupported hypotheses.
Resonance between two separately "prohibited"—and therefore spectroscopically

—
unknown transitions, one in the excited molecule and one in the quencher, which be-
come "permitted" by being coupled together, was suggested by Rollefson; the total
spin of the system can be preserved if one molecule goes from a triplet into a singlet
state, while the other simultaneously undergoes a reverse change.
Testing equation (23.16D) by comparing quenching eflaciencies in dif-

ferent solvents did not give satisfactory results.For example, the h
values obtained with nitrophenol and chlorophyll a, in a series of alcohols

(from methanol to octanol), were not inversely proportional to the viscosi-
ties of these solvents. This may mean that, although ki is a measure of
the probability of quenching by kinetic encounters, the frequency of such
encounters is not the simple function of viscosity assumed in eq. 23.16D.
Livingston and Ke found that the absorption spectrum of chlorophyll a
was not markedly affected by the presence of even a large amount of the
strongest quenchers listed in Table 23.IIIC (e. g., 0.3 mole/1, of C6H5NO2),
thus indicating that quenching was not due to a preliminary dark chemical
— —
reaction such as complex formation between chlorophyll a and the
quencher.
The considerable which the comparatively weak quencher,
effect,
phenylhydrazine, has on the absorption spectrum of chlorophyll h, was

noted before (chapter 21, page 649); no similar effect was observed with
chlorophyll a.
quenching could be conclusively proved only for the

Reversibility of
gaseous quencher, oxygen, since it could be easily removed. (It will be
recalled in this connection that the fluorescence of allomerized chlorophyll a
in methanol is only about one-half as intense as that of the original solu-
tion; in other words, a slow irreversible change is superimposed on the
reversible quenching by oxygen.) Dilution experiments with one weak
organic quencher, phenylhydrazine, indicated that quenching probably is
reversible in this case, too.
Franck and Livingston (1941) had discussed the possible existence of a residual
fluorescence which could not be further reduced by quenchers. (Such a "nonquench-
able" fluorescence is to be expected if the electronically excited molecule requires a cer-
tain time to assume the configuration suitable for chemical quenching; the fraction of
total fluorescence, emittedbetween excitation and the attainment of this configuration,
would then be "unquenchable".) In the system (chlorophyll a in CH3OH + nitro-
benzene), a weak fluorescence appeared even in a 4 M
solution of the quencher; however
it was too weak {F = O.OOSFo) to consider it as a definite confii'mation of this prediction.
Whatever the interpretation of the quadratic term in the denominator

of equation (23.16C), the large values of the linear term found with oxidiz-
ing molecules and the low values found with reducing molecules (including
the substrates whose autoxidation is sensitized by chlorophyll, such as
allylthiourea) undoubtedly are significant. Livingston and Ke stressed
a detailed similarity of the hst of strong quenchers in table 23.IIIC with
the hst of substances known to inhibit certain polymerization processes;

but rather than looking for a causative relation between the two effects, one
should perhaps consider both as conse(iuences of the same oxidative proper-
ties of the quenchers (or inhibitors).
Quenching of chlorophyll fluorescence by kinetic encounters with oxi-
QUENCHING AND ALLOMERIZATION 787
dants can be attributed, with a fair degree of certainty, to a (reversible)

oxidation of the excited chlorophyll molecule by the quencher perhaps —
the same reaction which causes the reversible bleaching of chlorophyll in
light (^'ol. I, page 486, and chapter 35). Thus, quenching experiments
confirm the repeatedly noted capacity of chlorophyll to serve as a photo-
chemical reductant. (In chapter 35, we will present evidence that it can
act also as a photochemical oxidant.)
Some of the effects described by Livingston (1948) clearly belong to a
different type —which we may
call "pseudo-quenching." These are
fluorescence changes caused by chemical reactions between nonexcited
chlorophyll molecules and the quencher (or stimulant). The most striking
results of this type When traces of iodine were
were obtained with iodine.
added to a solution began to change gradually;
of chlorophyll, fluorescence
minutes or even hours were needed to reach a steady state. This points to
a slow chemical conversion of chlorophyll to a compound with a different
capacity for fluorescence. Probably, the reaction is an irreversible oxida-
tion; perhaps, preceded by transient complex formation. As discussed
elsewhere (cf. page 613), the product appears to be similar to (or identical
with) allomerized chloro-phyll, as obtained by slow oxidation of alcoholic
chlorophyll solutions in air. According to Fischer (c/. Vol. I, page 459), this
ischlorophyll oxidized at carbon atom 10. Similar to allomerization in air,
the reaction with iodine (and a similar one with bromine) occurs only in
alcoholic solution (methanol or ethanol) but not in ether or carbon tetra-
chloride. In the case of chlorophyll a, the final product has about 55%
of the fluorescence intensity of the nonallomerized solution; in the case of
chlorophyll 6, it fluoresces twice as strongly as the initial compound.
Admixture of carbon tetrachloride to methanol lengthens the time
needed to complete the allomerization by iodine, from about 5 minutes for
chlorophyll a in pure methanol, to over an hour in a mixture of equal parts
of methanol and carbon tetrachloride.
Withchlorophyll 6 in methanol, fluorescence declines at first upon the
addition of iodine; but later (in about 30 minutes) it begins to increase
again and reaches, after 10-15 hours, a steady level about twice as high as
AVith bromine and chlorophyll 6, the minimum is reached
the original one.
about one minute), and the high steady level is approached in
faster (in
about an hour. The initial dip in fluorescence may be taken as sign of
—
complex formation the subsequent increase, as indication of the conver-
sion of chlorophyll to the allomerized form.
Higher amounts of iodine, and particularly of bromine, quench the
fluorescence of chlorophyll more or less completely, by causing a deeper
chemical change in the chlorophyll molecule.
Effects similar to those caused by small quantities of iodine or bromine
,
can be produced also by small amounts of salts, such as LaCls or CeCls in

the presence of air. These salts probably catalyze the allomerization of
chlorophyll by air, with the concomitant changes in the intensity of fluores-
cence (decrease with chlorophyll a, increase with chlorophyll h). These
catalyzed reactions, too, occur only in alcoholic solutions. In ethereal
solution, chlorophyll a is converted by LaCls into a yellow compound, and
all fluorescence soon vanishes.
Evstigneev and Krasnovsky (1948) and Evstigneev, Gavrilova and
Krasnovsky (1949^) also have investigated the quenching of chlorophyll
fluorescence by several substances (see footnotes to Table 23. HID), in
particular by oxygen. Their peculiar initial result already was mentioned
on page 648: they found the effect of oxygen to be strongly dependent
on the solvent. —
In polar solvents pyridine, ethanol, ethyl acetate,
—
acetone (and also in commercial benzene) the effect of oxygen was as
previously described: moderately strong quenching. In nonpolar sol-
vents —heptane, —
carbon tetrachloride on the other hand, an
toluene,
entirely different effect was found; fluorescence (of chlorophyll a or a +
b) decreased upon removal of oxygen (evacuation of the vessel by an oil
pump) by as much as a factor of two; it increased to approximately the

original level after readmission of air. (The fact that benzene behaved
like a polar solvent probably was due to an impurity.) Repeated evacua-
tion and aeration produced a gradually weakening effect —
a result which
could be attributed to superposition, upon reversible association of chloro-
phyll with oxygen, of an irreversible oxidation (allomerization). Later
(1949^), the same investigators reported that Avhat they first took for an
activating effect of oxygen was in fact an activating effect of water vapor
contained in the admitted air. Admission of air caused no activation of
fluorescence in moist solvents. As described on p. 771, Livingston at-
tributed activation to an enol-ketone transformation in the cyclopentanonc
ring, enhanced by the formation of hydrogen bonds, while Evstigneev and
co-workers thought that polar solvent molecules might have an affinity
for themagnesium atom (which, in the chlorophyll molecule, has two free
coordination places). Saturation of these affinities could stiffen the
molecule and delay internal dissipation of excitation energy. In agree-
ment with this concept, no activation was obtained with Mg-free pheo-
phytin, or phthalocyanin {cf. p. 772).
Coe (1941) suggested that "chemical" quenching of chlorophyll fluorescence by

"antioxidants" present in oils and fats, could be used practically as a measure of their
rancidity; but French and Lundberg (1944) found no evidence of such quenching by
cottonseed oil.
Kautsky, Hirsch and Flesch (1935) found, and Franck and Livingston
(1941) confirmed, that certain substrates whose photoxidation is sensitized
TRANSITION TO LONG-LIVED Af'TIVE STATES 789
})y chlorophyll, c. q., isoamylamino and allylthiourea, do not cause any
weakening of chlorophyll fluorescence. According to Kautsky, chloro-

phyll solutions in acetone, saturated with isoamylamine, fluoresce brightly;
chlorophyll solutions in pure isoamylamine are not only fluorescent, but
also show a red afterglow, lasting for about 0.01 sec. According to Franck
and Livingston, the fluorescence of a 10-* M chlorophyll sohition in ace-
tone, saturated with air and containing 0.5 mole/l. of allylthiourea, is only
15-20% weaker than the fluorescence of the same solution free of both oxy-
—
gen and allylthiourea despite the fact that the quantum yield of sensi-
tized photoxidation is, under these conditions, of the order of unity. This
shows that the sensitization of the reaction between allylthiourea (and
similar oxidizable substrates) and oxygen cannot be attributed to the inter-
action of excited chlorophyll molecules in the fluorescent state with either

oxygen or the oxidation substrate. In other words, sensitization is brought
about —in this particular case—predominantly or exclusively by molecules
whose energy would otherwise be dissipated without fluorescence (for
similar observations with other sensitizers, see Shpolskij and Sheremetev
1936).
To explain this phenomenon, one has to assume that the majority of
excited chlorophyll molecules do not fluoresce because they undergo trans-
formation into a still energy-rich but comparatively long-lived form. In
this form, they retain some of their original excitation energy as electronic
or chemical energy. Because of their long life, these activated molecules
have a good change of encountering oxygen molecules (or substrate mole-
cules, A), even when the latter are present in a very low concentration.
This explains why a high yield of sensitized autoxidation was sometimes
observed even at very low values of [O2] and [A].
Thus, experiments on the quenching of chlorophyll fluorescence by
oxygen and autoxidizable substrates bring us back to the problem of long-
lived activation state (or states) of chlorophyll, which we have discussed
once before when dealing with the mechanism of photochemical sensitiza-
tion by chlorophyll in vitro and in vivo (cf. Vol. I, chapters 18, page 483,
and 19, page 544).
Weiss and Weil-Malherbe (1944) suggested that self -quenching may ex-
plain the nonquenching of chlorophyll fluorescence by isoamylamine, with-
out the assumption of long-lived active states. On page 774, we noted
that the high efficiency of self-quenching, reported by these observers,
turned out to be an error, caused by self-absorption. But even if it
were as high as they suggested, it could not prevent sensitization from
competing with fluorescence. If, in the absence of the sensitization sub-
strate, 90% of excited chlorophyll molecules undergo self-quenching by
encounters with nonexcited chlorophyll molecules (Chi* + Chi), and 10%
—
emit fluorescence, the addition of a sul^strate that is oxidized with a quan-

tum yield of 50% should reduce both self-quenching and fluorescence in the
same proportion—i. e., to 45 and 5%, respectively.
Contrary to the suggestion of Weiss and Weil-Malherbe, self -quenching
could not explain also the dependence of the yield of sensitized autoxida-
tion on chlorophyll concentration (eq. 18.32).
The statement that stimulation of fluorescence indicates chemical
change (p. 781) does not apply to sensitization. That energy absorbed
by other pigments can be utilized for chlorophyll fluorescence, was first
observed in vivo (chapter 24). According to Duysens (1951), in a 10"^ M

solution of chlorophyll a +
6 in acetone, one-half of the quanta absorbed
by h are available for the fluorescence of a (by the same token, a must
quench the fluorescence of 6).
7. Long-Lived Active States and Afterglow of Chlorophyll
As stated in chapter 18, "long-lived activations" can sometimes be due

to chemical changes (as weU as to the formation of metastable electronic
states— an explanation advocated by Kautsky and more recently by G. N.

Lewis). Several of the quenching processes discussed earlier in this
chapter may lead to transient formation of unstable products. Metasta-
ble active products may occur in the course of physical energy dissipation
as well as in that of chemical quenching. In the first case, strong vibrations,
excited during "internal conversion," can induce internal chemical changes—
e. g., one or two hydrogen atoms may be transferred
to a different position
in the molecule, thus creating a metastable, tautomeric form. In the second

case— that of chemical quenching—metastable states may be produced
by reversible photochemical reaction with the solvent— e. an exchange of
g.,
electrons or hydrogen atoms. In this case, the long-lived, metastable

state of the pigment is an oxidized or reduced (rather than a tautomeric)
state. The active, oxidized or reduced product can be reconverted to the
original pigment either by reversal of the reaction by which it was formed
thus leaving no net photochemical change at all— or by other reactions
with the solvent, or with dissolved oxygen), thus leaving a sensitized
(e. g.,
photochemical change. All these possibilities were discussed in some de-

tail in Volume I (chapter 18).
The hypothesis that long-lived activated molecules are molecules in
metastable electronic states (Kautsky, G. N. Lewis) was dismissed as im-
plausible in Volume I, chapter 18 (page 486). Subsequent development of
this concept, supported by extensive experiments by Kasha and other
workers at Berkeley, makes it necessary to bring the subject up again here.
Both the ground state of a valence-saturated molecule, and the excited
states corresponding to intense absorption bands, usually are singlet
LONG-LIVED ACTIVE STATE AND AFTERGLOW 791
states {i. e., states with total electronic spin zero). Triplet states, with
a total electronic spin of one unit, can be obtained by reversing the spin of
one electron; but to do this in a closed shell, it is necessary to change also
its orbital eigenfunction, thus converting it from a bonding into
an anti-
bonding electron— in other words, dissociating or weakening a chemical
bond (e. g., breaking the second bond in a C=C, or double C=0 C=N
bond) The molecule in the triplet state thus partakes of the character of a
.
biradical.* The radiative return of such a molecule into the ground state,
with the emission of fluorescence, is "prohibited," because of the require-
ment that the spin must be conserved; the light quantum has no mecha-
nism for carrying the spin away. This makes "activated" molecules of the
triplet biradicaltype metastable— at least as far as termination of activa-
tion is concerned.
by fluorescence The singlet-triplet prohibition apphes,
however, strictly only when the electron spin does not interact with other
modes of motion of the electrons in the system, since such interactions give
the chance of disposing of the spin momentum by converting it into other
rotational momenta. One interaction, which is always there, is the cou-

pling of spin momentum with the rotational momentum of the electron
movement around the nucleus (orbital momentum). This interaction is

weak in light elements and increases with increasing atomic number. Other
interactions arise when the triplet molecule is exposed to external fields of
force (electric or magnetic). the case in condensed phases, where
This is
each molecule finds itself in the fields of force of the adjoining molecules.
Thus, the theoretical life-time of the metastable triplet molecules, as cal-
culated for an isolated molecule by considering only the coupling with the
orbital momentum, must be considerably reduced in condensed systems
in consequence of coupling with the medium. Furthermore, in addition
to radiative transfer into the ground state (fluorescence), metastable
molecules are exposed also to energy dissipation by internal conversion
—
into vibrational energy made irreparable, in a condensed phase, by loss
of vibrational quanta to the surrounding molecules. This is the same kind
of process by which metastable molecules are produced from the
excited
molecules in the fluorescent singlet state (transition A -^ T in schemes
23. lA and 23. IB). The latter transition occurs within <10-^ sec. (since
it could it be that a similar
successfully interferes with fluorescences);
transition, T -^ X,
delayed for a second or even a minute? (This is the
is
lifetime of long-lived activation in some dyestuffs, as derived from the

decay curves of phosphorescence.)
These were the considerations which have caused us in Volume I to
consider the hypothesis of metastable tautomeric states as more likely
to explain long-lived activation than the hypothesis of metastable elec-
* We mean by this term a radical with two free valencies, not a combination of
two radicals.
)
ironic states.The difference between the two hypotheses is that, in the

second one, the position of the atomic nuclei in the metastable state is
supposed to be the same as in the normal state or, at least, not separated —
from the normal position by a potential barrier. In the tautomeric state,
on the other hand, the atomic nuclei are rearranged so that a return into
the ground state is prevented by a potential barrier, and thus requires a
certain activation energy. For the rest, the tautomeric state, too, may
(o) Excitation
'^
(d) Emission -hv -.%,- (b) Toutomerizotion
(c ) Thermol excitotion
Scheme 23.IB
have the chemical nature of a biradical, with two free valencies, and the
spectroscopical nature of a triplet state, with a corresponding paramagnetic
moment.
The following two elementary transformations illustrate the difference
between pure electronic excitation to a triplet state and electronic excita-
tion coupled with tautomeric rearrangement:
(23.16E) H H H H
Ri —C=C —R2 ^
^
Ri —C— C— R-. (electronic tautomerization
I I
(23.16F) H H H
Ri —C=C —R2 ^
^
Ri —C — C —Ro I
(atomic tautomerization)
H I
The return into the normal state requires, in the second case, that the H
atom moves over to a neighboring carbon atom, swinging from one poten-
tial minimum into another over a barrier. This type of metastability
may therefore be longer lived than the first one, in which the return into
the ground state can be achieved by electronic rearrangement alone.
The concept of metastable triplet states as the origin of long-lived
fluorescence (phosphorescence) of dyestuff solutions has been fiu'ther de-
veloped in several papers from the Berkeley laboratories by Lewis, Kasha,
McClure and Calvin (1945, 1947, 1948). One interesting result was the
experimental confirmation of (lie paramagnetism of the ph()si)horescent
LONG-LIVED ACTIVE STATES AND AFTERGLOW 793
state (at least in one case —that of fluorescein in a rigid solvent). The ob-
served paramagnetic moment corresponds to that of the spin of two un-
paired electrons, as expected for a triplet spectroscopic state, and it was
argued that this provides a clinching argument for the Lewis-Kasha theory.
However, according to what we said above, paramagnetism does not prove
that the biradical is of a purely electronic nature, and not a tautomer of the
normal molecule. (Perhaps, one should call the metastablc molecules
envisaged by Lewis and Kasha "electronic tautomers" and contrast them
with ordinary or "atomic tautomers"; the term "mesomers" usually is
applied only to electronic structures of equal, or nearly equal, energy.)
Life-time calculations of metastable organic molecules have been made
in three ways —theoretically, on the basis of spin-orbit interaction alone
(this should give a high upper limit for actual life times in condensed sys-
tems!), and experimentally, either from the duration of phosphorescence,
or from the intensity of the (weak) absorption bands which have been
found to correspond to the phosphorescence bands in some organic com-
pounds. In general, the actual life-times of phosphorescence were not
shorter, but longer (by factors of the order of 10, 10-, or even 10^) than the
theoretical life-times, particularly in the case of aromatic compounds. The
life-times derived from the intensity of the absorption bands also often
were shorter than those observed by the phosphoroscopic method.
Whether these results indicate that in some molecules, at least, the metas-
table state corresponds to an atomic, rather than an electronic, tautomer
remains to be seen. A possible alternative explanation is that the life-
time calculations on the basis of the triplet-singlet exclusion rule alone give
too small values because this exclusion rule is reinforced, particularly in
aromatic systems, by additional symmetry considerations.
In the present chapter, we are concerned particularly with one aspect
of the problem of long-lived active states —that of the "afterglow." The
photochemically produced tautomeric products or the metastable triplet
molecules may have such high energy that, with the help of thermal energy
fluctuations, they can return, after a certain interval of time, into the orig-
inal electronically excited state and cause the emission of "delayed fluores-
cence" (also designated as "afterglow" or "phosphorescence"). This cycle
(c/. schemes 23.1 A and B) provides the most plausible explanation of phos-
phoresence of many dyestuff solutions. (Solutions of eosin, erythrosin,
rose bengal and many other dyes all show an afterglow lasting for 10"*
to 10~^ sec.) According to Kautsky, Hirsch and Flesch (1935), who
studied this effect in numerous dyes, the afterglow is extremely sensitive
to oxygen; a few millimeters pressure of this gas suffice to suppress it.
. :
In the case of chlorophyll solutions in organic solvent, the same authors

could find no afterglow, except when isoamylamine was used as solvent. The
interpretation of this result is uncertain. Perhaps, the tautomeric state,
T, does occur in chlorophyll, as in other dyestuffs, but the relative prob-
ability of its termination by direct transition into the ground state X
(shown by broken scheme 23. IB) is much larger than that of the
line in
return into the fluorescent state A. Thus, the intensity of red phos-
phorescence is practically zero—except in certain cases (such as that of
solution in isoamylamine) where, for an as yet unknown reason, the rela-
tive probability of the two competing processes is changed in favor of
phosphorescence
It is hardly a coincidence that isoamylamine the only medium in —
which red chlorophyll afterglow was observed—is a compound whose
photoxidation is sensitized by chlorophyll with a high quantum yield. It
was pointed out above that the sensitization process does not compete with
fluorescence (since neither isoamylamine nor oxygen, in the low concen-
trations used, has a marked quenching effect on chlorophyll fluorescence).
Therefore, sensitization must be brought about by long-lived active forms
of chlorophyll. If the same forms were also responsible for (red) phosphores-
cence, then clearly this phosphorescence and sensitization would be com-
peting; this is obviously not the case, since red phosphorescence occurs only
in medium (isoamylamine) where sensitization also takes place. This is
a
indeed a paradoxical result! One may try to find a solution by assuming
that the phosphorescence of chlorophyll in isoamylamine is a photochemi-
luminescence, with the light-emitting pigment molecules being formed in
the process of restoration of chlorophyll after its reversible oxidation (or
reduction). For example, using the sensitization mechanism (18.40)
one could write (with A for amine, and t for tautomer)
(23.17a) Chi + hf > Chi* (

> tChl)
(23.17b) Chi* (or tChl) +A > oA + rChl
(23.17c) rChl +M O2 > Chl*(+ I H2O)
(23.17d) Chi* > Chi + hf
(23.17) A + O2 ) oA( + ^H20)
Reaction (23.17c) is an exception from the rule that back reactions in

photochemical sensitizations occur unthout chemiluminescence. In chapter
24, we will quote evidence of a chemiluminescence accompanying the back
reactions in photosynthesis.
LONG-LIVED ACTIVE STATES AND AFTERGLOW 795
—
Another possible cause of phosphorescence with a spectrum somewhat
different from that of instantaneous fluorescence —
can be the direct, radia-
tive return of the metastable molecules from state T into the ground state
X, with the emission of a quantum. This is an alternative to the above-
mentioned, nonradiative return by internal conversion of electronic into
vibrational energy. The assumption that this type of phosphorescence
alone limits the life-time of the metastable state is the basis of the above-
mentioned calculations of Lewis, Kasha and McClure. Long-lived lumi-
nescence, with a frequency 2000-20,000 cm.-^ lower than that of direct
fluorescence, actually is known for many dyestuffs and other fluorescent
organic compounds, particularly at low temperatures in glassy solvents.
In the case of chlorophyll, a phosphorescence of this type would have to be
sought in the infrared.
Calvin and Dorough (1947) reported that in a mixture of chlorophyll a
and h, dissolved in a "rigid solvent" (EPA = mixture of ether, pentane
and alcohol solidified <-100° C. without crystalHzation), an afterglow
can be observed after illumination. Spectroscopic obser-
lasting 0.2 sec.
vation revealed a band beginning at 780-800 m/z and stretching into the
infrared. Similar results were obtained with zinc tetraphenylchlorin,
but not with copper tetraphenylchlorin— a difference ascribed by Calvin
to weakening, by the paramagnetic Cu'^+ ion, of the metastability of the
triplet state.
Livingston and co-workers (1948) found no such afterglow, at —180° or
-150° C. (2 X 10-^ mole/1, chlorophyll a in EPA). Experiments in other
solvents and at other temperatures also gave negative results— with
chlorophyll a +
& as well as a, and at concentrations from 10
~- to 5 X
10-^ mole/1., in air or in vacuum. According to Livingston, a personal
communication from the Berkeley group confirmed that the luminescence
of Chi (a +
h) at 800 m^, reported by Calvin and Dorough, probably had
been due to impurities. However, Berkeley observers asserted that chloro-

phyll b does have a weak infrared afterglow (r = 0.02 sec), starting at 860
m/i. This phosphorescence, if it exists, should be in direct competition
with photochemical sensitization by chlorophyll (Weiss, 1948).
8. Summary — A Scheme of Fluorescence and Sensitization
In summing up the discussion, we may

go back to Volume I (chapter
19) and reproduce again, in a somewhat amplified form, scheme 19.11
,
given there to describe the most Ukely mechanism of fluorescence and sen-
sitized photoxidation in chlorophyll solutions.* This scheme, amplified
include also internal conversion, self-quenching by collisions and photo-
to
chemical reaction with the oxidation substrate c. g., an amine— (but A—
simplified as far as the mechanism of sensitized photoxidation is
concerned)
is reproduced in scheme 23.11. To prevent the scheme from becoming too
Chl"
6 © ® <h © ©
(tO,) (+A)
tChl (tChI) k, oChKtHO,) rChl(+oA)
t + Oj)
( + A) (Chi) (+A) (+02)
Chi Chi Chi 2Chl 2Chl Chi Chi 2Chl Chi
(toA-hHOj) ( + "»') ( + oA)
Scheme 23.11. Fate of excitation energy in chlorophyll solutions containing o.xygen

^
and an autoxidizable substrate A
does not include reactions with the solvent it must, how-

complicated, it ;
ever, be kept in mmd that, as shown repeatedly in chapter 18, "pseudo-
monomolecular" reactions with the solvent may nearly always serve as
alternatives for truly monomolecular tautomerizations. The meaning of
the arrows in the scheme is: (1) internal conversion, (2) tautomerization
(followed by sensitized autoxidation of the substrate A, or by dissipative
return into the normal state Chi), (3) self-quenching, (4) fluorescence, (5)
sensitized autoxidation of A through primary reaction with O2, (6) sensi-
tized autoxidation of A through primary reaction with A. According to

the Lewis-Kasha theory, "tautomerization" may mean an electronic
(rather than nuclear) rearrangement, and "dissipation" may be achieved

by emission of phosphorescence.
Scheme 23.11 shows eight different ways by which normal Chi can be
re-formed after excitation, and four ways by which substrate A can be oxi-
*In the last two lines of page 546 in Volume I (first printing), a misprint
and an
omission must be corrected: The lines should read: "If, at [O2] =
10-1000 mm.,
A*[02] is not 'C k, the fluorescence yield, >p, nmst depend
on oxygen pressure in this
• to lO^" mole/1.. Aj !<),] i.s«; k, and Af, |(),] is»At',
."
rlnge; and if, at [Oil = 10 . .
— .
SCHEME OF FLUORESCENCE AND SENSITIZATION 797
dized to oA (two alternative direct reactions of Chi* and two alternative

reactions of the metastable tChl). It neglects all resonance effects.
The yield of fluorescence, ^, is according to scheme 23.11:
^'
(23.18) f =
(Av + A-. + kt) + A-JChI] + A-*[0.] + A-* [A]
(This equation is to replace equation 19.5.)

Some of the constants in (23.18) can be estimated.
kf is 1.2 X 10^ (inverse of the natural life-time of Chi*, as calculated
from the intensity of the red absorption band on page 634)
ki may —
be small compared to A;, in other words, tautomerization may
be a normal intermediate step of internal conversion. This is, however
not certain. The quantum yield 7 ^^ 1 was found (c/. Tables 18.11 and
18. Ill) at [A] ^
5 X 10~^ i. e., concentrations at which k% [A] may well
be high enough to make ki insignificant even if it is not small compared to
ki + ki 10^, if one assumes a fluorescence \aeld of ~ 10%

must be about
in the absence of quenching by O2 or A.
fcj should be >10^2 according to Weiss and Weil-Malherbe (c/. page
773), but more probably is about 10^ (the value obtained by Weiss and
Weil-AIalherbe for dyes other than chlorophyll). Figure 23.7 indicates
half-quenching at [Chi] = 1.5 X 10~2 mole 1., and thus fc, = 67 and
kc^^7 X 10^ However, the sigmoid shape of the curve points to resonance
transfer rather than collisions as the main quenching mechanism.
A;o is about 5 X
10^ calculated from a "half-quenching" concentration
of 2 X 10-2 mole/1,
(cf. Table 23.IIIC).
k\ could be calculated from Franck and Levi's curves for the quench-
ing of fluorescence by benzidine, if the concentration units used were known.
A'l values in Table 23.IIIC are bimolecular rate constants in sec.
(The X
1. X
mole"'^ the h, k* and A;a values in eciuation 25.18 are products of these
constants and the rate constant of monomolecular deactivation, kf -\- ki -\-
k,^ 10«sec.-i.)
We refrain from an attempt to deduce from scheme 23.11 an equation
for the quantum >aeld of sensitized autoxidation (to replace equation 19.6'),
because the result is much too complicated to allow comparison with the
experimental data, e. g., with Gaffron's equation (18.32) for the sensitized
autoxidation of allylthioui-ea. The "self-quenching" reaction of the tau-
tomer tChl:
(23.19) tChl + Chi » 2 Chi
has been added to account for the decrease in the yield of chlorophyll-
sensitized photoxidation with increased pigment concentration [Chi], in
: :
the [Chi] region where self-quenching of the short-lived state Chi* cannot
be significant (cf. Table 18. Ill, page 513, Vol. I). This "deactivation" of
tautomeric chlorophyll by normal chlorophyll can perhaps occur via dis-
mutation
(23.20) tChl + Chi > oChl + rChl
(as suggested, e. g., in equation 18.42b, page 519, Vol. I) followed by back
reaction
(23.21) oChl + rChl » 2 Chi
B. Fluorescence of Carotenoids and Phycobilins

in Vitro*
1. Fluorescence of Carotenoids
Carotenoids are usually described as nonfluorescent. According to

Willstatter and Stoll (1918), this is true both of the leaf carotenoids and of
the carotenoids of brown algae (e. g., fucoxanthol) . However, de Rogovski
(1912) asserted that he has observed a fluorescence of carotene in petroleum
ether, in the region 505-600 m/x, and Dhere and Castelli {cf. Dhere 1939)
stated that, at —180°, three separate fluorescence bands can be observed
in carotene solution in xylene. Klein and Linser (1930) mentioned a
green fluorescence of carotene solutions in alcohol. Strain (1936) found,
in the chromatograms of leaf extracts in petroleum ether, a fluorescent
and belonging to an unknown color-
layer situated below that of carotene a,
less substance, probably a hydrocarbon without sharp absorption bands in
the visible or near ultraviolet. Zechmeister and co-workers (see, for ex-
ample, Zechmeister and Sandoval 1945, 1946) found a fluorescent, colorless
polyene hydrocarbon with sharp absorption bands at 331, 348 and 367
mû (in petroleum ether) to be present in great abundance in extracts from
various plant organs (fruits, stems etc., but not chlorophyllous organs,
such as grass, leaves or green needles). This hydrocarbon, called phyto-
fluene (probably C40H64), may be a hydrogenation product (or precursor)
of the carotenes; it contains seven double bonds, with probably only five
of them conjugated.
Absence of fluorescence indicates, according to page 799, that the exci-
tation energy of carotenoid molecules in solution is dissipated within less
than 10~i^ sec.
FLUORESCENCE OF PHYCOBILINS 799
2. Fluorescence of Phycobilins
The phycobilins are usually described as brilliantly fluorescent. How-

ever, since no exact determinations of the yield of fluorescence exist, it
is impossible to judge whether the fluorescence is so much stronger than
that of chlorophyll, or whether its greater brilliancy is due to the fact that
the fluorescence bands of the phycobilins lie near the region of the greatest
sensitivity of the human eye, whereas those of chlorophyll are in the far red,
and partly in the infrared.
Association with proteins does not impair the fluorescence of phycobil-
ins. On the contrary, according to Lemberg (1930), the isolated pigments
fluoresce less strongly than the chromoproteids.
The fluorescence of red algae was first described by Stokes, in the same
paper in which he also reported the discovery of the fluorescence of green
leaves (c/. page 805). Since then,* the fluorescence of both the living
algae, and of their aqueous extracts, has repeatedly been observed, e. g.,
by Schiitt (1888), Hanson (1909), Turner (1916), Lemberg (1928), Dhere,
and Fontaine (1931), Roche (1933), Dhere and Raffy (1935), Van Norman.
French and Macdowafl (1948), Arnold and Oppenheimer (1949) and
French (1951).
Van Norman, French and Macdowall (1948) gave a photometric curve
for the fluorescence ofan extract obtained by grinding a species of the red
alga Iridaea under water and centrifuging at high speed. It shows a sharp
peak at about 580 m/i, clearly related to the first long-wave absorption
band of phycoerythrin at 566 m/x (cf. fig. 23. 9A, from French 1951).
A shoulder appears on the long-wave side of the 580 m/x fluorescence
band, indicating the presence of a second maximum at about 630 m/x.
This probably is the second (0-^1) fluorescence band of phycoerythrin
(leading to a vibrational ground state). The first fluorescence band of
phycocyanin (correlated with the first absorption band of this chromopro-
teid, the peak of which appears, in the same extract, at about 615 m^)
lies at 660 m^, according to the curve obtained by French (1951) by
subtraction of the phycoerythrin from the fluorescence

fluorescence
spectrum of the crude aqueous extract from a red alga (fig. 23. 9B) Earlier, .
Dhere and Fontaine (1931) gave 578 and 648 m/x, respectively, as the axes
of the fluorescence bands of the two phycobilins in aqueous extract. Dh^re
and Raffy (1935) noted a second phycocyanin band at about 728.5 m/x.
According to French's figures, the fluorescence bands of the phycobilins
are shifted to the red of the absorption bands, by 14 m/x in the case of
phycoerythrin, and by 45 m/x in tliat of phycocyanin.
FLUORESCENCE OF PIGMENTS IN VITRO CHAP. 23
800
<n
UJ
o
<
o
a.
o
400 500
WAVE LENGTH
23.9A
o r
UJ
_ Wo'er extract
z
UJ
u \
in
UJ
<r
o
3
600 650 700 750

WAVE LENGTH
23.9B
Fig. 23.9A. Absorption (-) and fluorescence (--) spectra of pure phycoerythrin
Courtesy C. French (1951).
S.
Fluorescence spectrum of phycocyanin as derived from the spectrum
of
Fig. 23.9B.
red alga by subtracting the fluorescence of phycoerythrin
a crude water exctract of a
Courtesy C. S. French (1951).
Arnold and Oppenheimer (1950) broke blue-green algae, Chroococcus

by squeezing through a syringe {cf. chapter 35) and separated the phy-
cobilin chromoproteid from the chlorophyll chromoproteid by fractiona-
tion with ammonium sulfate. The phycobilin fraction, resuspended in
water, showed a fluorescence band at 620-655 m^t. An estimate of the
yield of fluorescence gave a value of about 20%. The yield of fluorescence
is much lower (about 1.5%) in living Chroococcus cells. It increases upon
grinding of the cells under water, even without the separation of the phy-
cobihn from chlorophyll (cf. chapter 24, page 816). This is interesting
because the blue-green algae contain no chloroplasts; therefore, the "cell
juice," obtained by their grinding, should not differ so strongly in its proper-
ties from the contents of the living cell, as do the products of mechanical
destruction of chloroplast-bearing green cells. Arnold and Oppenheimer

suggested that the approximately tenfold increase in the yield of fluores-
cence upon grinding could be a pure dilution effect: In live cells, close
proximity of the average phycobilin molecule to one or several chloi-ophyll
molecules, permits a highly efficient resonance transfer of excitation energy
from phycobilin to chlorophyll (accounting for a high quantum yield of
photosynthesis in light absorbed by the phycobilins; cf. chapter 30, section
6). This leads to quenching of phycobilin fluorescence and substitution
of the (less intense) sensitized chlorophyll fluorescence. Dilution de-
creases the rate of resonance transfer and thus preserves the fluorescence
of the phycobihns.
Fluorescence of Pigments in Vitro
A. Fluorescence of Chlorophyll in Vitro
1834 Brewster, D., Trails. Roy. Sac. Edinburgh, 12, 538.

1852 Stokes, G. G., Trans. Roy. Sac. London, 1852, 463.
1914 Dh6r6, C, Co7npt. rend., 158, 64.
Wilschke, A., Z. wiss. Mikroskop., 31, 338.
1Q18 Willstatter, R., and StoU, A., Untersuchungen iiber die Assimilation der
Kohlensaure. Springer, Berlin, 1918.
1920 Stern, K., Ber. deut. botan. Ges., 38, 28.
1921 Stern, K., Z. Botan., 13, 193.
1926 Perrin, J., 2"^ congres chimie Solvay, Gauthiers-Villars, Paris, p. 322.
1927 Noack, K., Biochem'z., 183, 135.
Perrin, J., Compt. rend. acad. sci. Paris, 184, 1097.
1929 Perrin, F., Ann. phys., 12, 169.

1930 Dh6r6, C., Compt. rend., 192, 1496.
1931 Kautsky, H., and Hirsch, A., Ber. deut. chem. Ges., 64, 2677.
Dh^re, C, and Fontaine, M., Ann. inst. oceanogr., 10, 245.

Kautsky, H., and de Bruijn, H., Naturwissenschaften, 19, 1043.
1932 Perrin, F., Ann. phys., 17, 283.
1933 Kautsky, H., de Bruijn, H., Neuwirth, R., and Baumeister, W., Ber. deut.
chem. Ges., 66, 1588.

Knorr, H. V., and Albers, V. M., Phys. Rev., 43, 379.
1934 Knorr, H. V., and Albers, V. M., ibid., 46, 336.
Baas-Becking, L. G. M., and Koning, H. C, Proc. Acad. Sci. Amsterdam,
37, 674.
Albers, V. M., and Knorr, H. V., Phys. Rev., 46, 336.
Prins, J. A., Nature, 134, 457.
Franck, J., and Levi, H., Z. physik. Chem., B27, 409.
Bakker, H. A., Proc. Acad. Sd. Amsterdam, 37, 688.
1935 Knorr, H. V., and Albers, V. M., Cold Spring Harbor Symposia Quant.
Biol, 3, 98.
Albers, V. M., and Knorr, H. V., ibid., 3, 87.
Kautsky, H., Hirsch, A., and Flesch, W., Ber. deut. chem. Ges., 68, 152.
Wakkie, J. G., Proc. Acad. Sci. Amsterdam, 38, 1082,

Hubert, B., Rev. trav. botan. neerland, 32, 323.
Stern, A., and Molvig, H., Z. physik. Chem., A175, 38.
Dher6, C., and Raffy, A., Bull. soc. chim. biol., 17, 1385.
Weiss, J., Naturwissenschaften, 23, 610.
Zscheile, F. P., Protoplasma, 22, 513.
1936 Stern, A.,and Molvig, H., Z. physik. Chem., A176, 209.
Stern, A.,and Dezelic, M., ibid., A176, 347.
Franck, J., and Wood, R. W., J. Chem. Phys., 4, 551.
Biermacher, 0., Thesis, Univ. Fribourg (Switzerland) 1936.
Dher6, C, and Biermacher, O., Compt. rend. soc. biol, 122, 591.
Shpolskij, E., and Sheremetev, G., J. Phys. Chem. USSR, 8, 640.
1937 Kautsky, H., Biochem. Z., 291, 271.

Vermeulen, D., Wassink, E. C., and Reman, G. H., Enzijmologia, 4, 254.
Dh^r6, C., La fluorescence en biochimie. Paris, 1937.

Franck, J., and Herzfeld, K. F., J. Phys. Chem., 41, 97.
Rabinowitch, E., Trans. Faraday Soc, 33, 1225.
1938 and Wiedemann, E., Fortschr. Chem.
Stoll, A., organ. Naturstoffe, 1, 159.
Rothemund, P., J. Am. Chem. Soc, 60, 2005.
Smith, E. L., Science, 88, 170.
Stern, A., Z. physik. Chem., A182, 186.

1939 Meyer, K. P., Hev.l Phys. Acta, 12, 349.
Dh6r6, C., Fortschr. Chem. organ. Naturstoffe, 2, 301.
1940 Seybold, A., and Egle, K., Botan. Arch., 41, 578.
Rabinowitch, E., and Epstein, L. F., J. Am. Chem. Soc, 63, 69.
Knorr, M. V., Gibson Island A. A. A. S. Symposium on Photosynthesis
(unpublished).
Coe, M. R., Oil & Soap, 18, 227.

Franck, J., R., J. Chem. Phys., 9, 184.
and Livingston,
1942 Stewart, A., Knorr, M.
and Albers, V. M., Phys. Rev., 61, 730.
V.,
Fishman, M. M., and Moyer, L. S., /. Gen. Physiol., 25, 755; Science,
95, 128.
Weil-Malherbe, H., and Weiss, J., Nature, 149, 471.
Vavilov, S. I., and Feofilov. P. P., Cotnpt. rend. (Doklady) acad. sci. USSR,
34, 220.
Wassink, E. C, Katz, E., and Dorrestein, R., Enzymologia, 10, 285.
1943 Button, H. J., Manning, W. M., and Duggar, B. M., /. Phys. Chem., 47,
308.
and Harris, D. G., ibid., 47, 623 (1943).
Zscheile, F. P.,
Manning, W. M., and Strain, H. H., /. Biol. Chem., 151, 1.
Vavilov, S. I., /. Eksp. Teor. Phys. USSR, 7, 141.
1944 Vavilov, S. I., Compt. rend. (Doklady) acad. sci. USSR, 42, 331.
Vavilov, S. I., ibid., 45, 7.
French, C. S., and Lundberg, W. 0., Oil & Soap, 21, 23.
Weil-Malherbe, H., and Weiss, Chem. Soc, 1944, 544.
J., /.
1945 Lewis, G. N., and Kasha, M., /. Am. Chem. Soc., 67, 994.
1946 Forster, T., Naturw., 33, 166.
1947 Calvin, M., and Borough, G. B., Science, 105, 433.
Forster, T., Z. Naturforsch., 2h, 174.
Kasha, M., Chem. Revs., 41, 401.
Pekerman, F. M., Compt. rend. (Doklady) acad. sci. USSR, 57, 559.
1948 Forster, T., Ann. Physik, 2, 55.
Lewis, G. N., Calvin, M., and Kasha, M. (to be published).
Weiss, J. (personal communication).
McClure, B. S., presented at Symposium in Molecular Structure and
Spectroscopy, Ohio State Univ., June, 1948.
Livingston, R., First Annual Report, ONR Research Project 059,028.
Evstigneev, V. B., and Krasnovsky, A. A., Compt. rend. (Doklady) Acad.
Sci. USSR, 60, 623.
1949 P'ranck, and Livingston, R., Rev. Modern Phys., 21, 505.
J.,
Livingston, R., Second Annual Report, ONR Research Project 059,028.

Livingston, R., "Photochemistry of Chlorophyll" in Photosynthesis in
Plants. Iowa State College Press, 1949, pp. 179-196.
Livingston, R., Watson, W. F., and McArdle, J., J. Am. Chem. Soc, 71,
1542.
Livingston, R., and Ke, Chun-Lin, ibid, (to be pubUshed).
Vavilov, S. I., and Galanin, M. B., Compt. rend. (Doklady) acad. sci.
USSR, 67, 811.
Vavilov, S. I., Galanin, M. B., and Pekerman, F. M., Bull. (Izvestiya) acad.
sci. USSR, Physics ser., 13, 18.
Evstigneev, V. B., Gavrilova, V. A., and Krasnovsky, A. A., Compt. rend.

(Doklady) Acad. Sci. USSR, 66, 1133.
Evstigneev, B. V., Gavrilova, V. A., and Krasnovsky. A. A., ibid., 70, 261.
1950 Arnold, W., and Oppenlieimer, J. R., J. Gen. Physiol., 33, 423.
1951 Duysens, L. N. M., Nature (in press).
B. Fluorescence of Carotenoids and Phycobilins in Vitro
1888 Schiitt, F., Ber. deut. botan. Ges., 6, 36, 305.

1909 Hanson, E. K., New Pkytologist, 8, 337.
1912 de Rogowski, W., Thesis, Univ. Fribourg (Switzerland), 1912.
1916 Turner, B., /. Am. Chem. Soc, 38, 1402.
1928 Lemberg, R., Ann., 461, 46.
1930 Lemberg, R., Biochem. Z., 219, 255.
Klein, G., and Linser, H., Osterreichische botan. Z., 79, 125.
1931 Dhere, C, and Fontaine. M., Ann. inst. oceanogr., 10, 245.
1933 Roche, J., Arch. phys. biol., 10, 91.
1935 Dh^re, C, and Raffy, A., Compt. rend. soc. biol, 119, 232.
1936 Strain,H. H., Nature, 137, 946.
1939 Dhere, C, Fortschr. Chem. org. Naturstoffe, 2, 301.
1945 Zechmeister, L., and Sandoval, A., Arch. Biochem., 8, 425.
1946 Zechmeister, L., and Sandoval, A., J. Am. Chem. Soc, 68, 197.
1948 Van Norman, R. W., French, C. S., and Macdowall, F. D. H., Plant
Physiol, 23, 455.
1950 Arnold, W., and Oppenheimer, J. R., /. Gen. Physiol, 33, 423.
1951 French, C. S., and Kosk, V. M., Proc. Soc. E.vptl Biol (in press).
Chapter 24
FLUORESCENCE OF PIGMENTS IN VIVO *
Because of close relationship that exists between fluorescence and sen-

sitization (cf. chapters 18 and 19 in Volume I, and chapter 23 in this vol-
ume), the study of fluorescence of chlorophyll in the living plant can make
an important contribution toward the understanding of the mechanism of
photocatalytic action of this pigment in photosynthesis. Fluorescence is
a property of chlorophyll that can be —and has been —observed simultane-

ously with the measurement of photos\Tithetic activity. By measuring
the yield of fluorescence, one can obtain insight into the energy exchange and
dissipation processes in photosynthesizing cells, without interfering with
their life processes. No attempts have been made to observe changes in

the fluorescence spectrmn (or, for that matter, in the absorption spectrum) of
chlorophyll during photosynthesis, but this, too, may prove possible and
useful in future.
Many plant tissues fluoresce in ultraviolet light; but only those con-
taining chlorophjdl, bacteriochlorophyll or the phycobilins show a rather
weak, red or orange fluorescence when illuminated with visible light. The
fluorescence of the phycobilins (in blue-green and red algae) is more vivid
than that of chlorophyll, because it is stronger and the eye is more sen-
sitive to orange than to red light. Among green plants, the algae show
fluorescence more clearly than land plants, because light scattering (which
obscures fluorescence) is much weaker in their water-filled thalli than in
air-filled leaves.The fluorescence of leaves is so difficult to observe, that
after was discovered by Stokes in 1852, and also described by Simmler
it
in 1862 and Askenasy in 1867, other investigators, notably Lommel (1871),

Hagenbach (1870, 1872) and Reinke (1883), were unable to confirm its ex-
istence. Although Hagenbach (1874) and Reinke (1884) revised their
views later, the reality of leaf fluorescence remained subject to occasional
doubts for another quarter of a century, until the invention of the fluores-
cence microscope permitted the observation of the fluorescence of single
chloroplasts. (As early as 1883, Engelmann had tried, unsuccessfully, to
observe the fluorescence of chloroplasts in ultraviolet light under an or-
dinary microscope.) The picture of chloroplasts glowing with a crimson
fight on a faint milky background so thrilled the investigators who first
805
806 FLUORESCENCE OF PIGMENTS IN VIVO CHAP. 24
saw it— Tswett (1911), Lehmann (1914), Wilschke (1914) and Gicklhorn
(1914) —that
they gave enthusiastic descriptions of this phenomenon.
More recently, green leaves, green and colored algae and diatoms have all
been studied under the fluorescence microscope, by Lloyd (1923, 1924),
Testi Dragone (1927), Klein and Linser (1930) and Metzner (1937).
In the meantime, methods of macroscopic observation of plant fluores-
cence also have been improved, and Stokes' original results confirmed and
expanded. Notably Dhere and co-workers (see Dhere 1937, 1939) have
carried out numerous spectrophotographic investigations of plant fluores-
cence: Dhere and Fontaine (1931), Fontaine (1934) and Dhere and Raffy
(1935) studied brown, green and blue algae; Bachrach and Dhere (1931),
diatoms; and Dhere and Raffy (1935) and Dhere and Biermacher (1936),
green leaves. The investigations of Kautsky and co-workers (1932-1943),
McAlister and Myers (1940), Franck, French and Puck (1941), Shiau and
Franck (1947) and of the "Dutch group" (Wassink, Katz, Dorrestein et at.
(1939, 1942, 1945) dealt mainly with the alterations in the intensity of
fluorescence that accompany changes in the rate of photosynthesis.
French and co-workers (1948, 1951) and Duysens (1951) initiated a
very promising spectrophotometric investigation of fluorescence, particularly
of red algae.
1. Fluorescence Spectra of Plants
If we leave aside the phycobilin-carrying algae, the spectroscopic pat-

tern of the fluorescence of living plants is very simple. In solutions, both
chlorophyll a and chlorophyll h have a two-band fluorescence spectrum in
the visible region (cf. Table 23.1). In the living cell, these bands are
shifted so far toward longer waves that only one band of chlorophyll a
and one of chlorophyll h remain within the visible spectrum. Thus, the
visible fluorescence spectrum of green leaves and green algae consists of
only two bands.
Brown algae and diatoms contain no chlorophyll h; their fluorescence
spectrum therefore shows only one visible band. It was mentioned on
page 406 that Wilschke (1914) and Dhere and Fontaine (1931) observed
a second band in the fluorescence spectrum of heat-killed brown algae
and extracts from these organisms, and attributed it to a "chlorophyll
c" (chlorofucin) but no corresponding band was found by Dhere and
;
Raffy (1935) in the fluorescence spectra of living brown algae. Manning

and Strain (c/. page 614) concluded more recently that a chlorophyll c
actually exists in live diatoms; but they did not attempt to confirm this
by observation of the fluorescence band of this pigment in the spectrum of
the algae.
The positions of the visible fluorescence bands of the two chlorophylls in
FLUORESCENCE SPECTRA OF PLANTS 807
Table 24.1. The two different figures given in the

living cells are listed in
table for the leaves of Pelargonium illustrate the statement made on page
744 that the position of the band ''axis" depends on the sensitivity curve of
the photographic plate.
Table 24.1
Fluorescence Bands of Chlorophyll in Plants

—
808 FLUORESCENCE OF PIGMENTS IN VI VO CHAP. 24
close coincidence of the fluorescence band in vivo (No. 7) with the corre-
sponding absorption band (No. 6), and the "red shift" of the fluorescence
band in the Uving cell compared to its position in solution (Nos. 2 and 3
compared to No. 4).
oO o
o o mo o o
...
Wave
I ii_
lenqth, mu: mo m o o
Spectrum: ^
Reference
Fluorescence
Reference
Absorption
Fluorescence
Reference
oo o o tn
O O
o o o o
If) ID
r^ h- <0 Ô tf>
O
tf>
m o
sf
Fluorescence spectrum of living leaves of Pelargonium (Nos.

Fig. 24.1.
2, 3, and of chlorophyll in ether (No. 4), compared with the absorption
7)
spectrum (No. 6) (after Dhere and Raffy 1935).
Dhere and Raffy (1935) suggested that the second fluorescence bands of chlorophylls
a and b in vivo, may
account for the striking brightness
situated in the near infrared,
that green vegetation exhibits on landscape photogi'aphs on infrared-sensitive plates
(c/. fig. 22. 31 A). However, the fluorescence of living loaves is much too weak to produce
such a spectacular effect. Mecke and Baldwin (1937) disproved Dhere's theory by
showing that the vegetation remains dark when illuminated with infrared-free light and
photographed through a filter that transmits only the infrared. The brightness of green
plants in infrared light is thus due to lack of absorption, and not to fluorescence.
Dhere and Biermacher (1936) photographed the fluorescence of Pelar-

gonium leaves on plates whose sensitivity extended far into the infrared,
and foimd a new band, with an axis at 812 m/x, extending to 830 m/i. In
Table 24.11, the wave lengths of the peaks of the four known fluorescence
bands of the chlorophylls a and h in living cells are compared with the
wave lengths of the corresponding bands in ethereal solution.
Table 24.11 shows that, in the living cell, the fluorescence bands are
shifted by 5-15 m/x toward the infrared from their positions in ethereal
solution i. e., by about the same distance as the corresponding absorption
Table 24.11
Fluorescence Bands of Chlorophyll in Plants and in Ethereal Solution"

Comparison and 23.4 shows that the displacement of

of the figures 24.3
the fluorescence band is much
stronger in the purple bacteria than in
green plants. The fluorescence spectrum of live Chromatium shows only
one band, at 926 mju, while that of the extract contains two bands, at 806
and 695 mju, respectively. It was mentioned on page 751 that the first
and more intense of these two bands can be correlated with the main ab-
sorption band of extracted bacteriochlorophyll, at 770 m/i, but that the
correlation of the 695 m^l fluorescence band with the 605 m/z absorption
band is doubtful. Absorption spectra of live purple bacteria show two
(or three) absorption bands, at 800-870 and 800 mtx, respectively (c/. p.
702); but here again, only the one can be identified with the main
first
X-^ Z absorption band of dissolved bacteriochlorophyll (at 770 m^), while

the identification of the second one—which is comparatively weak and
variable in intensity—with the X -^ Y band at 605 mfx is uncertain (cf.
page 702). The relation between the fluorescence and the absorption
bands of bacteriochlorophyll is illustrated by Table 24.IIA. The table
shows that the fluorescence band I in vivo is shifted toward the infrared by
as much as 120 m^, compared to the position of the fluorescence band in
vitro, and by about 60 mn compared to the position of the
corresponding
absorption band in vivo.
Table 24.IIA
Spectra of Bacteriochlorophyll in Vitro and in Vivo
I, mti II, ni/i III, mil

Band {X -» Z) (X -* Y) ?
Absorption
860-870 — 800
Cell
Extract 770 605 —
Fluorescence
Cell 926 — —
Extract 806 695 (?) —
This tabulation indicates that, in the living cell, bacteriochlorophyll
fails to show one absorption band and one fluorescence band that are
found
in extracts, but shows one (or two) extra absorption bands, without
corresponding fluorescence bands, which have no counterpart in the
solution spectrum. Duysens (1951) confirmed that Chromatium and
Rhodospirillum show only one fluorescence band, correlated with the
absorption band at 890 m^. Absorption in the 800 and 850 m^ bands
contributes to the excitation of this fluorescence band; so does with a —
50-70% lower efficiency— the absorption by cartenoids. Excitation
energy is thus transferred from all pigments of the bacteria to the bacterio-
chlorophyll form having the lowest excitation energy.

—
The fluorescence of green bacteria (Chlorohium mirahle), probably due

to "bacterioviridin" (cf. Vol. I, p. 407), was observed by Buder (1913).
A fluorescence band at 635 m^, noted by French (1951) in a partially green leaf could
be due to photochlorophyll, whose absorption band lies at 620-630 ni/x in ether. French
estimated its position in vivo as 650 niju (from the action spectrum of chIoroph\'ll forma-
tion), but noted that this is incompatible with the location of the fluorescence at 635 m/x.
The fluorescence of the phycohilins in red and blue-green algae is of

great interest, because it permits a study of the interaction of two different
fluorescent pigments in one
cell. The first photograph of the fluorescence
spectrum of Rhodymenia, made by Dhere and Fontaine (1931), showed one
band in the orange (phycoerythrin) and one in the red (chlorophyll and
phycocjânin).
Van Norman, French and Macdowall (1948) determined fluorescence
curves of two red algae Gigartina and Iridaea; both showed three peaks,
at 575 myu (phycoerythrin, cj. p. 799), 055 m^ cj. p. 800) and
(phycocyanin,
700 m/x (chlorophyll, probably a -^ d, cf. beloAv). French (1951) gave fig.
24.4 for Porphyridium: Only chlorophyll fluoresces when cells are excited
with X 436 m^ or 450 m/x, i.e., by light absorbed by chlorophyll and caro-
tenoids only. Phycohilin bands develop with excitation by 470, 490, and
546 m/x but, even though most of the incident light is now absorbed by
phycoerythrin, chlorophyll fluorescence remains strong.
T~l I
I I I I
I
I I I I
I
I I I I I I I I I I I I I I I I
I
I I I I
I I
436 m;i (a) 546mjLi (c)
u
o
z (b)
UJ
o
CO
476 m/i
UJ
cr
o
3
I I I I I I I I I
J__L I I I I I I 1 I I I I I 1 I I I I I I I I I I I I
600 650 700 750 600 650 700 750

Fig. 24.4. Fluorescence spectra of a red alga when illuminated with equal energies of
different wavelengths. Courtesy L. N. M. Duysens.
812 FLUORESCENCE OF PIGMENTS I IV VIVO CHAP. 24
—
The chlorophyll a band in fig. 24.4 is in the usual position at 685 mix.
However, an additional band is indicated at 730 m/u; it can be attributed
to chlorophyll d. The weakness of chlorophyll d absorption (mere ripple
in fig. 22.20!) makes one suspect that chlorophyll d fluorescence is excited
mostly by energy transfer from other pigments. This is strikingly con-
firmed by the observation, reported by Duysens (1951), that an "uniden-
tified pigment"— presumably chlorophyll d — whose absorption, in Por-
—
;
FLUORESCENCE YIELD 7A^ VIVO 813
in a suspension of diatoms. In Chromatium (a purple bacterium) Vermeu-

len, Wassink and Reman (1937) at first found a much smaller yield
between 0.005 and 0.01%. These values are incredibly low for a "fluores-
cent" material. (Theoretically, the lowest yield to which fluorescence
could sink even in a "nonfluorescent" pigment is about 0.001%, since the
ratio of the period of a molecular vibration and the life-time of electronic
excitation is lO^^^-yiO"^-^ = 10~^) A redetermination of the yield of
fluorescence of Chromatium by Wassink, Katz and Dorrestein (1942) in
fact gave considerably larger figures —
of the order of 0.1%, i. e., similar to
those found in algae. Self-absorption may have been the cause or at —
least, one cause — ôf the error of the earlier determinations.
500 550 SCO 650

WAVE LENGTH, m/x
Fig. 24. 5A. Yield of fluorescence of Chlorella suspensions in relation to wave

length of exciting light (after Vermeulen, Wassink and Reman 1937). 4 sets of
measurements.
lOOp
450 500 550 600 650 700 750

WAVE LENGTH, m/i
Fig. 24. 5B. Yield of fluorescence of Chromatium suspensions in relation to

wave length of exciting light (after Vermeulen, Wassink and Reman 1937). 4
sets of measurements.
The yield of fluorescence in live blue-green algae (Chroococcus) was esti-

mated by Arnold and Oppenheimer (1950) by a rather crude method (visual
comparison with the intensity of light scattered by a block of magnesium)
—
they found it to be of the order of 1.5% about ten times higher than the
yield of fluorescence in green cells. Presumably, this fluorescence origi-
nates predominantly in phycocyanin, although chlorophyll, too, may con-
tribute to it.
Interesting results were obtained in the study of the effect of wave

814 FLUORESCENCE OF PIGMENTS 7A^ VIVO CHAP. 24
length of the exciting hght on the fluorescence of live cells. Vermeiilen,

Wassink and Reman (1937) found that the spectral distribution of the fluor-
escent light of Chlorella and Chromatium is independent of the wave length
of exciting radiation. The quantum yield of fluorescence (<^) also was ap-
proximately constant, between 442 and 624 mju in Chlorella, and between
450 and 750 ray. in Chromatium. However, a slow systematic decrease of
fp was observed in Chlorella at the shorter waves —
a trend that became
accelerated below 424 mû (fig. 24. 5A). In Chromatium., maxima and
minima of (p were observed in two or three places in the visible spectrum
(fig. 24. 5B). The Dutch investigators concluded from these observations
that the quantum yield of chlorophyll fluorescence in vivo does not depend
on wave length except when carotenoids interfere with the light absorption
by chlorophyll or bacteriochlorophyll. In green plants, this occurs only

below 520 mju the carotenoids of purple bacteria, on the other hand, have
;
absorption bands in the green, yellow and orange {cf. Table 21. IX and
fig. 22.27), and these bands could perhaps account at least for the first
minimum of the fluorescence yield noticeable in figure 24.5B at about 550

mju.
While qualitatively the conclusions of Vermeulen and co-workers ap-
pear plausible, quantitative considerations lead to some interesting compli-
cations. In Chlorella, for example, ^ declined, in the violet, by only 10 or.
20%, while figure 22.43 indicates that the carotenoids must account for at
leastone third the total absorption in this region! It thus appears as if
the chlorophyll fluorescence can be excited, with considerable probability,
also by the light absorbed by carotenoids! This hypothesis has been
strikingly confirmed by experiments with fucoxanthol-containing diatoms.
The results of these experiments, carried out by Dutton, Manning and
Duggar (1943), are shown in Table 24. III. They indicate that the yield of
chlorophyll fluorescence is the same, whether it is excited by red light, ab-
sorbed exclusively by chlorophyll, or by blue-green light (470 mû), three
quarters of which probably is absorbed by carotenoids, mainly fucoxan-
thol (see fig. 22.46 and figs. 30.9B and C. Table 24.III also contains
new results with Chlorella, which confirm the conclusions drawn above from
the earlier work of the Dutch observers. These results indicate that the
light absorbed by carotene and luteol is almost —
but not quite as ef-—
ficient in the excitation of chlorophyll fluorescence in green algae as the
lightabsorbed by fucoxanthol in diatoms. In striking contrast is the
result of the experiment withan acetonic extract from Nitzschia here, light
;
absorbed by the carotenoids is completely lost for fluorescence. The ex-

periment with acetonic solutions of chlorophylls a and h shows that the
quantum yield of chlorophyll fluorescence in solution does not increase with
SENSITIZED FLUORESCENCE IN VIVO 815
the wave length of exciting Hght between 436 and 578 mû. This proves
that an increased yield of nonsensitized fluorescence of chlorophyll in violet
light cannot be offered as alternative explanation of the results of the first
two experiments. Excitation transfer from carotenoids to chlorophyll

might become possible in sufficiently concentrated solutions; we men-
tioned on p. 790 that Duysens (1951) reported excitation transfer from
chlorophyll b to chlorophyll a in 10 ~^ M solution in acetone.
Table 24. III. Direct and Carotenoid-Sensitized Fluorescence of Chlorophyll
IN Diatoms (after Dutton, Manning and Duggar 1943)
carotenoids) at 436 mju, does not excite the fluorescence phycoerythrin or

phycocyanin (cui-ve a). Light absorbed mainly by phycoerythrin, on the
other hand, excites the fluorescence not only of both phycobilins, but also
of chlorophyll curves a, h, c. The hump on the red side of the chlorophyll
a band probably is due to chlorophyll d {cf. below).
Duysens' (1951) fluorescence spectra of Porphyra lacineata (fig. 24.5)
indicate that after excitation with 420 ra/x (absorbed by chlorophyll a and
the carotenoids) over 90% of excitation energy is transferred to an "un-
known pigment" (probably chlorophyll d); less than 10% of total fluo-
rescence is emitted by chlorophyll a, and only a negligible proportion by
phycocyanin. Excitation with 546 mju (light absorbed mainly by phyco-
erythrin) causes strong fluorescence of both phycocyanin and chlorophyll
a, and a comparatively weak fluorescence of "chlorophyll d." Duysens
interpreted these results as indicating the existence, in red algae, of two
kinds of pigment complexes: the largest part of chlorophyll a he suggested,
must be coupled with chlorophyll d, and transfer practically all excitation
energy to the latter pigment, although it is present in such a small amount
as to be hardly noticeable in the absorption spectrum at all {cf. p. 812).
A small part of chlorophyll a, not coupled with chlorophyll d, appears to
be associated with the phycobilins, and serves as ultimate recipient of the
major part of quanta absorbed by them. French's results indicate that
the extent of energy "leak" into chlorophyll d must vary Avidely from
species to species. In chapter 29, this hypothesis will be tied up with the
results of quantum yield determinations by Haxo and Blinks, who noted
a low photosynthetic efficiency of light absorbed by chlorophyll in some
red algae. In chapter 32, we shall explore whether the paradoxical fact
that fluorescence of chlorophyll a can be excited more strongly by light
absorbed by phycobilins than by light absorbed by chlorophyll a itself,
could be explained without Duysens' assumption of tAvo different pigment
complexes in red algae.
The general rule, indicated by the above-described fluorescence experi-
ments, is that plant cells contain one (sometimes, perhaps, two) pigment
complex energy absorbed by any one component tends to
in Avhich light
flow into the component with the lowest excitation level, and is therefore
remitted mainly as fluorescence of the latter — even if it is present in a very
low relative concentration.
This picture is supported by Duysens' observations (p. 810) that all
energy absorbed, in purple bacteria, by some carotenoids or by different

forms of bacteriochlorophyll, flows into the form of bacteriochlorophyll
that has the lowest excited level.
On page 801, we mentioned the increase in the intensity of the phyco-
bilin fluorescence observed by Arnold and Oppenheimer (1950) upon
:
EFFECTS OF HEAT AND HUMIDITY 817
breaking Croococcus under water, and their interpretation of this effect

cells
as a consequence of suppression of energy transfer from excited phycobilin

molecules to chlorophyll molecules, caused by dilution.
If we assume that all chloroph^dl in the cells is present in the same form,
then a fluorescence yield ip means the shortening of the normal life-time of

the excited state, to, to r = ^ro. In the case of chlorophyll in ChloreUa,
assuming ro = 8 X 10~^ sec. (page 634), we obtain:
(24.1) T = (1.5 to 3 X 10-3) X 8 X IQ-^ = (1.2 to 2.4) X lO-" sec.
and in the case of bacteriochlorophyll in Chromatiuni, assuming the same

value of To
(24.2) r = 7 X 10-3 X 8 X 10-8 = 0.6 X iq-io sec.
Two factors may determine the life-time of excited chlorophyll mole-

cules in live cells, and thus account for the above-calculated small values of
t: "normal" energy dissipation in the pigment-protein-lipide complex
(chloroplastin) and quenching (or stimulation) of fluorescence by metabolic
processes. The latter phenomena may themselves be of two kinds direct :
"photochemical quenching" by competition between sensitized photo-

chemical reaction and fluorescence, and indirect quenching (or stimulation)
of fluorescence due to the metabolic formation of substances that diminish
(or enhance) the fluorescence of chlorophyll. Observations described in
section 3 can be interpreted as revealing changes in the general structure
of the chloroplastin complex, while in section 4 —and, in more detail, in
chapters 27, 28 and 33 —we will discuss variations in intensity of fluores-
cence closely associated with participation of chlorophyll in photosynthesis.
3. Effects of Heat and Humidity on Chlorophyll Fluorescence in Vivo
has been found that, when chloroplast sediments (Noack 1927) or

It
(Seybold and Egle 1940) are placed in hot water, their fluorescence
live leaves
vanishes almost immediately; at the same time, the red absorption band
isshifted toward the shorter waves. This transformation occurs at a tem-
perature of 64-72° C. If the leaves are kept in hot water for several
minutes, fluorescence reappears, but the absorption band remains in the
siiifted position. Metzner (1937) probablj^ dealt with the same phenom-
(4ion when he described a "burst" of fluorescence caused by heating the
chloroplasts under the fluorescence microscope.
According to Seybold and Egle (1940), drying extinguishes the fluores-
cence of fresh leaves, but not that of leaves killed l)y boiling. The fluores-
:;
cence of some plants is highly sensitive even to minor changes in humidity
for example the fluorescence of Pleurococcus colonies on wood bark van-

ished after one or two hours in an atmosphere of less than 80% relative
humidity (at 25° C.) while the fluorescence of Mnium pundatum disap-
;
peared when the humidity declined below 85%. The fluorescence of the
leaves of Adiatum and Paretaria was found to be somewhat less sensitive,
but it, too, ceased to be visible after one or two days in an atmosphere of
75% relative humidity.
The fluorescence of sharply dried leaves cannot be restored by simple
wetting, but returns upon immersion into boiling water. A similar trans-
formation of the "sensitive" fluorescence of live cells into the "stable"
fluorescence of dead cells can be achieved by freezing or immersion into
ether. In the latter case, the fluorescence after the treatment is consider-
ably stronger than it was in the living state.
Seybold and Egle interpreted these results as indication that practically
all chlorophyll in leaves is present in a nonfluorescent (probably, protein-
bound) state, but that a small fraction of the pigment is dissolved in a
lipide phase, and therefore capable of fluorescence. They suggested that,
upon drying, the fraction of chlorophyll normally present in the lipide
phase is transferred into the colloidal aqueous phase, while, upon heating,
chlorophyll is first extracted from the lipide phase into the colloidal pro-
teinaceous phase (thus causing the fluorescence to disappear), but later
returns into the lipophilic material (concomitantly with the denatura-
tion of the proteins and melting of lipides) and thus again becomes fluores-
,
cent. (Metzner 1937 also had attributed the "burst" of fluorescence

caused by heating to the melting of the lipides.) Underlying this "two-
phase" hypothesis of Seybold and Egle was the conviction that all chloro-
phyll-protein complexes are nonfluorescent. However, while this seems
to be true enough of pure chlorophyll-protein precipitates (cf. page 775),
it does not apply to complexes which contain both proteins and lipides
(e. g., to "coacervates" of the type described by Hubert and Frey-Wyssling
cf. chapter 23, page 777). Seybold and Egle's argument is therefore not
convincing. The effects of heating and drying on chlorophyll fluorescence
in vivo can be explained in a much simpler way than suggested by Seybold
and Egle by assuming that the pigments normally contained in a weakly
:
fluorescent protein-chlorophyll-lipide complex lose the protection against

self-quenching (and therefore become nonfluorescent), when the lipides
melt in the heat and form a separate phase, but diffuse into this new phase
if the pigment-protein link is broken by denaturation {e. g., by somewhat
more prolonged heating) and thus again become fluorescent. The dis-
placement of the fluorescence bands of chlorophyll in living cells (by 5-15
mjLi toward longer waves from their position in organic solvents) agrees
with the assumption that fluorescence is emitted by the same chlorophyll

molecules responsible for the (similarly displaced) absorption bands.
VARIATIONS OF CHLOROPHYLL FLUORESCENCE 819
Seybold and Egle, on the other hand, had to attribute the fluorescence
bands to the fraction of chlorophyll dissolved in a lipide, and the absorp-
tion bands to the bulk of chlorophyll present in a protein-bound colloidal
state. Therefore their theory was predicated on the contention that the
fluorescence band of chlorophyll is shifted in lipides toward the longer
waves much more strongly than the corresponding absorption band. This
hypothesis was characterized as implausible on page 746.
To sum up, there seems to be no reason to attribute the fluorescence of
living plants to a small fraction of chlorophyll molecules, present in a
strongly fluorescent solution, rather than to the whole mass of the pigment
forming a weakly fluorescent complex with proteins and lipides (including
the carotenoids) . The close relationship between fluorescence intensity and
rate of photosynthesis, which will be discussed in the next section, also
indicates that fluorescence is a property not of a small fraction but of the
bulk of chlorophyll in the cell.
4. Variations of Chlorophyll Fluorescence Related to Photosynthesis
In the preceding section, we discussed chlorophyll fluorescence in vivo

in relation to what may be called the gross state of the green pigment in the
living cell — its high concentration and its simultaneous association, in the
"chloroplastin," \\ith proteins and lipides. The effects of drying, heating,
boiling or immersion in ether, described in that section, can be assumed to
be indicative of a partial or complete disintegration of the chloroplastin.
In the present section, we will deal with reversible changes in the yield
of fluorescence that are more or less closely associated with photosensitizing
activity and can be assumed to occur without essential changes in the com-
position and structure of chloroplastin.
Kautsky discovered in 1931 that rapid changes in the intensity of
fluorescence of leaves occur during the first seconds and minutes of illum-
ination after a period of darkness, and bear definite relation to the pre-
viously known changesof the rate of photosynthesis during this "induc-
tion period." Subsequent investigations by Kautsky and co-workers
(1931-1948), Franck and co-workers (1934-1949), McAhster and Myers
(1940) and of the Dutch group of investigators (Ornstein, Wassink, Katz,
Dorrestein et al. (1937-1949) have revealed many striking examples of close
interrelation between the intensity of fluorescence and the momentary
rate of photosynthesis. This relationship can be observed not only dur-
ing the induction period, but also in the steady state. Factors such as
light intensity, temperature, concentration of reactants that take part in
photosynthesis, presence of oxygen and various poisons and narcotics
are found to affect significantly the yields of both fluorescence and photo-
S3m thesis.
The close interrelation of the fluorescence of chlorophyll and its photo-
.
sensitizing activity (revealed through parallel measurements of the yields

of photosynthesis and fluorescence) has made fluorescence measurements
an important tool in the kinetic analysis of photosynthesis. We will
therefore restrict ourselves in the present chapter to some general considera-

tions of this relationship, postponing more detailed description of experi-
mental results and their interpretation to the several chapters in part IV
dealing with the effects of light intensity, temperature, carl)on dioxide and
other external factors, on the kinetics of photos^mthesis.
Fluorescence is one of the several ways in which excited chlorophjdl
molecules can dispose of their energy. Others include energy dissipation
(conversion into vibrational energy and ultimately into heat), and photo-
chemical reactions (either involving the chlorophyll molecule itself, or sen-
sitized by it). The intrinsic capacity of the excited chlorophyll molecule
to fluoresce (the monomolecular fluorescence constant kf, or its reciprocal,
the ''natural life time" of the excited state, t/) can be considered as con-
stant as long as the absorption spectrum of the chlorophyll molecule re-
mains essentially unchanged. The intrinsic capacity for energy dissipa-
tion (the monomolecular dissipation constant kt) and the rate of energy loss
through chemical reactions (rate constants ki, k^. , which can be mono-
molecular or bimolecular) are, on the other hand, subject to changes de-
pending on the association of the chlorophyll molecule with other molecules
before excitation, and on its encounters with other molecules during excita-
tion (as discussed in the sections of chapter 23 dealing with the quenching
and self-quenching of chlorophyll fluorescence in vitro). The variations of
chlorophyll fluorescence in vivo associated with variations in the rate of
photosynthesis must therefore be attributed to changes in the composition
or structure of the chlorophyll-bearing molecular complex (and consequent
alterations in the values of monomolecular constants of dissipation and
chemical quenching), and to changes in the probability of the chlorophyll
complex encountering, during the excitation time, molecules capable of
serving as effective "physical" or "chemical" quenchers (and consequent
alterations in the values of bimolecular constants of quenching)
The several more or less detailed interpretations of fluorescence changes
in photosynthesizing plants, which have been suggested, all are based on
these general ideas but differ in emphasis laid on one or the other specific
mechanism of quenching. Some (Kautsky; Wassink and Katz) attribute
the main function to "chemical quenching" by the reactants taking part
in photosynthesis, and consider each increase in fluorescence as evidence of
a decrease in the efficiency of the sensitized photochemical process (and
consequent decline of chemical quenching), and each decrease in fluores-
cence as evidence of increased efficiency of utilization of excitation energy
for the sensitized photochemical reactions (and consequent increase of
chemical quenching). Others (Franck) see the most important cause of
:
CHLOROPHYLL FLUORESCENCE AND PHOTOSYNTHESIS 821
changes in fluorescence intensity in the formation of chlorophyll complexes

with surface-active substances ("narcotics") which slow down energy dis-
sipation, and at the same time inhibit photochemical sensitization by pre-
venting photosensitive substrates from reaching the chlorophyll. This
amounts to a weakening of both processes (sensitization and dissipation)
which compete with fluorescence whereas in theories of the first-mentioned
;
type, onl}' one competing process (sensitization) is affected, while the other
two (dissipation and fluorescence), profit equally by the elimination of a
common competitor.
We will now describe more specifically the several suggested mechanisms
of interrelation of fluorescence and photosynthesis beginning with the pic-
ture used in Volume 1 (chapter 19).

In scheme 19. Ill (Vol. I, page 547) we attempted to represent the prob-
able relationship between sensitization and fluorescence of chlorophyll in
vivo. This scheme was formulated primarily for the interpretation of
sensitized photoxidation, but essentially similar conditions may be as-
sumed to prevail in photosjaithesis as well. The primary process was as-
sumed in chapter 19 to be a "tautomerization" of the complex X. Chi. HZ
(formed bj- association of chlorophyll with oxidant X and reductant HZ)
(24.3) X-Chl-HZ ^X-Chl*-HZ > HXChl-Z
In photosynthesis, this primary process must be followed by secondary,

catalytic reactions, in which HX is oxidized back to X (directh' or indi-
rectly) by the carbon dioxide-acceptor compound, {CO2} *, and Z is reduced
back to HZ (directly or indirectly) either by water (in ordinary photosyn-
thesis of green plants) or by reductants such as H2, H2S or thiosulfate (in
the photosynthesis of purple bacteria).
In this picture, variations in fluorescence can be related to those
in photosynthesis in both the above-mentioned ways by means of —
primary changes in the probabihty of sensitized chemical reaction, and
by means of primary change in the rate of dissipation of energy in the
chlorophyll-bearing complex. If the dissipation rate is constant,
fluorescence an indicator of the efficiency with which the excitation en-
is
ergy of chlorophyll is used for the primary photochemical process (equation

24.3) Whenever the latter process is retarded for one reason or another,
:
the sum of the probabilities of the two competing processes fluorescence —

and internal dissipation of the excitation energy increases correspond- —
ingly. Since fluorescence and internal energy dissipation are two alterna-
tive monomolecular processes, the yield of both will be changed in the same
proportion. Fluorescence thus becomes an index of the yield of the pri-
mary photochemical process, even though the absolute yield of fluorescence
* ir X = {CO2 , the secondary reaction is the replacement of a reduced by a
fresh molecule i CO2 !
•
—
( < 1%) is much too small to make it a significant competitor of this process.
For example, if the yield of the primary photochemical process drops from
80 to 40%, and the sum of the yields of dissipation and fluorescence there-
fore increases from 20 to 60%, the yield of each of these two processes will
increase by a factor of 3. If the yield of fluorescence was <p = 0.2% be-
fore it will become 0.6% afterward.

The occurrence of antiparallel changes of the yields of fluorescence and
photosynthesis was discovered by Kautsky in his investigations of the in-
duction period (for an example, cf. fig. 33.19A), and we now know that this
type of correlation is quite common, both in induction phenomena and in
the steady state. However, the relation between the yields of photosyn-
thesis and fluorescence is not always that of antiparallelism. Sometimes,
yield of photosynthesis changes strongly without an appreciable change
in 3deld of fluorescence (cf., for example, fig. 28.24 in which "light satura-
—
tion" of photosynthesis has no counterpart in the steadily increasing-
intensity of fluorescence).In other cases, e. g., in some types of induction
phenomena, photosynthesis and fluorescence both change in the same di-
rection (cf. fig. 33.22C). The picture of the mechanism of photosynthesis
used above to explain the usual antiparallehsm of photosynthesis and
fluorescence can, however, be used also to explain how exceptions from this
antiparallelism can arise.
In the first place, fluorescence competes only with the primary photo-
chemical reaction not \vith the over-all process of photosynthesis. The
rate of photosynthesis, as measured by the liberation of oxygen or consump-
tion of carbon dioxide, often is determined, not (or not only) by the ef-
ficiency of the primary photoprocess, but also by the rate of one or several
of the associated dark, catalytic reactions. Among these are reactions
that convert the primary photoproducts into the stable end products of
photosynthesis. AVhen these "finishing" reactions are too slow to keep
pace with the primary photochemical process (a situation that may arise,
for example, in excessively strong hght, or at low temperature, or in the
presence of certain poisons), the primary photoproducts will accumulate to
a certain extent, but will then disappear by back reactions. The quantum
yield of photos^mthesis will thus be reduced, but that of fluorescence need
not be affected at all, since the primary photochemical process which —
—
alone competes with fluorescence continues at full speed. This can
explain the occurrence of light saturation of photosynthesis without simul-
taneous increase in the yield of fluorescence (a phenomenon to which we
have referred above).
In Volume I (cf., for example, chapter 7) we have considered, in addition
to"finishing" dark reactions (which, as just stated, are likely to have no effect
on fluorescence at all), also catalytic reactions of "preparatory" character.
such as the binding of carbon dioxide by an "acceptor," to form a compound

designated as {CO2}. These reactions "prepare" the reactants for their
participation in the photochemical reaction proper. If one of the prepara-
tory reactions ceases to keep pace with the primary photochemical process
(equation 24.3), the conversion of the primary photoproduct HX-Chl-Z
back into the photosensitive form X- Chi -HZ will be retarded. If the
supply of the oxidant, {CO2}, is too small, while that of the reductant,
{H2O} (or of substitute reductants in bacterial metabohsm), is ample,
the chlorophyll-oxidant-reductant complex may
accumulate in the re-
duced form, HX- Chl-HZ. If is ample, but that
the supply of the oxidant
of the reductant is limited (a situation which can easily be realized in experi-
ments with bacteria), the complex will accumulate in the reduced form,
X Chi HZ Both forms are stable, because they cannot be transformed into
• •
.
the photosensitive form X- Chi- HZ by simple back reaction, and photostahle

because they cannot undergo the primary photochemical process (equation
24.3) The accumulation of either of them is likely to enhance fluorescence.
.
It seems, in fact, that all factors (such as COo-starvation or cyanide poison-

ing) which limit severely the carboxylation reaction CO2 -^ [CO2] normally
increase the yield of fluorescence, and that the same is true of the factors
limiting the supply of the reductant (H2, H2S or thiosulfate) in purple bac-
teria.
Considerations of this kind could explain why light saturation of
photosynthesis is accompanied by an increase in the yield of fluorescence
in some cases (namely, when saturation is caused by the limited velocity of
a "preparatory" reaction), and has no effect on the jdeld of fluorescence in
others (namely, when it is due to the limited rate of a "finishing" reaction).
Closer consideration of the picture also could explain why, when changes
in the yield of photosynthesis are correlated with changes of fluorescence,
not only does the absolute extent of the latter vary within wide limits, but
sometimes, even the sign of the effect is reversed, the usual antiparallelism
being replaced by a parallel increase (or decrease) of the quantum yields of
both photosynthesis and fluorescence:
Fluorescence and internal dissipation are affected in the same propor-
tion by a change in the rate of the primary photoprocess only if the factor
that caused this change does not affect the rate constant of internal dissipation.
There is no reason why this should always be true. The a priori prob-
ability (rate constant) of internal conversion may be quite different in
the complexes X- Chi -HZ, HX-Chl-Z, HX-Chl-HZ and X-Chl-Z. If
one of the "photostable" complexes, such as HX-Chl-HZ, dissipates

the excitation energy much more efficiently than the "photosensitive"
complex X- Chi -HZ, the fluorescence-quenching effect of its accumulation
ma}^ overcompensate the fluorescence-stimulating effect of the suppression
of theprimary photochemical process, and the net result will be a simul-

taneous decline in the yields of both fluorescence and photosynthesis.
(In other words, fluorescence, freed of one of its two competitors the —
—
primary photoprocess will face a stronger second competitor internal —
dissipation —and will suffer a net loss.) The energy-dissipating properties
of the chlorophyll-containing complexes may differ somewhat in different
species and even strains (otherwise, the yield of fluorescence would be ex-
actly the same in all plants) ; this may explain why the rationing of carbon
dioxide (or outright starvation) apparently has a different effect on fluores-
cence in leaves (investigated by Franck, French and Puck 1941 and Mc-
Alisterand Myers 1940), purple bacteria (investigated by Wassink, Katz
and Dorrestein 1942) and diatoms (investigated by Wassink and Kersten
1944). In the first case, denial oi carbon dioxide caused a considerable
increase of (p at high light intensities {cf. figs. 28.25, page 1048); in the
second case, it caused a slight increase of (p at moderate intensities and a
decrease at high intensities (fig. 28.30) in the third case, (p declined in
;
strong light in the presence of carbon dioxide, but remained constant in the
absence of carbon dioxide (fig. 28.28).
The primary photochemical process can be retarded not only by a defi-
ciency of reactants, {CO2} or thiosulfate, which are needed to restore

e. g.,
the photosensitive complex, but also by narcotization, that blankets the

complex and prevents its contact with the reactants. In this case, too,
one can expect a simultaneous effect on the probability of internal dissipa-
tion —this time in the direction of making the dissipation slower. Narcotics
are therefore likely to enhance fluorescence in a twofold way by prevent- :
ing "chemical quenching" by the primary photochemical process, and by

exercising a "protective" action of the type discussed in chapter 23, (page
776), i. e., by "wrapping in" the excited molecules, and weakening in this
way all energy-dissipating interactions with neighboring molecules.
According to Franck et al. (1941,1947,1949), formation of protective
"narcotic" layers can be brought about by supplying plants externally
with substances such as chloroform or urethan, and also by metabolic
—
reactions particularly those occurring under anaerobic conditions. Such
"internal" narcotization effects are supposed by Franck and co-Avorkers to
be responsible for the induction effects after an extended period of darkness
(see later in chapter 33) , for the inhibition of photosynthesis by anaerobic
incubation (cf. chapter 13, Vol. I, and chapter 33 in Part 2) and for the
"midday depression" (cf. chapter 26, page 873). In all these cases, cessa-
tion or retardation of photosynthesis is accompanied by enhancement of
fluorescence, and the effect on fluorescence often is considerably stronger
than could be attributed to the limitation of the primary photochemical
process.
In the case of inhibition of photosynthesis by anaerobic incubation in

the dark, the "narcotic" appears to be a fermentation product, an acid,
since its effect can be destroj'ed by neutrahzation. The "long" induction
period, and the midday depression, maj^ be due to the accumulation of
similar acids. The "short" induction period, on the other hand, must be
associated more directly with the intrinsic mechanism of photosynthesis.
It has been suggested (by Gaffron and Franck, see chapter 33) that, in
this case, a "narcotic" is produced as a result of partial oxidation of a
metabolite (sugar?) by the first oxidation product of photosynthesis
("photoperoxide" or, more generally, "oxj^gen precursor"); according to
Gaffron and Franck, a transient accumulation of this product occurs in the
first seconds of illumination, because of inactivation in the dark of the
enzjTne (or enzymes, which we called Ec and £J„ in chapter 7), required to
convert the oxygen precursor into free oxygen. Franck attributes to
similar "internal narcotization" also most, if not all, the fluorescence
changes produced by depriving purple bacteria of their specific reductants
(H2, H2S2O3, etc.). He sees the reason for the enhancement of fluorescence
caused by this treatment, not (or not primarily) in the stoppage of the
primar}^ photochemical process by lack of a reactant (and consequent in-
crease in both internal dissipationand fluorescence as suggested on p. 823),
but in the accumulation of photoperoxides (which in the normal course
of bacterial photosynthesis, are destroyed by the specific reductants), and
consequent production and deposition on chlorophyll of surface-active
(narcotic) substances. In this way, the rates of both the primary photo-
chemical process and the internal dissipation are reduced, and the chances
of fluorescence are correspondingly increased.
The effect on the yield of fluorescence of the deprivation of green plants
(or purple bacteria) of carbon dioxide (or of cjânide poisoning — both
treatments have the same primary result, namely reduction in the supply
of CO2 to the photochemical system) is less pronounced than the effect
{ 1
of the scarcity of the reductants; but, contrary to the belief of Wassink

and his co-workers, such an undoubtedly occurs (c/. chapter 27, p.
effect
940. Here, again, the enhancement of fluorescence could be attributed
either to declining rate of the primarj^ photochemical process (and conse-
quent increase in the probability of both internal dissipation and fluores-
cence) or (as suggested by Franck and co-workers) to the formation of
;
an int(nnal "nar(-otic" by pfwloxidalion, which is known to set in when

plants deprived of carbon dioxide are exposed to light. In anaerobic
purple bacteria, photoxidation cannot occur and CO2 has less effect on
fluorescence.
Franck sees in the formation of a protective layer of an internal narcotic
an important safety device the plants have developed to prevent destruc-
tive photochemical reactions from being sensitized by chlorophyll, when
the absence of reactants, or poisoning, prevents the use of absorbed energy

for constructive purposes (z. e., for photosynthesis).
While Franck's theory emphasizes the indirect mechanism of fluores-
cence changes associated with photosynthesis, the Dutch group (see
Wassink, Katz et at. 1938, 1942, 1949) use the simple concept of competi-
tion between the primary photochemical process and fluorescence (+ dis-
sipation). Because of the stronger effect on (p, in purple bacteria, of the
presence or absence of reductants (as compared to the presence or absence
of the oxidant, CO2) they assume that the primary process involves ex-
cited chlorophyll and the (enzymatically "prepared") reductants, but
does not involve CO2 (or the CO2} -complex). I
There is no doubt that simple competition between fluorescence and

photochemical primary process is insufficient to interpret all the experi-
mental evidence. Franck's concept of indirect action, on the other hand,
has been successfully applied to a wide range of phenomena and has thus
acquired a considerable degree of probability.
Wewill end this discussion here and leave the more detailed presenta-
tion and interpretation of experimental results to chapters 27-33 in part
IV, dealing with the kinetics of photosynthesis. Measurement of fluo-
rescence has become a tool in the study of the reaction kinetics of photo-
synthesis, and it would be inconvenient to separate the discussion of the ef-
fects of light intensity, or temperature, or carbon dioxide concentration on
the yield of fluorescence, from the discussion of the influence of the same
factors on the yield of photosynthesis.
Fluorescence of Pigments in Vivo
1852 Stokes, G. G., Trans. Roy. Soc. London, 1852, 463.

1862 Simmler, R. T., Ann. Phys. {PoggendorJ), 115, 593.
1867 Askenasy, E., Botan. Z., 25, 225.
1870 Hagenbach, A., Ann. Phys. {PoggendorJ), 141, 245.
1871 Lommel, E., ihid., 143, 568.
1872 Hagenbach, A., ibid., 146, 508.
1874 Hagenbach, A., ibid., Jubilee Vol., 303.
Engelmann, Th. W., Arch. ges. Physiol. Pflilgers, 32, 80.

1911 Tsvfett,M., ibid., 29, 744.
1913 Buder, J., ibid., 31 (80).
1914 Lelnnann, H., Z. iviss. Mikroskop.. 30, 417.
Wilschke, A., ibid., 31, 338.
Gicklhorn, J., Sitzber. Akad. Wiss. Wien, Abt. I, 123, 1221
1920 Stern, K., Ber. deut. botan. Ges., 38, 28.
1921 Stern, K., Z. Botan., 13, 193.
1923 Lloyd, F. E., Science, 58, 91, 229.

Lloyd, F. E., Nature, 112, 132.
Lloyd, F. E., Trans. Roy. Soc. Can., (Ill) 17, 129.
1924 Lloj'd, F. E., Science, 59, 241.
1927 Noack, K., Biochem. Z., 183, 135.
Testi Dragone, G., Atti reale accad. naz. Lincei [VI], 6, 179.
1930 Klein, G., and Linser, H., Osterreichische boton. Z., 79, 125.
1931 Kautsky, H., and Hirsch, A., Natunnssenschaften, 19, 964.
Dhere, C., and Fontaine, M., Compt. rend. soc. biol, 107, 1098.
Dhere, C., and Fontaine, M., Ann. inst. ocmnogr., 10, 245.
Bachrach, E., and Dhere, C., Compt. rend. soc. biol, 108, 385.
1932 Kautsky, H., Hirsch, A., and Davidshofer, F., Ber. deut. chem. Ges., 65,
1762.
1934 Kautsky, H., and Hirsch, A., Biochem. Z., 274, 423.
Kautsky, H., and Spohn, H., ibid., 274, 435.
Fontaine, M., Revue algologiqiie, 7, 189.
Baas-Becking, L. G. M., and Koning, H. C., Proc. Acad. Sd., Amsterdam,
37, 674.
Franck, J., and Levi, H., Z. phys. Chem., B27, 409.
Kautsky, H., and Hirsch, A., ibid., 278, 373.
Dher^, C., and Raffy, A., Bull. soc. chim. biol, 17, 1385.
Dh6r^, C., and Raffy, A., Compt. rend., 200, 1146.
Dhere, C., and Raffy, A., Compt. rend. soc. biol, 119, 232.
1936 Kautsky, H., and Marx, A., Naturwissenschaften, 24, 317.
Kautsky, H., and Flesch, W., Biochem. Z., 284, 412.
Dhere, C., and Biermacher, 0., Compt. rend., 203, 412.
Biermacher, 0., Thesis, Univ. Fribourg (Switzerland).
Kautsky, H., and Hormuth, R., Naturwissenschaften, 24, 658.
Franck, J., and Wood, R. W.^ /. Chem. Phys., 4, 551.
1937 Kautsky, H., and Marx, A., Biochem. Z., 290, 248.
Kautsky, H., and Hormuth, R., ibid., 291, 285.
Dhere, C., La fluorescence en biochimte. Paris, 1937.
Metzner, P., Lecture before Botanical Society Darmstadt, quoted from
Frey-Wyssling, Submikroskopische Morphologic des Protoplasmas, 1938.

Vermeulen, D., Wassink, E. C., and Reman, G. H., Enzymologia, 4, 254.
Mecke, R., and Baldwin, W. C. G., Naturwissenschaften, 25, 305.
1938 Wassink, E. C., Vermeulen, D., Reman, G. H., and Katz, E., Enzymologia,
5, 100.
Ornstein^ L. S., Wassink, E. C., Reman, G. H., and Vermeulen, D.,
Enzymologia, 5, 100.
Kautsky, H., and Eberlein, R., Naturivissenschaften, 26, 576.
1939 Dh^re, C., Fortschr. Chem. org. Naturstoffe, 2, 301.
Wassink, E. C., and Katz, E., Enzymologia, 6, 145.
1940 Seybold, A., and Egle, K., Botan. Arch., 41, 578.
McAlister, E. D., and Myers, J., Smithsonian Inst. Pub. Misc. Collections,
99, No. 6.
1941 Kautsky, H., and Zedlitz, W., Naturwissenschaften, 29, 101.

Franck, J., and Herzfeld, K. F., /. Phys. Chem., 45, 978.

Franck, J., French, C. S., and Puck, T. T., ihid., 45, 1268.
1942 Franck, J., Am. J. Botanij, 29, 314.
Wassink, E. C, Katz, E., and Dorrestein, R., Enzymo ogia, 10, 269.
Dorrestein, R., Wassink, E. C, and Katz, E., ibid., 10, 355.

1943 Button, H. J., Manning, W. M., and Duggar, B. M., J. Phys. Chem., 47,
308.
Kautsky, H., and Franck, U., Biochem. Z., 315, 139, 156, 176, 207.
1945 Wassink, E. C, and Kersten, J. A. H., Enzymologia, 11, 282.
1947 Shiau, Y. G., and Franck, J., Arch. Biochem., 14, 253.
1948 Kautsky, H., and Franck, U., Nature, 35, 43, 74.
Van Norman, R. W., French, C. S., and Macdowall, F. D. H., Plavt
Physiol, 23, 455.
1949 Franck, J., "The Relation of Fhiorescence in Plants to Photosynthesis,"
in Photosynthesis in Plants, Iowa State College Press, Ames, 1949, p.
293.
Katz, E., "Chlorophyll Fluorescence as Energy Flow Meter in Photosyn-
thesis," ibid., p. 287.
1950 Arnold, W., and Oppenheimer, J. R., J. Gen. Physiol., 33, 423.
1951 French, C. S.,and Koski, V. M., Proc. Soc. Exptl. Biol, (in press).
Duysens, L. N. M., Nature (in press).
PART FOTTR
KINETICS OF PHOTOSYNTHESIS
INTRODUCTION
The investigation of reaction kinetics, i. e., of the dependence of rate

on various external factors, has acquired a greater importance in the study
of photosynthesis than in that of other biochemical processes. Faced with
the failure of the usual qualitative methods of biochemistry to disentangle
the complex mechanism of photosynthesis, many investigators turned to
rate measurements, hoping that the number, sequence and character of
the partial processes involved in photosynthesis could perhaps be derived

from such studies. The results did not quite satisfy expectations, for
reasons not difficult to understand. What one measures as the "yield of
photosynthesis" isthe net result of the operation of a complex mechanism.
No simple kinetic equation can account for all the factors that influence
this yield. It is comparatively easy, after having made a series of kinetic
measurements on a selected object, to invent a model that would interpret
these particular observations, or even to write down equations fitting the
experimental results more or less closely. The literature on photosynthesis

abounds in such formulations. Their limited significance is illustrated by
the fact that practically nobody ever uses equations derived by somebody
else; instead, every investigator starts anew, often without as much as
referring to his predecessors, and —what is —
even more unfortunate without
making an attempt to correlate his formulae with any experimental results
but his own.
Photosynthesis is such a complex and heterogeneous process that it is
probably impossible to make a complete analysis of its mechanism merely
by measuring the rate of the over-all process under different conditions.
However, this does not mean
that kinetic measurements of photosynthesis
are useless but rather that they are most useful when combined with other
;
biochemical and biophysical methods of approach, such as the use of poisons

and narcotics, provision of substitute reductants and oxidants, or partial
tearing down of the photosynthetic apparatus (e. g., by the separation of

chloroplasts from cytoplasm). Several investigators have already used
measurements an auxiliary
of the intensity of chlorophyll fluorescence as
physical tool to supplement chemical rate measurements; it may prove
possible to make similar use of absorption and fluorescence spectra, of
radiochemical and perhaps also electrochemical measurements, to provide
"running comment" on the state of the photosynthetic apparatus during
831
832 INTRODUCTION TO PART FOUR
photosynthesis. In such an integrated study, kinetic measurements will

remain one of the most important components; but the ambition to ob-
tain, by their means alone, a quantitative interpretation of the whole
phenomenon of photosynthesis will be replaced by more limited aims.
.
Chapter 25
METHODS OF KINETIC MEASUREMENTS*

It is not our purpose to give here a detailed description and apprecia-
tion of the experimentalmethods used in the quantitative study of photo-
synthesis, but merely to indicate the ])rinciples on which these measure-
ments have been (or can be) based.
Photosynthesis by green plants consumes carbon dioxide, water and
light; it produces oxygen, carbohydrates and chemical energy. This gives
six possible objects of quantitative study. However, one of the reaction
— —
components water is present so abundantly in living organisms that the
determination of its consumption is practically impossible (except, per-
haps, by means of isotopic tracers). In the photosjmthesis of bacteria
and "adapted" algae (c/. Vol. I, chapters 5 and 6), on the other hand, the
consumption of the reductant (H2, HoS, H2S2O3 etc.) can be measured as
easily as that of the oxidant (CO2)
1. Material
Since Warburg's fundamental investigations (1919, 1920), unicellular

algae, particularly the two species Chlorella vulgaris and Chlorella pyrenoid-
osa, have become the favorite objects of quantitative studies, because of
the ease with which they can be cultured and handled in the form of sus-
pensions. The methods of culture of Chlorella and similar algae cannot be
discussed here reference must be made to special literature such as Kiister
;
(1921), Pringsheim (1924, 1926), Pearsall and co-workers (1937, 1940) and
Myers et al. (1944, 1946). One observation, hoMever, must be mentioned:
It has been found (Pratt and co-workers 1940 to 1945) that growing
Chlorella cultures produces a substance that acts as an inhibitor of further
growth (and incidentally also as an inhibitor of photosynthesis; cf. chapter
26, page 880).
Chlorella suspensions have provided the material for the studies by
Warburg and his school, and by Noddack and co-workers in Germany, as
well as by Emerson, Franck, Gaffron, Daniels, Manning and their co-work-
ers in America. The algae used are green, roughly spherical, unicelhilar
organisms, about 5 m in diameter, containing a single, bell-shaped chloro-
* Bibliography, page 85.5.
833
834 METHODS OF KINETIC MEASUREMENTS CHAP. 25
Table 25.1
Characteristics of Two Chlorella pyrenoidosa Suspensions
(after Noddack and Eichhoff 1939)
MATERIAL 835
})hyll) increased rapidly at first — in agreement with Noddack and Eich-

hoff's observations —and then approached constancy {cf. chapter 31 for
more detailed discussion of the relation of the rate of photosynthesis to
chlorophyll content).
Suspensions like those in Table 25.1, placed in vessels 1-3 cm. deep,
absorb 20-50% of incident white light. If complete absorption of visible
light is desired (as in Warburg and Negelein's procedure), suspensions
containing more than lO'^ cells per milliliter must be used in vessels of the
same depth.
The photosynthetic efficiency of Chlorclla suspensions has been related
to many factors, such as the intensity of light (Warburg) and the rate of
1.3
1.2
5
a>
1.0
(n 0.9
^ 0.8
0.7
0.6
0.5
0.4
steady; while, in a 0.1 M KHCO3 solution, there resulted an increase in

rate by about 30% within six hours, followedby several hours of steady
oxygen production and then a rapid decline. In a mixture of 0.035 M
KHCO3 and 0.065 M
NaHCOs, the rate remained unchanged for about
fifteen hours, after which the stimulating effect of the potassium salt ap-
parently wore off, and the rate declined rapidly (fig. 25.1). In all these
experiments, the cells spent about one half of the total time in light and
one half in darkness.
Among other algae used in quantitative photosynthetic studies were the
Chlorophyceae: Scenedesinus {cf., for example, Gaffron 1942), Hormidium
flaccidum (van der Honert 1930 and van der Paauw 1932), Stichococcus
hacillaris (Aufdemgarten 1939); the Rhodophycea, Gigartina harveyana
(Emerson and Green 1934) the Cyanophycea, Chroococcus (Emerson and
;
Lewis 1942); the brown alga, Fucus serratus (Steemann-Nielsen 1942);

and the diatoms, Nitzschia dosterium (Barker 1935, Button and Manning
1941), A^. dissipata (Wassink, Kersten 1945), Navicula minima (Tanada
1951).
Advantages of algae as material for photosynthetic measurements (e. g.,
the convenient use of manometric methods) are shared to some extent by
the higher aquatic plants. Among them, Elodea has been the most popular
in photosynthetic studies, its widespread occurrence in stagnant
because of
waters. Other aquatic plants used in photosynthetic work were Cahomba
caroliniana (Smith 1937) and Potomageion (Gessner 1937).
Detached leaves provided the material for most of the earlier investigations
of photosynthesis they were used by Blackman and co-workers, by Brown
;
and Escombe (1905) and by Willstatter and StoU (1918) in their pioneer
investigations of the quantitative aspects of photosynthesis. Because of
the interruption of natural translocation processes, the time course of
photosynthesis in detached leaves may differ from that in similar leaves
attached to the stem (Vol. I, p. 332).
Whole land plants, enclosed in glass vessels, were long used in investiga-
tions of the rate of photosynthesis under field conditions. A group of
workers at the Smithsonian Institution in Washington {cf. Hoover, Johns-
ton and Brackett 1933 and McAlister 1937) showed that this method can
give results equivalent, as to precision and consistency, to those derived
from experiments with algae. The material used in their studies were
single young wheat plants.
The culture of purple bacteria that can be used for studies of bacterial
photosynthesis has been described by van Niel and co-workers (1931,
1944), French (1937), Gaffron (1933-1935) and the Dutch group (Eyniers,
Wassink, Katz, Dorrestein et at. 1938, 1942) the bacteria used included
;
Rhodospirillvm rubrum, Streptococcus varians, and strains of Chromatium

and Rhodovihrio.
^
.
LIGHT MEASUREMENTS <S37
2. Light Measurements
111 many older investigations, the influence of light intensity on photo-

s\Tithesis was studied by illuminating the plants with "white light" (of the
sun, or of an incandescent lamp) and introducing gray filters, or altering
the distance between light source and plant. The intensity was then given
in relative units (e. g., "Ho of full sunlight" or "lamp at 30 cm. distance")
;
Other obsen'ers determined the intensity of illumination by visual com-

parison with a standard light source, and expressed it in meter candles
(also called lux, or lumen per square meter) or foot candles (1 foot candle =
10.764 meter candles). These figures cannot be used for the calculation of
the incident energij, unless the spectral distribution is known. A knowl-
edge of the so-called "color temperature" of the light source (the tempera-
ture that a black body must have to produce radiation of the same color)
provides some helpful information. However, no light source is a black
body; and, even if it were, the spectral distribution of the light it supplies
is modified by passage through air, glass or other material media. The
figures given below can therefore be used only for approximate calculations.
The maximum illumination from direct sunlight at noon in summer (at
sea level at the latitude 42° N) about 85,000 lux* (8000 foot candles),
is
its color temperature (cf. Tajdor and Kerr 1941), about 5400° K. Under
these conditions, the average light flux is 2.0 X 10"- cal. or 8.5 X 10^ erg/
(cm.^ sec.) (including the infrared) or 10 erg/(cm.^ sec. lux). The diffuse
light from the clear blue sky increases the illumination by about 21,0CG
lux (2000 foot candles). The sky light has a much higher color temperature
—
than the sunlight over 10,000° K. for clear blue sky, decreasing to 7000°
K. for hazy or overcast sky (see fig. 22.52). At sea level, when the sun is
in the zenith, about 40% of the incident radiation belongs to the spectral
region below 700 mju (photosynthetically active radiation) and about 60%
to the region above 700 m^ (photosjnithetically inactive, extreme red and
infrared radiation). Thus, in sunlight, one lux is equivalent to 4 erg/ (cm.
sec.) of pliotos\Titheticalh^ active light.
The characteristics of incandescent lamps (gas-filled tungsten filament
lamps) in table 25. TI are taken from Hardy and Perrin's The Principles
of Optics (1932).
About 30% of the lamp light below 760 mju belongs to the region 700-
760 m/x, which is scarcely used at all by green plants, so that the proportion
of photosynthetically active energyin medium power lamps
is only 7-8%
(100-200 watts) and 9-10% power lamps (500-1000 watts). In
in high
other words, an illumination of one lux from a 100 watt lamp corresponds
to about 5.0 erg/(cm.- sec.) and the same illumination from a 500 watt lamp,
to about 4.5 erg./(cm.2 sec.) of photosynthetically active light.
* We will use the abbreviation klux for 1000 lux.

Table 25.11
Characteristics of Incandescent Lamps
LIGHT MEASUREMENTS 839
tween these units is shown in Table 25. III. However, without information
or
as to spectral composition, the indication of the energy flux in ergs,
calories, is even less revealing than that of the intensity of illumination in
lux, because 60% of direct sunlight and about 95% of the energy flux from
incandescent lamps belong to the far red and infrared, and are not used
by plants for photosynthesis. Unless the proportion of these radiations is
known, quoting the energy flux may easily give an entirely erroneous con-
cept of the quantity of light available for photosynthesis.
sumed per quantum absorbed) can be obtained over a considerable range

of wave lengths and intensities. The convenience of this actinometer is
the possibility of using it in conjunction with Warburg reaction vessels in
a manometric sj^stem. Assuming equal absorption of light in the reaction
300 400 500 600 700

WAVE LENGTH, m/i
Fig. 25. 2A. Spectral sensitivity curve of a selenium barrier layer cell (Wes-
ton Photronic Cell).
20
.^ 16 G.E. Pj
C 22(Cs)
D
> 12
>
i B
G E. ly
lu
V) 405 (No)
4 -
RC.A. 910 (photomultiplier)
200 300 400 500 600 700 800

WAVE LENGTH, m/i
Fig. 25.2B. S'lHictral .sen.sitivit.y curves of three typical vacuum-type photocells.
vessel and in the actinometer vessel, and arranging for identical light fluxes
to reach both vessels (e. g., by placing them side-by-side in a uniform light
field, or by alternating the reaction vessel and the actinometer vessel in

—
the same position), quantum yield determinations can be made simply by

comparing the pressure changes in the two vessels. Average quantum
yields can be determined in this way for prolonged periods of illumination,
or for illumination with diffuse light,more easily than with instruments
which measure momentary light intensity, and require a collimated beam.
This actinometer promises to become a very useful tool in the study of
photosynthesis; hut despite its convenience it, too, must l)o used with cau-
tion, and checked from time to time against a physical light -measuring in-
strument such as a thermopile or bolometer. The nature and mechanism
of the reaction in the Warburg-Gaffron-Shocken actinometer is unknown,
and the exact dependence of its rate on light intensity, wave length, nature
of the solvent, presence of impurities, oxygen pressure, concentrations of
the reductant and the sensitizer, and rate of stirring, remain to l)e investi-
gated. Available measurements indicate that the quantum yield declines
10""'
slowly with increasing illumination in the "middle range" (3-7 X
einstein/(sec. cm.-)), but changes faster both at the higher light inten-
sities (>10 X lO-i*^ einstein/(sec. cm. 2)) and at very low light intensi-
ties (<3 X 10-1" einstein/(sec. cm. 2)). It remains to be seen, however,

whether the factor determining the change is intensity of the beam i. e.,
—
energy per unit cross section or its energy (more probably, energy ab-
sorbed in unit volume of the liquid). Some decline in quantum yield with
increasing illumination may be caused by exhaustion of oxygen in the il-
luminated layer; however, this is likely to account only for a part of the
observed trend. Another possible cause of this decline is competition of
back reactions between the intermediate oxidation and reduction prod-
ucts, with the "forward" reaction which leads to the consumption of oxy-
gen. If they are bimolecular in respect to the intermediates, the back re-
actions must be favored by a higher concentration of the latter and there-

fore can become more effective at light intensity increases.
It must be recalled that photometers with selective spectral sensitivity
cannot be relied upon not only in the determination of absolute light inten-
sities, but also in the comparison of two light sources or in the determina-
tion of the proportion of light absorbed by passage through a colored sys-

tem. Unless the absorption is very weak throughout the spectrum, the
spectral composition of the transmitted light will be different from that of
incident light, and the selectively sensitive instrument will react differ-
ently to these two fluxes. It will tend to exaggerate the absorption if the
latter takes place in the region of maximum sensitivity, and underestimate
it if it occurs in the region of low sensitivity. The steeper the spectral
sensitivity curve, the larger will be the errors that occur in absorption
measurements nonmonochromatic light. Selenium barrier layer cells,
in
for example, cannot be used for such measurements even in "monochro-
matic" red light, isolated b}^ filters (or monochromators with wide slits),
since their sensitivity droj s by a factor of 10 between 600 and 700 m^
(fig. 25.2A).
For all these reasons, if white light is used for photosynthetic work, the
best way of characterizing its intensity is to measure it by means of a ther-
mopile protected from infrared light by a suitable filter. This will give an
adequate picture of the quantity of light available for photosynthesis, and
enable one to determine correctly the proportion of this light absorbed by
the plants.
The desire for greater sensitivity often will force the investigator to use
a photoelectric cell, instead of a thermopile, despite all the shortcomings
associated with its selective sensitivity be done only in full
; this should
realization of the errors that can be introduced in this way. Only in work
with truly monochromatic light are the photocells entirely reliable (as-
summg that the linearity of their response has been ascertained by fre-
quent comparison with a thermoelement).
In addition to the problem of a reliable photometric instrument, dif-
ficulty arises in the determination of the quantity of light absorbed by
leaves, algae or cell suspensions, because of the scattering phenomena dis-
cussed in chapter 22.

The scattering by cell comparatively weak, and that by
suspensions is
leaves can be reduced by injection with water, or— still better— with gly-
cerol (by evacuation under the liquid), thus eliminating the most effective
source of scattering— the liquid-air interfaces (c/. fig. 22.8 and 9). How-
ever, figure 22.2 shows that, even in Chlorella suspensions, enough scatter-
ing is present to cause a marked error in the determination of absorbed light

—
energy an error of only a few per cent in the region of strong absorption,
but of 100% or more in green or in the far red, where tme absorption is very
weak.
In measurements in these spectral regions, as well as in precision ex-
periments in other parts of the spectrum, it is necessary to measure the
total scattered flux the diffusely transmitted and diffusely reflected
{i. e.,
fluxes, Ta and Ra, together with directly transmitted and specularly

re-
flected fluxes Ts and Rs) and to use the complete formula:
(25.2) A = h - Ts - Td - Re - Rd = h -S
for the determination of the absorbed light energy, A.
The neglect of both T^ and R^ in the determination of A must lead to
entirely erroneous results, because, for all leaves and Ts thalli, is only a
fraction — —
and often a small one of the total transmitted flux.
The work with leaves requires a consideration of scat-
fact that optical
tering was clear to Maquenne (1860) and Simmler (1862) but some inves- ;
tigators—not only botanists like Sachs (1864) and Detlefsen (1888), but
even physicists like Vierordt (1871) and Lazarev (1924, 1927)— thought
that they could neglect it. In most measurements, however, an attempt
was made to include at least the diffusely transmitted light, Ta, by the
simple device of placing a large collecting surface immediately behind
the absorbing system. Seybold (1932) pointed out that this procedure
brings the risk of measuring the thermal radiation of the tissue together
with the transmitted flux. (A similar error could be caused by fluorescence,
but the latter usually can be neglected.) To avoid errors, one may inter-
pose an infrared-absorbing filter between the leaf and the collecting ther-
mopile.
The measurement of the diffusely reflected flux Ed requires more elabo-
rate devices and has often been omitted. The resulting error in the deter-
mination of the absorbed intensity can be considerable, since leaves of
land plants reflect about as much, or more, light as they transmit namely, —
from 10 to 15,% of (infrared-free) white light (c/. page 683). Submerged
algae or water-filled leaves have a lower reflectance —
they transmit about
20% and reflect from 5 to 10% of white However, diffuse reflection
light.
cannot be entirely neglected even when working with algal suspensions, as

shown by the results of Noddack and Eichhoff (1939) in figiu-e 22.2. The
sharp reflection peak at 180° is due to the walls of the vessel; but, in addi-
tion to this specular reflection, the figure shows a small, but not negligible,
diffuse reflection integrated over all angles, it adds 3 or 5% to the trans-
;
mitted flux and reduces correspondingly the absorbed energy, A.

In work with cell suspensions in spherical or cylindrical vessels, the dis-
tinction between reflected and transmitted light becomes irrelevant, and an
integral measurement of light scattered in all directions, S, can be sub-
stituted for the separate measurements of R and T. A small vessel con-
taining the suspension can be placed inside an "integrating" box or cell,
or in the focal point of a mirror, illuminated by a narrow beam of light
entering through a hole in the mirror, and the light scattered in all direc-
tions can be collected and measured. For the determination of /, a "white"
scatterer can be substituted for the suspension cell. A device of this type
was Noddack and Eichhoff's (1939) "ellipsoid photometer," in which the
light scattered by a small cell was collected on a thermopile sensitive to
light falling from all directions. The scatterer was placed at one focus of
an ellipsoidal mirror, and the collector at the other.
In speaking of the methods of determining the hght energy absorbed by cell suspen-
sions, we must also mention Warburg and Negelein's method of total absorption (1922,
1923). These authors used a very concentrated Chlorella suspension; the vessel had a
silvered back wall so that no light was transmitted; and the absence of diffusely re-
flected hght was ascertained by experiment. (The correctness of this last assertion was
questioned by Mestre 1935, and this criticism is supported by the above-mentioned re-
sults of Noddack and Eichhoff.) Thus, \\'arl)urg and Xogelein assumed, simply, A = /
It was mentioned on page 673 that, inworking with solutions, the neces-
sity of estimating R is usually avoided by using a blankcell, whose reflec-
tion is assumed to be equal to that of the solution cell. Many authors have
hoped to get around the necessity of measuring Ra for leaves or thalli in a
similar way, by using as "blanks" plant tissues deprived of pigments.
This idea has been carried out in different ways: Reinke (1886) used algal
thalli from which the pigment had been extracted by alcohol; Linsbauer
(1901), Brown and Escombe (1905), Seybold (1932, 19331-2) and Meyer
(1939) compared the transmission by green parts with that by white parts
of variegated leaves; Wurmser (1921) determined the transmission of
thalli before and after bleaching by prolonged illumination. However, the
interpretation of results obtained in this way presents considerable dif-
ficulties.It has already been said (page 673) that equation (22.2b) is
only an approximation, although a satisfactory one, even in the work with
transparent media. Weigert (1911) thought that it could also be used, as
such, for leaves, and applied it to the data of Brown and Escombe; but
his calculation led to absurdly low values of A, and its fallacy has been
pointed out by Willstatter and StoU (1918) and Warburg (1925).
In all precision experiments on light absorption by plants, measure-
ments of the three quantities I, T and R cannot be avoided. The determi-
nation of T and R can
be carried out either by means of integrating devices
that collect the reflected and the transmitted light, or by differential "gonio-
photometric" methods, i. <?., by determining scattering as a function of
the angle between the incident and the scattered beam.
3. Measurements of Oxygen Evolution
Oxygen produced can be identified and measured by

in photosynthesis
different chemical or physicochemical methods, either in the liquid phase
containing the aquatic plants, or in the gas phase. Because of the low
solubility of oxygen in water, methods of the first kind (e. g., the potentio-
metric determination of the oxygen concentration in solution) are suitable
only for the measurement of small effects, e. g., for the observation of the
photosynthetic activity in thefirst minutes of illumination {cf. chapter 33).
We cannot deal here with the analytical technique of the determination

of oxygen. White phosphorus, organic oxygen absorbers (such as pyrogallol
or the leuco dyes), copper, sodium hyposulfite and chromous chloride are
among the reagents used for this purpose. For solutions, Winkler's
method is perhaps the most popular one; it utilizes oxygen (through the
intermediary of the system manganous chloride-manganic chloride) for the
liberation of an equivalent quantity of chlorine, which can easily be de-
MEASUREMENTS OF OXYGEN EVOLUTION 845
termined by titration with potassium iodide and thiosulfate. If pyrogallol

or the leiico dyes (indigo white, leuco methylene bhie) are used for oxygen
determination, the progress of oxygen hberation can be followed colori-
metrically or spectrophotometrically. The same methods are applicable
to the com-ersion of hemoglobin into oxyhemoglobin —
a method of oxygen
determination introduced into photosynthetic studies by Hoppe-
first
Seyler (1879) and used more recently by Hill (1937, 1939). Osterhout
(1918) suggested the use of hemocyanine-containing crab blood (which be-
comes blue same purpose.
in the presence of oxj'gen) for the
Twô physiological methods of ox3^gen detection wei-e discovered by
Beijerinck (1901) and Engelmann (1881, 188G and 1894), respectively.
Beijerinck's method utilizes the bioluminescence of certain bacteria (e. g.,
Micrococcus phosphorens) wliich becomes visible in the presence of ex-
,
tremely small quantities of oxygen (5 X 10 ~^ mm. O2 pressure above the

liquid, or 1 X 10 ~* m./l. in water, according to Harvey and Morrison
1923). Engelmann's method utilizes motile bacteria (e. g., Proteus vul-
garis), which come to rest in oxygen-free medium, but begin to move about
in the presence of traces of oxygen. This method has been sharply criti-
cized by Pringsheim (1886) for an answer to this criticism, see Engelmann
;
(1887). With the help of motile bacteria, the photosynthetic activit}^ of a

single cell can be observed under the microscope. Luminescent bacteria
have been used, e. g., in attempts to decide whether single isolated chloro-
plasts liberate oxygen in light (c/. Vol. I, chapter 4, page 62).
The chemical and biochemical methods are difficult to adapt to a con-
tinuous control of the rate of photos}Tithesis. Physicochemical methods
therefore early attracted the interest of workers in this field. In modern
quantitative studies of metabolic processes, manometric measurements
have acquired a predominant importance; biochemists have found that
almost every biochemical reaction can be conducted so as to cause absorp-
tion or liberation of a gas and means of meas-
this often ])rovides the best
uring its rate. The and carbon
reactions of hemoglobin with oxygen
monoxide were the first for which this method was developed bj^ Haldane
and Barcroft; applications to respiration and photosynthesis came
next. Since the time of Sachs (1864), a cmde method of measuring the
volume of liberated oxygen was known and widely used "bubble count-
—
ing." In quiet sohitions of a given surface tension, the gas bubbles de-
taching themselves from the leaves have an approximately uniform size,
so that the rate of gas formation can be calculated by multiplication of the

number of bubbles formed per unit time by the volume of a single bubble.
This method is simple and sensitive, but obviously fraught with errors,
caused bj'' the differences in wettability of leaf surfaces, coalescence of small
846 METHODS OP KINETIC MEASUREMENTS CHAP. 25
bubbles to larger ones, effect of convection currents or stirring on the size

of bubbles and similar complications. A number of authors (Kohl 1897
Kniep 1915, Wilmott 1921, Bose 1924, Arnold 1931, among others) have
Fig. 25. 3A. "Warburg apparatus" for manometric measurement of photosynthesis.
Scale In Ccnfimetcrs
Fig. 25. 3B. Two "Warburg vessels" with equal liquid volume but differ-
ent gas volume (after Emerson and Lewis 1941).
strived to improve this method and to make the bubble counting automatic;
a discussion of these attempts can be foimd in Spoehr's book (1926, page
231). An important objection has been raised by Gessner (1937): Con-
stant size bubbles can only be formed in quiet water, where a photosynthe-
sizing plant rapidly becomes surrounded by a layer of water that is alkaline,

deficient in carbonic acid and supersaturated with oxygen^three factors
each of which may strongly affect the rate of photosynthesis.
Perhaps the most serious source of error is the fact, studied by Iîep
and Minder (1909), that the gas bubbles emerging from cut stems of water
plants contain not pure oxygen produced by photosynthesis but a mix-
ture of oxygen with air from intercellular spaces. The nitrogen content
of the bubbles varies with the rate of bubbling. As the air is drawn from
the intercellular spaces by the stream of photosynthetic oxygen, new air
diffuses into them from the medium, so that even after an extended period
of illumination the bubbles still contain some nitrogen.
The manometric method was introduced into the investigation
precise
of photosynthesis by Warburg in 1919 (see figure 25. 3 A). Since one
mole of carbon dioxide is consumed for each mole of oxygen liberated, the
manometric method can only be used if the carbon dioxide consumption is
completely or partially eliminated as a source of pressure changes. This
can be achieved by the use of a large volume of liquid in contact with a
small volume of gas, or by means of an alkaline buffer. In the first case,
the comparatively high solubility of carbon dioxide in water enables the
plants to use dissolved gas for a considerable time while only slowly re-
ducing the pressure of carbon dioxide in the gas phase. The use of buffers
enhances this stabilizing effect of the aqueous phase, though at the cost of
introducing a "nonphysiological" pH.
In the application of manometric technique to respiration, practically
complete elimination of carbon dioxide as source of pressure changes could
be achieved by binding this gas chemically outside the cell suspension.
For this purpose, the reaction vessels were provided with a central well (or
side tube) containing alkali. A similar procedure cannot be applied to
photosynthesis because the latter requires the presence of carbon dioxide
in the suspension. The alternative of eliminating oxygen from the gas
phase (for example, by placing white phosphorus, or pyrogallol, in the
side tube) and measuring pressure changes due to carbon dioxide alone,
also does not recommend itself, because the rate of photosynthesis is sensi-
tive to oxygen pressure and often is strongly reduced by anaerobic condi-
tions (cf. chapter 13, page 326). Furthermore, elimination of oxygen
makes the application of the respiration correction difficult if not impossible
since in the dark, respiration is totally suppressed, whereas, in light, some
oxygen produced by photosynthesis is drawn into the respiration cycle
before reaching the external absorber.
We are thus leftwith the choice of using cells suspended in a carbonate
buffer, thusworking at a high "unphysiological" pH, or employing an acid
solution with an unphysiologically high carbon dioxide content (e. g., phos-
848 METHODS OF TvINETIC MEASUREMENTS CHAP. 25
phate buffer equilibrated with air containing 1-5% CO2; without this
high initial concentration, the carbon dioxide suppl.y in the carbonate-free
solution will be rapidly exhausted in hght).

From the point of view of interpretation of manometric data, alkaline-
buffered solutions are more convenient than acid solutions, since oxygen
alone is responsible for all the pressure changes observed above these solu-
tions.
Ordinaril3% mixtures of 0.1 molar solutions of sodium bicarbonate and
potassium carbonate are employed (Table 8. V) Using one sodium and one
.
potassium salt seems to be somewhat preferable to using two sodium or

two potassium salts (c/. page 835). A mixture of 85 parts bicarbonate and
15 parts carbonate at 20° C. is in equilibrium with free carbon dioxide at a
partial pressure of about 1.9 mm. (c/. Tables 8. II and 8.V; it has to be
taken into account that the solubility of CO2 in 0.1 molar salt solution is
~10% lower than in distilled water). Ten cubic millimeters of carbon
dioxide can be withdrawn from 1 cc. of this mixture without appreciably
altering the partial pressure of CO2 in the gas. Mixtures containing more
carbonate and less bicarbonate have higher buffering capacities, and lower
concentrations of carbonic acid.
As mentioned before, the drawback of carbonate buffers is their high
pH (about pH 9 for the above-mentioned buffer No. 9). Some algae are
damaged by short exposure to even less alkaline buffer mixtures {pR 8.5).
Chlorella pyrenoidosa, on the other hand, can be kept even in the more
alkaline carbonate mixtures for many hours without signs of damage to the
photosynthetic apparatus. Respiration in carbonate mixtures is some-
what slower than respiration in neutral or acid media. Despite absence of
visible damage, the maximum quantum yield of Chlorella photosynthesis
seems to be somewhat lower in alkaline carbonate than in acid (phosphate)
buffers; whether the reduction is <20% (Rieke, Emerson and co-workers)
or as high as 50% (as suggested by Warburg) is a matter of controversy
(c/. chapter 29, pages 1096 and 1107).
When it is definitely desirable to avoid alkalinity, measurements of

photosynthesis usually are made in slightly acid medium (e. g., pH 5, ^
obtained with IVH2PO4), saturating this medium with air enriched with
from 1 to 5% carbon dioxide. Both oxygen and carbon dioxide are ex-
changed with the gas phase above these solutions, and the observed pres-
sure changesmust be apportioned to the two gases by means of a rather
delicate calculation.
For each individual 'Warburg vessel," filled to a certain mark with
water, a "vessel constant," Kq,, can be determined, giving the increase in
pressure caused by the production in this vessel of one cubic centimeter of
oxygen; a similar constant, Xco„ can be calculated for carbon dioxide.
: . :
(If the liquid volume is sufficiently large compared with the gas volume,
KcOi will be much smaller than Kq,.)
The knowledge of these constants and assumption of a photosynthetic
Qp = AO./— ACO2 (compare chapter 3, Vol. I), permit one to
quotient,
calculate the rate of photosynthesis from manometric readings in a single
vessel. doubts arise as to the value of the photosynthetic quotient, the
If
latter can be treated as a second unknown, and an additional equation for
its determmation can be obtained by using a second Warburg vessel of dif-
ferent dimensions the same vessel filled to a different

(e. g., level, or a vessel
\Adth equal liquid volume but an enlarged gas volume; cf. fig. 25. 3B).
The pressure increase in the first vessel is
= KÂ02 + Kco.ACO, = AO, -

(25.3a) Ap' (^K^,
^^
and that in the second vessel
= AO2 -
(25.3b) Ap" (a'o,
^^
If the K values are known, the two equations permit the calculation of the
two unkno\^Tis, AO2 and Qp (Warburg)

Of course, this purely manometric determination of the rate of photo-
synthesis is only reliable if it is definitely known that no other gas except
oxygen and carbon dioxide is involved in the gas exchange. After Gaffron
(1942) became suspicious that this is not the case with certain anaerobically
incubated algae, he introduced chemical absorbers for oxygen, carbon di-
oxide and hydrogen into the side arms of the manometer, and proved in
this way the occurrence of hydrogen fermentation and photochemical
hydrogen consumption by these algae {cf. Vol. I, chapter 6).
Two conditions must be strictly fulfilled if manometric measurements

are to be reliable. In the first place, when pressure changes of the order
of 0.1 mm. Hg mm.
Brodie solution) are to be measured, while the total
(1
pressure is of the order of 100-1000 mm. Hg, maintenance of constant
temperature becomes very important. The reaction vessels must be
placed in a precision thermostat, and shaken vigorously to ensure continu-
ous thermal equilibrium. needed also to satisfy a
Vigorous shaking is
second condition — between free and

rapid establishment of equilil)riTnn
dissolved gases. This is essential both to prevent a lag in the manometric
readings and to make sure that no depletion of carbon dioxide occurs in
the lowest layer of the reaction vessel, where illumination (through the
flat bottom!) is strongest, and carbon dioxide consumption bj^ photosyn-
thesis is fastest. It is thus necessary both to mix the liquid rapidly

(to equalize carbon dioxide concentration) and to splash it vigorously (to
.
increase and renew the liquid-air interface), thus accelerating the release of
oxygen into the gas space.
Because of these complications, direct chemical methods for determina-
tion of carbon dioxide and oxygen appeal to some investigators as more
satisfactory than pressure measurements; the latter give only indirect
evidence as to the identity of the gases causing the pressure changes. In
spite of this, the manometric techniques have been resorted to again and
again because of certain advantages not available with chemical analysis.
The manometer measures change in pressure, regardless of total pressure of
the gas in question. Thus, 10 cu. mm. of carbon dioxide can be determined
with equal precision (assuming constancy of temperature), regardless of
whether the total amount of gas present is 50 or 500 cu. mm. This is not
true of direct chemical methods. The manometer has a fast response, so
that measurements can be made over short periods of light or darkness.
The two-vessel method further requires exact identity of physiological
processes and identical time course of pressure equilibration in the two
vessels.
For the most precise manometric work, a differential manometer may be
substituted for the usual open-type manometer (Warburg 1926). This
eliminates the disturbing influence of barometric pressure. Differential
manometers can be conveniently read to 1/100 of a millimeter with a cathe-
tometer. This technique has been especially developed for measurements
of the quantum requirement of photosynthesis (c/. chapter 29)
Several methods of magneiometric oxygen determination (based on
paramagnetism of the O2 molecule) have been developed. Pauling's
magnetic oxygen meter (Pauling, Wood and Sturdivant 1946) is fabricated
by Beckman, Inc. Its range (0-1 atm. O2) makes it not directly applicable
to precision measurements of photosynthesis.
A new method for continuous determination of oxygen content in solu-
tion was introduced in 1938, based on the measurement of conductivity.
It is a form of the so-called polarographic analysis, which has found
numerous applications in modern analytical chemistry. The essential de-
vice a small-surface cathode, in a solution such that the passage of the cur-
is
rent involves cathodic reduction of dissolved oxygen to H2O2. The maxi-

mum current that can pass through a cell with such an anode is determined
by by diffusion, and is therefore propor-
the supply of oxygen to the anode
tional to theoxygen concentration.
An apparatus for polarographic oxygen determination in biological
studies was developed by Petering and Daniels (1938) and applied by
Petering, Duggar, and Daniels (1939) to the determination of the quantum
yield of photosynthesis in Chlorella. The range of determinable concen-
trations is from about 5 X
10 ~^ m./l. upward to saturation. (Water satu-
rated with air at 25° contains 2.4 X 10"* m./l. O2.) A similar method was
used by Blinks and Skow (1938^) for the investigation of induction phe-
nomena in photosynthesis. They found it advisable to replace the usual
CARBON DIOXIDE CONSUMPTION 851
dropping mercury electrode by a stationary mercury electrode, or by a

Pt point electrode. (Concerning a rotating electrode, see Kolthoff 1940.)
Kaiitsky discovered the extreme sensitivity of the phosphorescence
of certain dyestuffs, e. g., trypaflavine adsorbed on silica gel, to traces of oxy-
gen. Franck and Pringsheim (1943) investigated this quenching effect
quantitatively and found that 50% quenching is obtained at a partial
pressure of 5 X 10~^ mm. O2. Pollack, Pringsheim and Terwood (1944)
and Franck, Pringsheim and Lad (1945) applied this method to some
problems of photosynthesis, e. g., oxygen production by single flashes of
light. Figure 25.4 shows the results obtained with a 2 second flash and
several 0.03 second flashes of illumination in a suspension of Chlorella.
The reason why the "oxygen bursts" recorded in the figure last for 1 minute
ormore is that the oxygen produced durng the flash is only gradually
4 5
TIME, mia
Fig. 25.4. Oxygen production by Chlorella in single light flashes (measured

by the phosphorescence method) (after Pollack, Pringsheim and Terwood
1944). Large peak: 2 sec. illumination; small peaks: 0.03 sec. illumination.
carried away by the stream of nitrogen from the cell suspension into the
vessel containing the phosphorescent gel. The apparatus, as constructed
by Pringsheim and co-workers, could be used for the measurement of rates
down to 5 X 10 ~^ cc. 02/min. However, the method is only useful under
anaerobic, or almost anaerobic conditions, since, in the presence of more
than 10~^ mm. O2, the quenching becomes practically complete.
4. Measurements of Carbon Dioxide Consumption
Like the liberation of oxygen, the consumption of carbon dioxide in

photosynthesis can be measured either by the traditional methods of
chemical analysis, or by physicochemical methods. Absorbers for carbon
dioxide are well known; any alkaline material (e. g., lime or baryta) can
be used for this pui-pose, and the quantity of absorbed carbon dioxide
can be determined gravimetrically, titrimetrically, electrometrically or

manometrically. The tests are made either in samples of the air or sohi-
tion taken from the reaction vessel before and after a period of photosyn-
more conveniently, in circulating gas, before and after its pas-
thesis, or,
—
sage through the reaction chamber a method first introduced by Kreusler
Source lamp _,/ Entrant slit of spectograph
___^^ _^ 1-Concave mirror
Flat mirror'
to B
Plant growth
chamber
-Nutrient
supply
Fig. 25.5. Apparatus for continuous spectroscopic measurement of carbon dioxide

exchange in plants (after McAlister 1937).
(1885),and used also in the classical work of Willstatter and StoU (1918)
It isconvenient to use methods of analysis not requiring the taking of
samples, e. g., to measure the conductivity of the absorbing solution in
equilibrium with the gas (cf. Newton 1935, and Clark, Shafer and Curtis
1941). The smallest amounts of carbon dioxide that can be determined
in this way are of the order of 10"'' g.
Several methods of continuous determination of carbon dioxide in sohi
tion have been suggested. In working with (unbuffered) solutions, the
CARBON DIOXIDE CONSUMPTION 853
consumption of carbon dioxide can be followed acidimetrically, c. g., by-

means of an appropriate color indicator (Osterhout and Haas 1918; Oster-
hout 1918), or a glass electrode (Blinks and Skow 1938).
Two sensitive, purely physical methods have been used for the deter-
mination of carbon dioxide in the gas phase. They were based on spec-
trophotometry and thermal conductivity, respectively. McAlister (1937)
built an apparatus (fig. which the carbon dioxide content of cir-
25.5) in
culating gas was recorded continuously by means of an infrared-sensitive
spectrophotometer, using the strong carbon dioxide absorption band at

4.2 to 4.3 M- The apparatus was later altered to register changes in carbon
dioxide concentration (uistead of concentration itself) cf. McAlister and
;
Myers (1940).
A simplified
method of estimation of small quantities of carbon dioxide
in the air by infrared absorption, without the use of a monochromator
(after eliminating water vapor as the only other infrared-absorbing com-
ponent), was described by Dingle and Pryce (1940). Scarth, Loewy and
Shaw (1948) improved this apparatus, increasing considerably its sensitiv-
and adapting it for the determination of both carbon dioxide and water.
ity,
They used it to follow the course of photosynthesis and of transpiration of

leaves.
Harder and Aufdemgarten (1938) and Aufdemgarten (1939) described
an automatic carbon dioxide recorder, in which the rate of cooling of an
electrically heated wire was used to determine the content of the ambient
gas in carbon dioxide. (Changes in the concentration of carbon dioxide
affect the heat conductivity more strongly than equivalent changes in oxy-
gen concentration.) Some registration curves obtained with this instru-
ment are reproduced in figs. 33. 10A,B and 1 1 A,B. An improved apparatus
^\as described by van der Veen (1949).
5. Measurements of Carbohydrate Production and Energy Conversion
Two methods — analytical determination of the carbohydrate produc-

tion, and determination of the heat of combustion of the synthesized or-
ganic material —
were much used in the earlier work on photosynthesis.
They however, hardly suitable for exact kinetic investigations. As de-
are,
scribed in chapter 3 (Vol. I, page 35) the amount of analytically determin-
able carbohydrates found in the plant after a prolonged period of photo-
synthesis often considerably smaller than was expected from the rate of
is
consumption of —
carbon dioxide a result attributable to rapid secondary
transformations of the primary product, and possibly also to direct photo-
synthetic production of compounds other than carbohydrates.
The combustion of organic material is an appropriate method for the
:
determination of total yield of photosynthesis over the whole growth per-

iod,and is widely used in field experiments but it is obviously unsuitable
;
for exact kinetic detemiinations, in which the organic material present at

the beginning of the experiment, cannot be neglected.
However, a different method of measurement of photosynthesis, also
based on the determination of energy conversion, appears feasible, although
it probably cannot compete in simplicity and exactness with the methods
based on the determination of carbon dioxide or oxygen

Of the total light energy ( A//) absorbed by the plants, one part ( A//c) is
transformed into chemical energy (latent heat of combustion of the syn-
thesized organic material) and the rest ( A//,) is converted into heat. We
stated above that the exact determination of Ai/^ is difficult because of the
initialweight of organic material but the determination of A//^^ is possible.
;
It can be carried out, without affecting the plants, by placing them in a

sensitive calorimeter Ai/^ can then be calculated by subtracting Ai7, from
;
A/jT. Magee, DeWitt, Smith and Daniels (1939) measured the heat pro-
duced in a small quartz vessel in a thermostated container in consequence
of the absorption of a certainamount of light by a suspension of Chlorella
cells, and compared with the amount of heat produced in the same ap-
it
paratus when the light is absorbed by India ink or another inert absorber.
In these experiments, the rate of energy absorption was of the order of
1000 erg/sec, and AHi was found to be smaller than AH by about 20%.
Similar methods were developed by Arnold and by Tonnelat (c/. chapter 29,
page 1122).
6. Application of Isotopic Indicators
The use of isotopic indicators —stable isotopes such as H-, C^'' or 0^*,
—
and radioactive isotopes such as H^, C'^ and C^^ may make possible the
development of several new sensitive methods for measuring the yield of
photosynthesis. All that seems to be needed is to supply the plants with
carbon dioxide or water enriched in one of these isotopes and measure
either the accumulation of this isotope in the plant cells, or its disappear-
ance from the supplied material. However, the phenomena of isotopic
exchange and isotopic discrimination make the task less simple than it may
appear at first.
Apphcations of the isotopes H^, C^^ C^* and O^^ to the solution of some
qualitative problems in the chemistry of photosynthesis (such as the forma-
tion of a carbon cUoxide acceptor complex, and the origin of the oxygen
evolved in photosynthesis) were described in Volume I (see particularly
pages 54, 202 and 241). New investigations with C^^ as tracer, aimed at
the identification of the organic intermediates of the reduction of carbon
dioxide, will be described in chapter 36.
The techniques of labehng and counting have been described in several

reviews, for example, in the monographs by Kamen (1947) and Calvin,
Heidelberger, Reid, Tolbert and Yankwich (1949).
Methods of Kinetic Measurements
1860 Maquenne, L., An7i. agron., 6, 321.
1862 Simmler, R. G., Ann. Phys. (Poggendorf), 115, 593.

1864 Sachs, J., Botan. Z., 22, 353, 361, 369.
1871 Vierordt, K., Die AnWendungen des Spedralapparates. Jena.
1879 Hoppe-Sejder, F., Z. physiol. Chem., 2, 425.
1881 Engelmann, T. W., Botan. Z., 39, 441.
1885 Kreusler, U., Landw. Jahrb., 14, 913.
1886 Reiiike, J., Botayi. Z., 44, 161, 177, 193, 209, 225, 241.
Engelmann, T. W., ibid., 44, 43, 64.
Pringsheim, N., Botan. Zentr., 28, 93.

Pringsheim, N., Ber. dent, hotan. Ges., 4, XC.
1887 Engelmann, T. W., Botan. Z., 45, 100.
1888 Detiefsen, E., Arb. botan. Inst. Univ. Wurzhurg, 3, 534.
1894 Engelmann, T. W., Arch. ges. Physiol. Pfliigers, 57, 375,
1897 Kohl, F. G., Ber. deut. botan. Ges., 15, 111.
1901 Beijerinck, M. W., Proc. Acad. Sci., Amsterdam, 4, 45.
Linsbauer, K., Botan. Zentr. Beihefte, 10, 53.
1905 Brown, H. T., and Escombe, F., Proc. Roy. Soc. London, B76, 29.
1909 Kniep, H., and Minder, F., Z. Botanik, 1, 619.
1911 Weigert, F., Z. unss. Phot., 11, 381.
1915 Kniep, H., Jahrb. iviss. Botan., 56, 460.
1918 Willstfitter, R., and StoU, A., Vntersuchungen uber die Assimilation der
Osterhout, W. J. V., J. Gen. Physiol, 1, 17.
Osterhout, W. J. V., ibid., 1, 16.
Osterhout, W. J. V., and Haas, A. R. C., Science, 47, 420.

Osterhout, W. J. V., and Haas, A. R. C., Proc. Natl. Acad. Sci. U. S., 4,
85.
1919 Warburg, 0., Biochem. Z., 100, 230.
1920 Warburg, 0., ibid., 103, 185.
1921 Wilmott, A. J., Proc. Roy. Soc. London, B92, 304.
Kiister, E., Kidtur der Mikroorganismen. 3vd ed., Teubner, Leipzig and
Berlin, 1921.
Wurmser, R., Arch. phys. biol., 1, 3.
1922 Warburg, and Negelein, E., Z. physik. Chem., 102, 236.
O.,
1923 Warburg, and Negelein, E., ibid., 106, 191.
0.,
Harvey, E. N., and Morrison, T. F., J. Gen. Physiol., 6, 13.
1924 Boê, J. C., The Physiology of Photosynthesis. Longmans, Green, 1924.
856 METHODS OF TCINETIC MEASTTREMENTS OHAr. 25
Lazarev, P. P., J. Applied Phijs. USSR, 1, 142.

Pringsheim, E. G., "Algenkiiltur," in Abderhalden's Handbuch der bio-
logischen Arbeitsinethoden, Sectiou 11, Part 2, Urban und Schwarzen-
berg, Berlin and Vienna.
Warburg, 0., Biochem. Z., 152, 51.
1926 Spoehr, H. A., Photosynthesis. Chemical Catalog Co., New York, 1926,
p. 231.
Pringsheim, E. G., Beitr. Biol. Pflanz., 14, 283.
Warburg, 0., Uber den Stoffwechsel der Tumoren. Springer, Berlin, 1926,
1927 Lazarev, P. P., Biochem. Z., 182, 131.
1930 van der Honert, T. H., Rev. trav. botan. neerland., 27, 149.
1931 Arnold, A., Planta, 13, 529.

van Niel, C. B., Arch. Mikrobiol., 3, 1.
1932 Hardy, A, C, and Perrin, F. H., The Principles of Optics. McGraw-Hill,
New York, 1932.
van der Paauw, F., Rec. trav. botan. neerland., 29, 497.
Seybold, A., iUd., 18, 479.
1933 Seybold, A., ibid., 20, 577.
Hoover, W. H., Johnston, E. S., and Brackett, F. S., Smithsonian Inst.

Pub. Misc. Collections, 87, No. 16.
Gaffron, H., Biochem. Z.. 260, 1.
1934 -
Emerson, R., and Green, L., /. Gen. Physiol, 17, 817.
Gaffron, H., Biochem. Z., 269, 447.
1935 Barker, H. A., Arch. Mikrobiol, 6, 141.
Mestre, H., Cold Spring Harbor Symposia Quant. Biol, 3, 191.
Newton, R. G., Ann. Botany, 49, 381.

Gaffron, H., Biochem. Z., 275, 301; 279, 1.
1937 McAlister, E. D., Smithsonian Inst. Pub. Misc. Collections, 95, No. 24.
Smith, E. L., J. Gen. Physiol, 20, 807.
Gessner, F., Jahrb. loiss. Botan., 85, 267.
Hill, R., Natiire, 139, 881.
French, C. S., /. Gen. Physiol, 20, 711; 21, 71.
Pearsall, W. and Loose, L., Proc. Roy. Soc. London, B121, 451.
H.,
1938 Emerson, R., and Green, L., Plant Physiol, 13, 157.
Petering, H. G., and Daniels, F., /. Am. Chem. Soc, 60, 2796.
Blinks, L. R., and Skow, R. K., Proc. Natl Acad. Sci. U. S., 24, 413.
•
Blinks, L. R., and Skow, R. K., ibid., 24, 420.
Harder, R., and Aufdemgarten, H., Nachr. Ges. Wiss. Gdttingen, 3, 191.
Eymers, J. G., and Wassink, E. C, Enzymologia, 2, 258.
1939 Aufdemgarten, H., Planta, 29, 643; 30, 343.
Roy. Soc. London, B127, 192.
Hill, R., Proc.
Magee, J. L., De Witt, T. W., Smith, E. C, and Daniels, F., J. Am. Chem.
Soc, 61, 3529.
Petering, H. G., Duggar, B. M., and Daniels, F., ibid., 61, 3525.
Eichhoff, H. J., Biochem. Z., 303, 112.
Noddack, W., and Eichhoff, H. J., Z. phydk. Chem., A185, 241.
Emerson, R., and Lewis, C. M., Am. J. Botany, 26, 808.
1940 McAUster, E. D., and Myers, J., Smithsonian Inst. Pub. Misc. Collections,
99, No. 6.
Kolthoff, I. M., and Laitinen, H. A., Science, 92, 152.
Pearsall, W. H., and Bengry R. P., Ann. Botany, 4, 365.
Pratt, R., Am. J. Botany, 27, 52.
Pratt, R., and Fong, J., ibid., 27, 431.
Sargent, M. C, Plant Physiol, 15, 275.
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1941 Clark, D. G., Shafer,J., and Curtis, 0. F., Plant Physiol, 16, 643.
Emerson, and Lewis, C. M., Ayn. J. Botany, 28, 789.

R.,
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1942 Emerson, R., and Lewis, C. I\L, J. Gen. Physiol, 25, 579.
Gaffron, H., ibid., 26, 195.
Pratt, R., Aju. J. Botany, 29, 142.

Steemann Nielsen, E., Dansk Botanisk Ark., 11, No. 2.
1943 Pratt, R., Am. J. Botany, 30, 32, 626.
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1944 Pollack, M., Pringsheim, P., and Terwood, D., J. Chem. Phys., 12, 295.
van Niel, C. B., Bad. Revs., 8, 1.
Myers, J., Plant Physiol, 19, 579.

Myers, J., and Clark, L. B., J. Gen. Physiol, 28, 103.
1945 Pratt, R., Oneto, J. F., and Pratt, J., ibid., 32, 504.
Franck, J., Pringsheim, P., and Lad, D. T., Arch. Biochem., 7, 103.
Wassink, E. C, and Kersten, J. A. H., Enzymologia, 12, 3.
1946 Myers, J., /. Gen. Physiol, 29, 419, 429.

Pauling, L., Wood, R. E., and Sturdivant, J. H., /. Am. Chem. Soc, 68,
795.
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1947.
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1949 Warburg, O., and Schocken, V., Arch. Biochem., 21, 363.
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P. E., Lsotopic Carbon. Wiley, New York, 1949.
van der Veen, R., Physlologla pluiUaruin, 2, 217.
1950 Ban', A. C, Ilium. Eng., 65, 37.
1951 Tanada, T., Am. J. Botany, 38, 27().
Chapter 26
EXTERNAL AND INTERNAL FACTORS IN PHOTOSYNTHESIS
1. The "Cardinal Points" and the "Limiting Factors"
Even when studying simple reactions in vitro, the physical chemist is
rarely able to control all the conditions that affect the reaction rate. Con-
sequently, seldom will two investigations of the velocity of a reaction result
in agreement in more than the order of magnitude. Beside the readily con-
trollable external factors, such as temperature, pressure and light intensity,
the rate often depends on factors as elusive as the state of the walls of the
vessel, or the presence of minute impurities. It is therefore easy to judge
the difficulties encountered in the kinetic study of a complex chemical
process in a living organism. The rate of such a process depends on many
physiological factors that do not enter ostensibly into the kinetic equations.
Among phenomena, photosynthesis is perhaps the most sensitive
all life
to slight variations in the structure and composition of the biocatalytic

apparatus. No wonder that doubts have been expressed as to the very
possibility of deriving significant kinetic relationships from the quantita-
tive study of this phenomenon.
At first, considerable optimism prevailed in this respect. The develop-
ment of natural science in the nineteenth century led to the belief that bio-
mathematical laws, and several
logical processes follow relatively simple
attempts have been made to formulate such laws for the production of or-
ganic matter by plants.
The dependence of photosynthesis on external
earliest discussions of the
factors were based on the concept of the three "cardinal points" (Sachs
1860). According to this concept, which was widely accepted until the
turn of the century, biological processes get under way at certain minimum
values of the relevant external variables (such as temperature, pressure,
humidity, light intensity etc.), reach the highest rate at certain optimum
values of these variables, then decline, and cease altogether after the max-
imum tolerable values have been exceeded.
Twenty years before the enunciation of the principle of the three car-
dinal points, Liebig (1840, 1843) had formulated a simple rule for the effect
of various elements on the yield of field crops. He postulated that these
858
)
THE "cardinal POINTS" AND THE "LIMITING FACTORS" 859
by the quantity of the one nutrient element (such as

yields are determined
potassium, phosphorus or nitrogen) present in the lowest concentration
(relative to its optimum quantity). This postulate became known as the
"law of the minimum."
Sixty years later, Blackman (1905) suggested that a generalized form
of this law can be applied to photos\Tithesis, and can explain the obsen-a-
tions in this field better than the concept of the three cardinal points. He
took from Liebig the idea that the rate of a biological process (in this case,
photosynthesis) is determined, under given conditions, by a single "limit-
ing" factor; but, in addition to the supply of material ingredients (the
only kind of factors with which Liebig was concerned), Blackman con-
sidered also tem-pcraUtre and light intensity as potential "limiting factors."
Fig. 26.1. Photosynthesis according to the concepts of "cardinal points'

{MOM') and "limiting factors" {ABCD).
He suggested that the rate of photosynthesis increases with the increase in

value of any one of these factors {Fi) as long as this factor is "slowest"
and ceases to be dependent on Fi when one of the other factors (F2, F3, . . .
becomes limiting. In other words, the plot of the yield of photosynthesis,

P, versus a variable Fi (at constant values of all the other kinetic factors)
was postulated by Blackman to have the shape ABC (fig. 26.1), instead of
the shape MOM' required by the optimum theory. Blackman could not
deny that extreme conditions generally inhibit biological processes — in
other words, that the horizontal plateau BC in figin-e 26.1 cannot extend
indefinitely,but that sooner or later, P must begin to decline (as indicated
by the broken line in the figure). However, he attributed this decline to
destnictive phenomena {e. g., freezing of cell water, or denaturation of pro-
teins) not inherent in the kinetic mechanism of photosynthesis itself. Ac-
cording to Blackman, these inhibitions merely limit the range in which the
"law of limiting factors" can be verified; their superimposed character is
indicated by the fact that, whenever they come into play, the rate, instead
860 EXTERNAL AND INTERNAL FA f 'TORS CHAr. 20
of being determined uniquely by the momentary values of the variables

Fi, F2, . . ., becomes time-dependent, thus
revealing a progressive (and often
irreversible) destruction of the biochemical apparatus.
Compared with the hypothesis of the three cardinal points, Blackman's
principle of limiting factors represented substantial progress in the inter-
pretation of the kinetics of photosynthesis. Since its first enunciation in
1905, has been accepted and widely used by students of photosynthesis.
it
Unfortunately, Blackman was not satisfied with the improvement of the

general qualitative picture: In the belief that the efficiency of biological
processes must be subject to simple quantitative ndes, he insisted on treat-
ing the concept of "limiting factors" as an exact law of nature. He postu-
lated that the fimctious P = /(F,) must have precisely the shape shown in
Increase
of F2
Fig. 26.2. Kinetic curves of the first type (Jlackman t3T)e).
figure 26.1, i. e., consist of a linear ascending part, terminated by a sharp

break and followed by a horizontal plateau. In order that the same rule
l)e true also for the dependence of photosynthesis on all the other factors,
F2, Fi, he had to assume that for different values of a parameter, F2,
. . ,
—
.
the yield P as a function of Fi is represented by a set of broken fines such

as ABC, ABDE, ABDFG. .in figure 26.2—which coincide at the low Fi
.
values (part AJ5 in the figure) and are distinguished only by the position
of the break, where the ascending part goes over into the horizontal plateau.
This means that the rate of photosynthesis was assumed to be proportional
to the one factor that is limiting under the given conditions, and entirely
independent of all the other factors.
Only in the case of temperature as the "limiting factor" did Blackman
admit the possibility of a different shape of the ascending part of the curve,
an exponential (instead of a linear) rise, in agreement with the general ex-
perience in the field of temperature dependence of chemical reactions.
In the belief that the law of limiting factors must be strictly obeyed,
—
Blackman and his pupils made attempts to fit the experimental data into
this oversimplified theoretical picture. Others objected to this, and a con-
troversy arose, with the result that articles "for" or "against" Blackman's
theory have been appearing in botanical journals for now over forty j'êars.
This protracted and largely unnecessary controversy has hampered rather
than helped the penetration into plant physiology of the general principles
of i-eaction kinetics a.nd photochemistry (such as the law of mass action,
Boltzman's and Arrhenius' activation equations and the quantum principle
of photochemistry), which alone can provide adequate basis for the kinetic
treatment of any chemical reaction, whether in vitro or in vivo. It will ))e
shown below that, from the point of view of these principles, Blackman's
"law" is only an idealization, which can be more or less closely approxi-
mated under certain special conditions.
Brown and Heise (1917) and Brown (1918) were among the first to
criticize the way in which Blackman supported the law of limiting factors
by reinterpretation of the observations of earlier investigators, and to point

out that even Blackman's own measurements did not conform strictly to
the type of figure 26.2. Some subsequent investigations, e. g., that of
van der Honert (1930), produced curves that so closely approached the
"Blackman type" (c/., for example, fig. 27.2) that the authors believed the
law of limiting factors to be strictly valid under ideal experimental condi-
tions {e.g., uniform illumination of all cells; cf. page 864). Other, equally
reliable measurements gave, however, an entirely different picture
families of P = /(Fi) curves that diverged from the very origin {cf., for
example, fig. 27.1). On the basis of measurements of this type, Bose (1924)
went to another extreme and suggested, as an alternative to Blackman's
postulate, that the effect of a certain change in a factor, Fi, on the yield of
photosynthesis, isindependent of the prevailing values of all the other fac-
tors, F-2, Fs . . . (while according to Blackman this effect should depend en-
tirely on whether Fi is the "Hmiting factor" or not). Bose's postulate
requires that the curves representing the yield of photosynthesis in relation
to a factor Fi at different values of F2 have shapes of the type shown in
figure 26.3. Bose's "law" was derived from a very small number of meas-
urements, and is merely another approximation, applicable to certain con-
"Blackman behavior."
ditions opposite to those that favor the
"Blackman type" curve systems have widely separated saturation
plateaus, but coincident initial slopes; "Bose type" cunê systems also
have separated saturation plateaus, but distinct initial slopes. We will
often refer to Blackman type curve systems as the "first type," and to Bose
type curve systems as the "second type." A third type of kinetic curves,
also encountered in photosynthesis, is characterized by initial divergence,
but final convergence in a common saturation plateau (as in fig. 26.4).
862 EXTERNAL AND INTERNAL FACTORS CHAP. 26
The conditions that bring about the three types of curves will be discussed
later in this chapter.
While the limitations of Blackman's law of limiting factors were de-
bated, Liebig's notion of the "absolute minimum" of one nutritive factor,
from which Blackman's concept was derived, also was found to be too
rigid, and attempts were made to change it so as to admit the possibility of
simultaneous sensitivity of a crop to several nutrient factors, each of which
was said to be in a "relative minimum." New analytical formulations
of the minimum law, suggested, e. g., by Mitscherhch (1909, 1916, 1919,
1921) and Baule (1918, 1920), led to yield curves that approached the
maximum asymptotically, without a sudden break.
Similar compromise solutions were suggested for photosynthesis.
Harder (1921), Lundegardh (1921, 1924) and Singh and Lai (1935) con-
Fig. 26.3. Kinetic curves of the Fig. 26.4. Kinetic curves of tlie third t3Tie.
second type (Bose type). Arrow indicates increase of F-2.
eluded from their measurements that the rate of photosynthesis may de-
pend on several factors at the same time; when one "factor" gradually
ceases to be "limiting," the influence of another one increases. This con-
cept stands midway between the two postulates of Blackman and Bose;
attempts have been made to use it for a general formulation of the kinetic
relationships in photosynthesis.
However, because of the diversity of factors and conditions encoun-
tered in photosynthesis, it is unlikely that any analytical approximation
willprove equally satisfactory for all the cases studied. Liebig's law of
the minimum —
applied to one kind of factor only nutrient elements; it
was not unreasonable to suggest that it represents an equally good first
approximation for all such factors, and that an improved mathematical
formulation may provide a generally useful second approximation. Such
hopes are much less justified in the case of photosynthesis, where Blackman
included under the heading of "limiting factors" such heterogeneous mag-
nitudes as the concentration of a reactant (CO2) the amount of the sensi-

,
tizer (chlorophyll), the rate of supply of light energy, and temperature.

Maskell (1928-), in a paper from Blackman's laboratory, made one
more attempt to justify Blackman's law in its original form. He suggested
that the cause of the apparent deviations from this law lies in "mutual in-
teraction of factors." Here is one example of what he meant: In the
"carbon dioxide-limited state," the rate-determining carbon dioxide con-
centration must be that prevailing in the immediate neighborhood of the
chloroplasts. Because of the consumption of carbon dioxide by photo-
synthesis, this concentration is not necessarily identical with the outside
concentration (in the atmosphere or in the ambient solution) but depends ,
on the rate of diffusion of carbon dioxide from the medium to the chloro-
plasts, and thus on the aperture of the stomata. Consequently, if illumina-
tion causes changes in this aperture, it may indirectly affect the rate of
photosynthesis even in the "carbon dioxide-limited" state, not because the
rate is "truly" sensitive to both light intensity, /, and carbon dioxide con-
centration, [CO2], at the same time, but because illumination influences the
effective value of the factor [CO2]!
However, the insistence that a "true" must exist under
luiiiting factor
all conditions is alien to reaction kinetics. The

between the "law
relation
of limiting factors" and the general concepts of reaction kinetics was first
clearly stated by Romell in 1926. He pointed out that Blackman's term
"slowest factor" is meaningless, and that one can only speak of a slowest
process in a sequence of processes. The rate of a simple homogeneous re-
action usually is a function of all the relevant factors, e. g., the concentra-
tions of all reactants, temperature a photochemical process) light
and (in
intensity. "Limitation" effects of the type suggested by Blackman can oc-

cur only if the reaction, the over-all rate of which is being measured, con-
sists of several successive steps, with one step supplying the reactants for
the next one. If is slow, it becomes a "bottleneck,"
the supply process
and the velocity of the over-all reaction may become independent of all fac-
tors which do not affect this one "limiting" or "rate-determining" step. A
simple example is provided by many photochemical reactions, where the
supply of light-activated molecules is the "bottleneck," or limiting process.
Whenever "Blackman behavior" is observed in practice, it can be assumed
that one is dealing with a series of consecutive reactions that includes (at
least) one step of limited maximum efficiency. The rate of the over-all
process then cannot exceed the maximum rate of passage of the system
through this "bottleneck."
The bottleneck stage of photosynthesis may be the supply uf light en-
ergy, or thesupply of a reactant or the removal of a reaction product. We will

consider these three cases more closely in section 3 of this chapter. We will
—
see there that, even when a "bottleneck" reaction does exist, the relation-
ships postulated by Blackman are only approximated, but never exactly
fulfilled.
2. Photosynthesis Not a Homogeneous Reaction
In the kinetic treatment of photosynthesis, it is necessary to keep in

mind that photosynthesis is not a homogeneous reaction. This statement is
true in a twofold sense : In the first place, the photosynthetic apparatus is
—
a colloidal system with a definite structure probably containing rows of
oriented pigment molecules adsorbed on interfaces between proteinaceous
and lipide layers (c/. the discussion in chapter 12, Vol. I). In the second
place, the supply of light energy and of the reactants to different parts of
this structure is not uniform, particularly when photosjoithesis proceeds at
a high rate.
Let us consider these two aspects of the problem more closely.
The reactions involved in photosjaithesis are, at least in part, surface
reactions. It is furthermore likely that they can be called topochemical
reactions —meanmg that they occur not by encounters of moleculesmoving
about at random in two-dimensional adsorption layers, but by a more pur-
poseful mechanism, which requires the reacting molecules to take definite
paths past a number of catalytic "reaction centers." The products formed
in one reaction center are directed to the next one, so that back reactions
of unstable, intermediate products of oxidation and reduction products are
prevented by their spatial separation. Equations based on the law of mass
action can be applied to this type of reaction only with considerable reser-
vations.
The second aspect of the inhomogeneous character of photosynthesis
the nonuniform supply of reactants and energy to the different regions of
the photosynthetic apparatus —
was repeatedly discussed in the literature,
for example,by Schanderl and Kaempfert (1932) and Katz, Wassink and
Dorrestein (1942). Even if the chlorophyll molecules and the catalyst
molecules participating in photosynthesis were available uniformly in all
—
parts of a chloroplast which is in itself uncertain the supply of carbon —
dioxide, as well as that of light quanta, vary from place to place.
is likely to
In strong light, and with a limited supply of carbon dioxide, the chloro-
phyll molecules closer to the source of supply may use up most of the avail-
able reduction substrate and leave only little for the less favorably situated
pigment particles; in other words, the numerical value of the concentra-
tion factor [CO2] in kinetic equations may vary from place to place. A
similar consideration applies to the light mtensity factor, /: Because of
the high optical density of chloroplasts —particularly for red and blue-
PHOTOSYNTHESIS XOT A HOMOGENEOUS REACTION 865
violet light {cf. figure 22.35, p. 698)—the rate of light absorption is likely
to be much slower for chlorophyll molecules situated deep in the body of a

chloroplast (or on the "shady side" of it) than for those located on the light-
exposed surface. Thus, inhomogeneity cannot be avoided even by using
dilute cell suspensions in which illumination is the same for all cells, but
not for all chlorophyll molecules in them. In denser suspensions, only a
uniform time average of illumination of all cells can be achieved, and this only
by very intense stirring. In the thalli (^f multicellular algae, or in the
leaves of the higher plants, the disparity between the rates of light absorp-
tion in different cells cannot be corrected at all. The absorption in the
866 EXTERNAL AND INTERNAL FACTORS CHAP, 26
teria), can be recalculated to represent P in relation to the average light

intensity, /, actually falling on an individual algal or bacterial cell (see
p. 1009 and figs. 28.22A, B and C).
3. Some General Kinetic Considerations
Some investigators, who realized the inevitable distortion of light curves

and carbon dioxide curves of photosynthesis by the "depth effects" dis-
cussed in the preceding section, assumed that in the measure in which these
effects can be eliminated (experimentally, by the use of very dilute systems,
or mathematically, by applying adequate corrections for inhomogeneity)
the kinetic curves would approach the ideal "Blackman type." Undoubt-
edly, the elimination of depth effects shortens the transitional region be-
tween the ascending part of the curves and the saturation plateau; but
figure 28.22C shows that it does not make the breaks sharp. Only a frac-
tion of deviations from "Blackman behavior" can be attributed to inhomo-
geneity; even with all "depth effects" eliminated, the kinetic analysis of
photosynthesis will still have to contend with kinetic curve systems of all
three types exemplified by figures 26.2, 26.3 and 26.4.
The common feature of all these curves is the occurrence of saturation,
i. e., of states in which the rate of photosynthesis is independent of the vari-
able Fi.The saturation level may depend on the parameter F2 —as in the
Blackman type and Bose type curve systems (figs. 23.2 and 23.3) —or it may
be independent of F2, in which case curve systems of the "third type"
(fig. 23.4) are observed.
(a) Sources of Saturation in PhotosTjnthesis
The over-all rate of a process consisting of a series of successive chemical

or physical stages —sometimes referred to as a catenary series — cannot ex-
ceed the rate of any of its individual steps. "Saturation" of such a process
with respect to a given kinetic variable, Fi, is therefore reached whenever
the over-all rate becomes equal to the maximum rate of a single step (a
step which in itself must be independent For example,
of this variable).
under given conditions of external carbon dioxide pressure and temperature
(perhaps also humidity and other factors affecting the colloidal structure of
the cell), carbon dioxide can be supplied to the photosynthesizing cells at
not more than a certain maximum rate, which is reached when the station-
ary concentration, [CO2], at the site of photosynthesis is zero, and the dif-
fusion gradient between the medium and the chloroplast has therefore the
maximum possible value. This maximum rate of carbon dioxide supply by
diffusion is independent of illumination (except for possible indirect rela-
tions of the type mentioned on page 863). Therefore, "light saturation"
SOME GENERAL KINETIC CONSIDERATIONS 867
should occur whenever the over-all rate of photosynthesis approaches the

maximum rate of carbon dioxide supply by diffusion. In this considera-
tion, the diffusion of carbon dioxide could be replaced bj^ a preliminary
chemical reaction the rate of which is proportional to the concentration,
[C02],e-g-, the formation of the compound !C02} from carbon dioxide and
an "acceptor," which was postulated in chapter 8 (Vol. I). In this case,
light saturation is determined by the maximum possible rate of formation
of {CO2} that is reached when all acceptor molecules are free, (i. e., when
all complexes CO2 { 1 are utilized for photosynthesis practically instantane-
ously after their formation). Similarly, "carbon dioxide saturation" must
occur whenever the over-all rate of photosynthesis approaches the rate of
supply of light energy, and the quantum yield assumes its highest possible
value.
There are other factors, besides carbon dioxide supply and the supply
of light energy, which also can impose "ceilings" on the over-all rate of pho-
tosynthesis and thus cause saturation phenomena. This role can be played,
e. g., by the concentration of any one of the several catalysts participating
in photosynthesis (including the "photocatalyst" chlorophyll). For ex-

ample,if one reaction step in the "catenary series" of photosynthesis is the
monomolecular transformation of a catalyst-substrate complex:
(Catalyst + Substrate) > Catalyst + Product
the maximum rate of this reaction step is reached when all the available
catalyst molecules are loaded with substrate molecules. When photosyn-
thesis proceeds at a rate that requires such maximum utilization of one cata-
lyst, variations in most kinetic variables (such as the concentrations of the
reaction partners or of the other catalysts, or light intensity) cannot increase
the rate any further. Only a rise of temperature can lift the "ceiling" im-
posed by the maximum velocity of such a catalytic transformation. The
part of a "rate-limiting" catalyst can be assumed by any of the several
catalysts the existence of which was postulated in Volume I {cf. chapters
6, 7and 9) —for example, the "carboxylase" Ea, the "stabihzing catalyst"
(a "mutase" Eb, or the "deoxygenases" Ec and Eq. Chlorophyll can
?)
play a similar role, c. g., if the primary photochemical process involves a

chemical change of this pigment, and a certain time is required for its restor-
ation. "Acceptors" or "carriers," such as the carbon dioxide acceptor
postulated in chapter 8 (Vol. I) are catalysts, too, and the available quan-
tity of any such auxiliary compound also can serve as a "limiting factor"
in photosynthesis.
All these factors can — and most of them probably do —contribute to
the limitation of the rate of photosynthesis under different conditions, thus
causing the "saturation" of this rate wath respect to vacious kinetic vari-
.
868 EXTERNAL AND INTERNAL FACTORS CIL\P. 26
ables. There is no theoretical reason to expect, and no actual experience

to indicate, that saturation with respect to, say, light intensity, or carbon
dioxide concentration, is always caused by the same "limiting factor." To
the contrary, clear indications can be found of saturation phenomena caused
by slow diffusion, slow carboxylation, various catalytic deficiencies, limited
lightsupply and limited supply of reductants (in bacterial photosynthesis)
One may consider this as evidence of good adjustment of the photosynthetic
process as a whole, since it means that the different parts of the complex
mechanism of photosjTithesis have approximately the same maximum
capacity. One understands, in the light of this multiplicity of possible
rate-limiting steps, why repeated attempts to represent the kinetics of
photosynthesis by means of models consisting of a small number of reac-
tions, e. g., of one light reaction and one dark reaction only, could not have
led to more than very limited success.
Saturation with carhon dioxide is reached at pressures three or four times
higher than the partial pressure of carbon dioxide in the free atmosphere
(c/. Table 27.1); while saturation with light is reached at light intensities
equivalent to 10-100% of full midday sunlight
Table 28.1) and thus (c/.
about equal to the average light intensity to which freelj^ growing plants
are exposed in nature. The optimum temperature of photosynthesis is some-
what above the average summer temperature, at least in temperate zones.

An approximate adjustment of the photosynthetic mechanism to natural
conditions is thus obvious. Perhaps, this adjustment was achieved in
times when both the average temperature and the carbon dioxide content
of the atmosphere were somewhat higher than they are now. However,
this inference is by no means certain nature has seldom been able to de-
;
velop ideal solutions of its adaptation problems, and is usually satisfied

with more or less rough approximations. The present kinetics of photo-
synthesis may have been the best plants were able to evolve in response
to the now prevailing climatic conditions.
(h) Origin of Kinetic Curve Systems of Different Types
The distinctive feature of "Blackman type curves," described on page

860, are closely coincident initial, linear parts of the cui'ves. This charac-
teristic distinguishes them from the two other t}ês of curve systems, repre-
sented in figures 26.3 and 26.4. On the other hand, Blackman curves
(fig. 26.2) and Bose curves (fig. 26.3) have in common a wide spacing of
saturation plateaus, v.hile curves of the "third type" in figure 26.4 all ap-
proach a common saturation level.
Coincidence or separation of the saturation levels depends on whether
saturation is imposed by the same parameter ¥-2, to which the set of curves
SOME GENKRAL KINETIC CONSIDERATIONS RHO
refers, or by some other parameter, Fg, F4 For example, in a set of

. . .
curves, P =/[CO.], for various values of light intensity, I, the saturation

levels will be well separated (as in figs. 26.2 and 26.3) if saturation is im-
posed by the rate of light supply, but will coincide (as in fig. 26.4) if satura-
tionis due to the limited rate of a dark, catalytic reaction.
Coincidence or divergence of the initial slopes depends on the qualitative

and quantitative relationships between the variables Fi and F2. If both
these variables affect the rate of the same reaction step, the rate will gener-
ally depend on both of them. For example, if Fi is the concentration,
[CO2], and F2 is temperature, and if the slope of the ascending part of the
curves P = f [CO2] is determined by the velocity of carbon dioxide m\)p\y
by diffusion, this slope will depend on temperature. If, on the other hand
(a) the factors Fi and F2 affect different steps in the "catenary series"
(e. g., if Fi is light intensity and F2 is temperature) and (6) the

relative
values of Fi and F2 are such that the process affected by F2 is far below its
maximum rate when that affected by Fi approaches its maximum rate-

then (and only then) the reaction rate will be a function of Fi alone, and
practically independent of F2.
The second condition (b) is important. Contrary to the way in which
the concept of "limiting factors" or "rate-determining reaction steps" often
is used, the existence of a reaction step of limited maximum efficiency af-
fects the rate of the over-all reaction long before the rate actually "hits
the ceiling." Therefore, if several reaction steps have maximum rates
which are not too different in their order of magnitude, the rate of the over-
all reaction must be affected by all these "potential bottlenecks" and not
only by the "narrowest" one.

This fact was recognized in the alterations of Liebig's "law of the mini-
mum," which we have mentioned on page 862, and some quantitative illus-
trations of it will be given later in our analytical discussions. To make it
plausible, we may use a mechanical analogy; the flow of water through
a system of pipes with several strictions depends on the diameter and length
of all of them, and not only on the one that has the greatest flow resistance.
In the plot of P against Fi, for different values of F2, we can expect all
cui-ves to be to the right of, and below, two "limiting" lines: one a slant-
ing "roof" imposed by the maximmn rate of the "slowest" reaction step
the rate of which is more generally, is a function of
proportional to Fi (or,
Fi) ; and the second a horizontal "ceiling" imposed by the maximum rate
of the slowest reaction step the maximum rate of which is independent of
Fi. For example, in the case of curves P = f [CO2], for different tempera-
tures, the two limiting curves may be determined by the maximum rate of
diffusion of carbon dioxide and the rate of light absorption, respectively
(cf. fig. 26.6). These two limiting lines together form a typical "Blackman
—
curve"; but they are merely which the actual kinetic curves
limits within
are confined. The point we are trying to make —
in advance of its analyti-
cal proof — is that it would be wrong to imagine that the existence of the
"roof" OA and the "ceiling" BC will leave unaffected the curves situated
entirely within the "permitted area" OXC, and merely force back into this
area the curves (or section of curves) that would otherwise cross the limit-
Fig. 26.6. "Roof" and "ceiling." OA, maximum rate of a diffusion step
(proportional to [CO2]); OA maximum rate of carboxylation (proportional
',
to [CO2]); BC, maximum rate of primary photochemical process = rate of (
light absorption) (independent of [CO2]); B'C, B"C", maximum rates of

catalytic reactions.
ing lines. To the contrary, the existence of the "roof" and the "ceiling"
as well as that of the potential, higher "roofs"and "ceilings" {OA' B'C, ,
B"C" in fig. 26.6) —

push down and toward the right even the kinetic
will
curves that would not have approached the limiting values if the limitations
were absent.
We will have opportunity for analytical proof of these statements in
subsequent chapters. For example, in chapter 27 we will derive "carbon
dioxide curves" of photosynthesis from simple models of the mechanism of
entry of carbon dioxide into the photosynthetic reaction and we will find ;
there that the sequence of the two reactions:

: . :
SOME GENERAL KINETIC CONSIDERATIONS 871
(26.1) CO: +A .
' ^ ACO2 — > A + {HCO2}
a reversible binding of carbon dioxide by an acceptor A followed by

(i. e.,
reduction of the complex ACO2 by a photochemically produced reductant

{H}) leads to a "Bose type" system of curves P = /[CO2] (where Pis the
rate of the over-all reaction). If carboxylation is so rapid (compared with
photosjTithesis) that its equilibrium not disturbed even in strong light, is
these "carbon dioxide curves" will be hyperbolae, which diverge from the
origin, and remain in a constant ratio up to saturation. Saturation corre-
sponds, in this case, to complete carboxylation of all the available acceptor,
and is therefore reached at the same value of the variable [CO2] in all
curves. The saturation rate rises with increasing concentration of the
reductant {H} (and therefore also with light intensity, since wê assume
that {
H } is produced by light)
We will further see that, if carboxylation is a slow process the rate of

which is proportional to [CO2], a [C02]-proportional "roof" is imposed on
the rate of the over-all reaction, namely
(26.2) Pmax. = A-„Ao [CO2]
where Ao is the total concentration of the acceptor*, and ka the rate

constant of carboxylation {cf. equation 26.1). We will show that, because
of this "roof," the curves P = f [CO2], which would otherwise begin with
the slope kÂo {i. e., which would stay just inside the "permitted area")
will be reduced to an initial slope half as large, i. e., kaAo/2. More gener-
ally, curves that, without limitation, would have begim with a slope akÂo
will be reduced to an initial slope kÂoa/(a 1). Thus cui-ves that in +
the case of rapid carboxylation would begin with slopes between 10 kÂo
and 100 kaAo, would all be confined, in consequence of slow carboxylation,
to slopes between 10/11 fcÂo and 100/101 fc„Ao, and would thus present a
"Blackman picture." If the carboxylation product, ACO2, in equation
(26.1) has toundergo a monomolecular transformation before it can react

with {H}, this would impose a [C02]-independent ceiling on the rate of the
overall reaction, namely
(26.3) Pmax. = kiAo
where ki is the rate constant of the postulated monomolecular transforma-

tion, and Ao the total available quantity of A. As a result of this "ceiling,"
the curves P = f [CO2], which would otherwise reach a saturation value of
P = kiAo, will be reduced to a saturation level half as high, P = A;iAo/2.
* We omit square brackets in the designation of constant concentration, i. e., we
write Ao, Chlo, etc., instead of [A]o, [Chl]o, etc.
—
The curves that would other\vise reach a saturation value high above fciAo
willbe crowded into a narrow space immediately below fciAo, and the curves
with saturation values below fciAo, will all be depressed. A curve the satu-
ration level of which would otherwise be iSfciAo will be reduced by the im-
position of the ceiling to a saturation level of ^kiAo/(0 !)• It follows +
from this formula that, even if the saturation rate without the limitation
were only one tenth of the maximum rate of the postulated monomolec-
ular transformation of ACO3, it would nevertheless be reduced by 10% by
this "potential bottleneck."
4. Internal Factors and the "Physiological Concept" of Photosynthesis
The on page 860, that the rate of photosynthesis

belief, referred to
must obey a simple and rigid kinetic law has been partly responsible for the
conclusion, reached by some plant physiologists, that it does not obey any
recognizable kinetic law at These physiologists, disillusioned by the
all.
over-simplifications in which the preceding generation had indulged, turned

their attention to the complex relations between photosynthesis and other
phenomena of plant life, such as nutrition, respiration, growth or aging.
Factors often referred to as "internal" or "plasmatic" are supposed to be
responsible for these relations. Stressing the prime importance of these
factors in the regulation of photosynthesis, Kostychev and his pupils
minimized or denied the direct influence of the easily regulated "external"
factors, such as light intensity, CO2 concentration and temperature.

Their revolt against the apphcatioa of kinetic laws to photosynthesis and similar
processes would perhaps be less violent if these physiologists would have reaUzed that the
"law of hmiting factors" is by no means the last word in the physicochemical approach
to photosynthesis, that, in fact, the concept of "hmiting factors" is foreign to reaction
kinetics. The no other kinetic laws are possible led Chesnokov and Bazyrina
belief that
(19.30) to argue that since, according to Harder and Lundegardh, the factors "carbon
dioxide concentration" and "light intensity" are not "truly limiting" (z. e., that often
tne change in either of these factors can affect the rate) the "true" limiting factors must
be sought inside the plant. It did not occur to them that photosynthesis may have no
"true" limiting factor at all.
Kostychev, in an article called "A New Concept of Photosynthesis"
(1931), suggested that "external" factors affect photosynthesis mainly, if

not exclusively, in an indirect way, by stimulating or inhibiting certain
unknown ])lasmatic activities. All the conclusions obtained by Blacknian
(and others lêfore and on the basis of the "ph\sieochenii(ud"
after him)
approach were rejected Ijy Kostychev as spurious. A similar point of view
was taken by van der Paauw (1932) and by Kostychev's co-workers,
Chesnokov and Bazyrhia (1930, 1932), who sought to prove by experiments
—
that two external factors carbon dioxide concentration and temperature
have no direct effect on the rate of photosynthesis at all, and that the third
—
RATK UNDER CONSTANT CONDITIONS 873
one —light intensity —affects this process only partly by direct action, and
partly through plasmatic stimulation.
In the U.S.S.R., there is a tendency now to consider this point of view as the only
one in accordwith dialectic materialism, and attempts to isolate photosynthesis from
other functions of the Uving organism and study it as an independent photochemical
reaction are criticized as "mechanistic."* Such dogmatic assertions, practically
banished from physics and chemistry, but still recurring in biological sciences, particu-
larly in the U.8.S.R., are strangely beside the point. A reaction in a living organism
is distinguished from that in a test tube by the complexity of the system in which it
takes place. This complexity is due to three causes: the impossibility of separating the
reacting system from other components of the organism; its inhomogeneous structure;
and its complex and largely unknown chemical composition. Unprejudiced experiments
alone can prove whether, despite these handicaps, direct relationships can be established
between external kinetic variables and the rate of the specific process under investigation.
If this proves possible, a promising approach to the understanding of the process is
opened, and it would be foolish to refuse to use it because of dogmatic objections.
5. Rate of Photosynthesis under Constant Conditions.

Midday Depression and Adaptation Phenomena
Obsei-vations that lend support to the "physiological" concept of photo-

synthesis include, among others: the difference in photosynthetic activity,
under identical external conditions, of plants grown in various habitats;
the adaptation of photosynthetic activity to changed conditions (stronger

or weaker light, higher or lower temperature cf. for example, Harder 1933
and Brilliant 1940); the effects of aging; and the changes of photosyn-
thetic activity under constant external conditions (fatigue, midday depres-
sion etc.) Not only do plants of different species behave differently under
.
identical external conditions, but variations are found also between "sun
plants" and "shade plants" of the same species, "sun leaves" and "shade
leaves" on the same branch and even between different parts of the same
leaf (c/. Drautz 1935). The photosynthetic activity of a plant often
changes strongly in the course of a smgle day, not to speak of a whole season.
Obsei-vations of diurnal changes have played an especially important role
in the development of Kostychev's "new concept" of photosynthesis.
Offhand, one would expect photosynthesis to increase steadily after
sunrise until the light intensity has reached the saturating value, and then
remain more or less constant, unless cloudiness decreases the illumination
below the saturating intensity, until the evenmg decline sets in. The actual
behavior of the plants often follows, however, a much more complicated
* An monograph, Photosynthesis as Life Process of the Plant, by Miss
interesting
Brilliant (1947)seems to be the only comprehensive review of photosynthesis published
in Russian in recent years. It contains a survey of about 200 Russian and 350 other
papers, many of them not utilized in the present book.
pattern. Thoday (1910) discovered that the production of organic material

by leaves may show a temporary decHne in the middle of the day; the
plant takes an "afternoon nap" (c/. fig. 26.7). McLean (1920) observed
that this decline may even result in the release of carbon dioxide.
The gas exchange measurements Kostychev and co-workers (1926-
of
1931) (cf. also Chesnokov, Bazyrina and co-workers 1932), carried out
under a large variety of climatic conditions, from Central Asia to the Arctic
Sea, and with algae as well as with land plants, showed that the phenom-
enon of "midday rest" is widespread in the plant world, but that it assumes
8 II 13 14 17 20
TIME AFTER MIDNIGHT, hr.
Fig. 26.7. Diurnal course of photosynthesis of two leaves of Erio-

botrya japonica under natural conditions (after Kursanov 1933):
solid line, May 30; broken line, June 6.
the extreme form of a reversal of photosynthesis and evolution of carbon

dioxide only under special conditions, particularly in hot climates.
The diurnal course of photosynthesis has received the attention also of numerous
other investigators, among whom we may mention Geiger (1927), Montfort and Neydel
(1928), Maskell
(1928i), Hiramatsu (1932), Harder, Filzer and Lorenz (1932), Bosian
(1933), Kursanov von Guttenberg and Buhr (1935), Monch
(1933), Stalfelt (1935),
(1937), Filzer (1938), Neuwohner (1938), Neubauer (1938), B. S. Meyer (1939) and
Bohning (1949).
Harder (1930) and co-workers, Schoder (1932) and Drautz (1935), as

well asNeuwohner (1938), have attempted to explain the diurnal curves by
combined variations of several external factors (light intensity, humidity
and temperature) They succeeded only partially, and had to admit that a
.
considerable part of the observed variations remained unexplained and had

to be attributed to unknown "plasmatic" factors. This is particularly
clearly demonstrated by the observations of Filzer (1938), who found that
leaves, picked from trees at different times of the day and then investigated
under constant conditions in the laboratory, showed the same periodic
changes in photosynthetic production as did leaves left attached to the
plant and exposed to the natural change of night and day. Similarly, Mas-
MIDDAY DEPRESSION 875
kell (1928^) found that detached cherry laurel leaves, illuminated with con-
stant light for 24 hours,showed a deep depression of photos>Tithesis during
the night hours; thus, not only the "midday nap," but also the "night
sleep" appears to be influenced by internal factors.
Geiger (1927), Maskell (19281-2) and St^lfelt (1935) considered the
closure of the stomata as the immediate cause of the middaj^ depression.
Maskell (1928^) observed that the nightly depression of photosynthetic
activity of steadilj^ illuminated leaves can be avoided by increasing the
jmrtial pressure of carbon dioxide ("pressing carbon dioxide through half-
closed stomata"), and that steadily illuminated leaves of Hydrangea
(the stomata of which are almost rigid) showed only a slight decline of
photosynthesis during the night hours. Both Maskell (1928^) and St§,lfelt
(1935) found a parallelism between the average aperture of the stomata and
the rate of photosynthesis (of. chapter 27, page 910).
It thus seems plausible that the diurnal rhythm of photosynthesis of

the higher land plants is by stomatal move-
to a large extent conditioned
ments. The question remains, however, what causes the stomata to close
at certain times of the day, even though the illumination and the carbon
dioxide supply are kept constant?
One "internal factor" that has been much discussed in connection with
this problem is the accumulation of (soluble or insoluble) carbohydrates.
(Concerning the effect of excess carbohydrates on the rate of photosynthe-
see chapter 13, Vol. I.)
sis, The midday depression may be a pause during
which these materials are translocated or partially combusted. This ex-
planation, first accepted by Kostychev, Kudriavtseva, Moisejeva and
Smirnova (1926) and Kostychev, Bazyrina and Chesnokov (1928), was
later rejected by Chesnokov and Bazyrina (1930^), w^ho found that plants
with entirely different diurnal course of translocation may nevertheless
show the same diurnal course of photosynthesis. It was on the basis of
results such as this that Kostychev (1931) finally reached his extreme con-
clusion concerning the purely physiological regulation of photos\Tithesis.
Against these findings of Kostychev and co-workers, Kursanov (1933),
von Guttenberg and Buhr (1935) and Monch (1937) confirmed the existence
of a relation between the accumulation of sugars and starch and the diurnal
rhythm of photosynthesis. However, according to von Guttenberg and
Buhr (1935) and Neuwohner (1938), no smgle ex-planation can be made to
fit all cases of midday depression. In some cases (e. g., in young leaves in
spring) it is clearly traceable to the choking of the photosynthetic ap-
paratus with carbohydrates. In other cases {e. g., in summer leaves on hot
days) the loss of water and the ensuing closure of stomata provide the most
plausible explanation. Accumulation of half-oxidized products which
"narcotize" the photosynthetic apparatus, as suggested in Franck's theory
of induction (chapter 33) , is another type of mechanism which must be taken

into consideration. Drop of the CO2 content of the air (Bohning 1949) and
enhanced CO2 supply through the roots (p. 910) also have been blamed for
themidday depression.
The phenomenon of midday depression was found by Montfort and
Neydel (1928) also in stomata-free ferns, and by Kostychev and Soldaten-
kov (1926), Kursanov (1933) and Neubauer (1938) in algae.
Gessner (1938) found no pronounced midday decline of photosynthesis
in the higher aquatic plants (Elodea, Potomageton etc.), except with shade-
adapted species or individual plants in which it could be interpreted as
"inhibition by excess light" {cf. Volume I, page 535). Although minor
fluctuations of the rate remained unexplained, the rate of photosynthesis in
Gessner's submerged plants generally followed the changes in the intensity
of illumination. The maximum of photosynthesis was often found in the
early afternoon rather than at noon, but this could be explained as a tem-
perature effect.
Chesnokov, Giechikhina and Jermolayeva (1932) found that respiration, too, has a
complicated diurnal rhythm. Because of this, the true rate of photosynthesis cannot be
obtained by applying a uniform respiration correction to the net rate of oxygen liberation
measured at different times of the day. They also found that the respiration of leaves
often is much stronger than was generally assumed before. In young leaves, in particu-
lar, the rate of respiration may approach that of photosynthesis. This explains why
the rate of carbon dioxide liberation by some plants during the midday depression was
found to be almost as large as the rate of the carbon dioxide consumption by photosyn-
thesis before and after this rest period.
However interesting the phenomena of the diurnal rhythm of photo-

synthesis, and similar "physiological" effects (such as aging, fatigue, etc.)
may be, the primary question for a kinetic study of photosynthesis is not
whether these variations can be explained, but whether they can be elimi-
7iatcd, and photosynthesis made to proceed at an even and reproducible
rate. It is difficult to realize such steadiness in field experiments. Boysen-

Jensen and Miiller (1929), Boysen-Jensen (1933) and Mitchell (1936) said
that, if conditions are reasonably constant, rate of photosynthesis in natural
surroundings remains steady but Maximov and Krasnosselskaja-Maximova
;
(1928) and Waugh (1939) observed that the photosynthetic production of

leaves on the tree fluctuated, under constant external conditions, in suc-
cessive four minute periods, by as much as =*= 100% of the hourly average.
Kostychev (1931) concluded from these observations that measurements
of photosynthesis over short periods have no meaning, and that the
minimum time over which photosynthesis should be measured is a whole
day!
Stocker, Rehm and Paetzold (1938) found that rapid changes of the
rate of jjhotosynthesis under natural conditions (fluctuation period:
.
RATE FLUCTUATIONS 877
several minutes) are associated with similar fluctuations of the carbon

dioxide concentration in the air.
Scarth, Loewy and Shaw(1948) observed that the photosynthesis of

detached leaves, determined by measuring the infrared absorption of
carbon dioxide, occasionally showed unexplained, regular fluctuations
(with a period of the order of 1 hour)
When plants are investigated under natural conditions, the apparently
erratic behavior can be attributed to the difiiculty of controlling all the
relevant factors. However, considerable doubt has also been expressed
as to the capacity of plants to carry out photosynthesis at a constant rate
under controlled conditions in the laborator>\ Experiments with lower
plants, e. g., unicellular algae, such as Chlorella, have given comparatively
satisfactory results: If certain prescriptions concerning culture and treat-
ment were adhered to, these algae could be relied upon to maintain a con-
stant and reproducible rate of oxygen production for several hours (leav-
ing aside the short time induction phenomena to be discussed in chapter
33). According to Pratt (1943^), when alkaline buffers are used, the con-
stancy of the rate depends on the nature of the cation present: Thus, in
0.1 M NaHCOs, the rate declined during the first 10 hours and then be-
came steady; M KHCO3, increased during the 10 hours, remained
in it first
steady for the next 5 hours and then declined rapidly; in 0.065 M Na-
HCOs + 0.035 M KHCO3, the rate remained steady for the 15 hoursfirst
and then began to decline (see fig. 25.1).

Noddack and Eichhoff (1939) stated that for a given suspension of
Chlorella, the rate of photosynthesis is reproducible within ±10%, and
that these variations can be further reduced by preliminary adaptation of
the cells to the light intensity in which they are to be studied.
Experiments with higher land plants or aquatic plants have been con-
tradictory, and at first rather discouraging. Tnie, Willstatter and StoU
(1918) found that detached leaves, properly supplied with water and car-
bon dioxide, maintain a constant rate of photosynthesis (within a few per
cent) for 4 or 6 hours, even in strong light (40,000 lux) but Harder (1930,
;
1933), Arnold (1931) and Jaccard and Jaag (1932) asserted that strong
trends as well as irregular changes develop in the photosynthesis of aquatic
plants kept imder constant external conditions. Arnold obsei^ved, e. g.,
that in moderately strong light (18,000 lux) the rate of photosynthesis of
Elodea dropped, in 2 or 3 hours, to one fifth or one tenth of its origmal
value; at 4000-6000 lux, it increased for the first 2 or 3 hours and then
decreased slowly only at 2000-3000 lux did it remain approximately con-
;
stant for several hours. Harder (1930, 1933) made similar observations
and stated that light intensity must be measured relative to the intensity
to which the plants have been "acclimated" before the experiment. By
.
changing the ratio of these two intensities, he obtained the family of curves
—
represented in figure 26.8 some showing a steady increase in photosynthe-
sis with time, others exhibiting an equally steady decline. He interpreted
these time phenomena in terms of three plasmatic effects: "activation,"
Fig. 26.8. Changes of photosynthesis

with time in aquatic plants under constant
conditions. Numbers indicate increasing
ratios between the conditioning and the
illuminating intensity (after Harder 1930).
"deactivation" and "exhaustion" (the last two being distinguished by the

duration of the dark period required for recovery)
Experiments by Gessner (1937) and Steemann-Nielsen (1942) indicated,
however, that only the phenomenon of Induction (Harder's "activation")
is of fundamental nature (it will be dealt with in chapter 33), whereas
—
"deactivation" and "exhaustion" can be avoided at least as far as water
—
plants in light of about 40,000 lux are concerned ^by preventing the stag-
nation of water, and the consequent dwindling of the carbon dioxide supply.
^^
o.... s
3
TIME, hr.
Fig. 26.9. Constant photosynthesis of a light-adapted plant (L) and a

shade-adapted plant (S) of Elodea canadensis (after Gessner 1937).
That stagnant water in the immediate neighborhood of assimilating plants

can easily be depleted of carbon dioxide (even if it contains reserves in the
form of carbonates and bicarbonates) was first i)roved by the calculations
of Romell (1927). Figure 26.9, taken from Gessner, shows the time course
RATE UNDER CONSTANT CONDITIONS 879
two twigs of Elodea canadensis, one adapted to strong

of photos3^lthesis of
and another to weak Hght, in a steadily renewed medium. Both show
light
good constancy for many hours of uninterrupted illumination (after an
induction period in the shade plant)
initial Some of Gessner's experiments
.
were extended over 6 days, with rate variations remaining within ±25%.
If to these results of Gessner with aquatic plants we add those of War-
burg and his successors with unicellular algae, and of Hoover, Brackett,
and Johnston (1933), Mitchell (1936) and Bolming (1949) with higher
land plants, there appears to be no fundamental difference between plant
300-
N
O
n
E
e 200-
in
>-
<n
o
h-
o 100 -
X
Q.
U.
o
UJ
a:
20 40 60 80 100 120 140

INCIDENT INTENSITY
Fig. 26.10. Gas exchange of Chlorella cells from cultures of

different ages (in air, temperature 29° C.) (after Wassink and
Katz 1939).
classes with respect to their capacity to carry out uniform photosjiithesis

for considerable periods of time.
Experiments with carefully treated plant material show a simple, di-
rect and reversible response of the rate of photosynthesis to at least two
external factors, light intensity and concentration of carbon dioxide (and
within certain narrow limits, also to changes in temperature).
Obviously the "internal factors" are in no way eliminated, even in such
selected material. Their importance is revealed in the induction phenom-
ena, in the permanent effects of age and previous treatment and in the
fluctuations of the rate, by as much as 10 or 20%, which occur without any
apparent external reason. Different plants or algal suspensions of the same
species (even when s^'own under apparently identical conditions) may differ
in photosynthetic production by a factor of 2 or 3. Effects of age appear
not only in the higher plants (see the comparison of young leaves with ma-
ture leaves in table 28.V, after Willstatter and Stoll, 1918) but also in cul-
tures of unicellular algae (c/. fig. 26.10; van Hille 1937, 1938, Wassink
and Katz 1939 and Pratt 1943). We
cannot hope to eliminate these
internal factors in the study of photosynthesis, but it is possible to keep
them a,]iproximately constant, at least for the duration of an experiment.
Furthermore, there is not much point in treating these internal factors as
mysterious ''plasmatic stimulations" or "inhibitions." It is reasonable to
expect that some of them will be traced to accumulations or ehminations of
—
certain chemical compou7ids catalysts, poisons or metabolites while —
others will be found to be connected with changes in the physical structure
of the photosynthetic apparatus, for example, with the swelling or shrink-
ing of the protoplasm or changes in permeability of cell membranes. In
chapter 33 we will discuss the most extensively studied example of the ac-
tion of "internal factors"—the induction phenomena, and will attempt to
mterpret them as a result of deactivation, in the dark, of certain catalysts
required for photosynthesis, combined with the accumulation of metab-
olites possessing narcotizing properties.
6. Aging and Self-Inhibition
The aging effect, in Chlorella in particular, shown in figure 26.10, has

been correlated by Pratt (19431'^) Avith the gradual accumulation of a
growth-inhibiting substance of definite chemical and biological properties.
Since this is the first case in which an "internal factor" in photosjmthesis
was identified as a chemical entity, the observations of Pratt will be
described insome detail.
The production of a growth-inhibiting factor in Chlorella cultures was
discovered by Pratt in 1940 (c/. chapter 25, page 833). Later (1942) he
observed that this "factor" is a substance that can be extracted from "aged"
cells, and whose action can then be demonstrated on young cultures.
Its
molecules are ~ 15 A in diameter; it is soluble in 95% ethanol, ether, pe-
troleum ether and water and destroyed by heat. It is more effective in

is
neutral than in acid solution, and can more easily be extracted from alka-
line than from acid aqueous solutions. It has considerable antibiotic
effect on bacteria.
The water-soluble growth-inhibiting substance (prepared by extracting
dried cells of50-60 day old Chlorella vulgaris cultures with 95% ethanol,
evaporatmg to dryness and extracting with water) was tested for its effect
on photosynthesis of Chlorella cells from 4 day old cultures (Pratt 1943).
AGING AND SELF-INHIBITION 881
Figure 26.11 A shows that the rate declined Imearly with the logarithm of
the amount of extract added. Experiments at various light intensities
(fig. 26.1 IB) showed that the inhibitor affects the saturation rate in strong
light rather than the quantum yield in weak light, i. e., it acts like a catalyst
1.0 -
0.9-
on
LlI
> 0,
Hi
fig. is sufficient for saturation).

26.1 IB, Figure 26.12 shows the dedine
of the ratewith advancing age; after 30 days, the rate was only one third
—
the original in fair agreement with Wassink and Katz's results in figure
26.10. Respiration also declined during the same period, but a httle less
than photosynthesis (to about one half the initial rate). The concentra-
tion of chlorophyll and the size of the cells showed no marked change.
The influence of the inhibitor produced by aged cultures can be recog-
nized even in the "second generation". The cultures prepared by inocula-
tion with a 3 day old culture showed, after 5 days' growth, a 10% higher
rate of photosynthesis than a similar culture prepared by inoculation with
material from a 23 day old culture. During the 5 days' growth, the num-
ber of cells has increased by a factor of 20, but the effect of the inhibitor
was noticeable even after this dilution.
Pratt, Oneto and Pratt (1945) found that in Chlorclla cultures grown
in continuous light in inorganic medium, the inhibiting agent ("chlorel-
Hn") accumulated in the medium rapidly in the first 3-4 days; its con-
centration decreased sharply in the next 2-4 days, and then grew again,
reaching a constant level in about 2 weeks. The decline in chlorellin con-
centration occurs at the period when the increase in the number of cells
per unit volume is most rapid. Possibly, rapidly dividing cells use up all
the available chlorellin for their own metabolism (assuming it is a useful
product) ; alternatively merely a poison, not only for bac-
(if chlorellin is
teria but also for the algae themselves), these cells may produce an anti-
dote to this poison.
"Chlorellin" production was also studied, in co-operation with Pratt
and his group, by Spoehr and co-workers (1944, 1945, 1946). They worked
on improvement of the methods of cultivation of algae and extraction of
the antibiotic, first using cell-free culture medium, and later, the cells
themselves. The quantity of antibiotic extractable from dried cells was
found to increase by allowing the undergo oxidation (by grind-
latter to
ing and exposing to light in air). Brown, partly crystalline material, ex-
tracted from dried cells with 80% methanol containing 2% KOH, and
transferred into petroleum ether after acidification, is nonantibiotic; it
becomes colorless and antibiotically active by oxidation in light. It thus
seems that living cells contain no antibiotic material the latter is formed ;
by oxidation, both in the medium and in dried and ground cells (it remains
to be proved that the two antibiotic products are identical). The cell-
—
derived antibiotic is a lipoid probably a mixture of unsaturated fatty
acids. Pure acids of this type (linoleic, elaidic, etc.) also show no anti-
biotic activity before exposure to oxygen and but acquire antibiotic
light,
properties after such exposure. Autoxidation of unsaturated compounds

is known to produce peroxides whose bactericidal effect is well established.
—
However, the antibiotic activity of "chlorellin" is not affected by thiourea

and potassium iodide, which destroy labile peroxides. Spoehr and co-
workers noted considerable similarity between "chlorellin" and the first
antibiotic, discovered fifty years ago (but never chemically identified)
the pyocyanase from Pseudomonas ruginosa. In both cases, the material
seemes to be a mixture of several unsaturated fatty acids.
Further chemical study showed that the mixture obtained by photox-
idation of the fatty acids from Chlorella contained short-chain acids (about
Cii) separable by distillation, and showing a relatively strong antibacterial
activity.
The studies of Pratt and Spoehr offer a promising approach to the
understanding of at least some of the phenomena in photosynthesis usually
attributed to not further identifiable "plasmatic" or "internal" factors.
External and Internal Factors in Photosynthesis
1840 Liebig, J., Die organische Chemie in ihrer Anwendiing auf Agricultur und
Physiologie. First ed., Vieweg, Braunschweig, 1840.
1843 Liebig, J., Ann. Chem. (Liebig's) 46, 58.
1860 Sachs, J., Jahrb. vdss. Botan., 2, 338
1905 Blackman, F. F., Ann. Botany, 19, 281.
1909 Alitscherlich, E. A., Landw. Jahrb., 38, 537
1910 Thoday, D., Proc. Roy. Soc. London, B82, 443.
1916 Mitscherlich, E. A., Landw. Jahrb., 49, 335.
1917 Brown, W. H., and Heise, G. W., Philippine J. Sci., C12, 1, 85.
1918 Willstatter, R., and StoU, A., Untersuchungen ixber die Assimilation der
Brown, W. H., Philippine J. Sci., C13, 345.
Baule, B., Landw. Jahrb., 51, 363.
1919 Mitscherlich, E. A., Landw. Jahrb., 52, 279.
1920 Baule, B., ibid., 54, 495.
McLean, F. T.,Ann. Botany, 34, 367.
1921 ^Mitscherlich, E. A., Landw. Jahrb., 56, 71.
Harder, R., Jahrb. iviss. Botan., 60, 531.
Lundegardh, P., Svensk. botan. Tid., 15, 59.
1924 Lundegardh, P., Der Kreislauf der Kohlensdure in der Natur. G. Fischer,
Jena, 1924.
Bose, J. Ch., Physiology of Photosynthesis. Longmans, Green, London,
1924.
1926 Romell, L. G., Jahrb. wiss. Botan., 65, 739.
Kostychev, S., Kudriavtseva, M., Moisejeva, W., and Smirnova, M.,
Planta, 1, 679.
884 EXTERNAL AND INTERNAL FACTORS CH\P. 26
Kostychev, S., and Solclatenkov, S. V., ibid., 2, 1.
1927 Geiger, M., Jahrb. unss. Botan., 67, 635.

Romell, L. G., Flora, 121, 125.
1928 Maximov, N. A., and Krasnosselskaja-Maximova, T. A., Ber. deut. botan.
Ges., 46, 383.
INIaskell, E. J., Proc. Roy. Soc. London, B102, 467.
^laskell E. J., ibid., 488.
Montfort, C, and Neydel, K., Jahrh. wiss. BoUui., 68, 801.
Kostychev, S., Bazyrina, K., and Cliesuokov, V., Planta, 5, 696.
1929 Boysen-Jensen, P., and Miiller, D., Jahrb. wiss. Botan., 70, 493.
1930 Kostychev, S., and Berg, V., Planta, 11, 144.
Kostychev, S., and Kardo-Sysoeva, H.. ibid., 11, 117
Kostychev, S., Cliesnokov, V., and Bazyrina, K., ibid., 11, 160.
Harder, R., iftw/., 11,263.

Chesnokov, V., and Bazyrina, K., ibid., 11, 457.
Chesnokov, V., and Bazyrina, K., ibid., 11, 473.
van der Honert, T. H., Rec. trav. botan. neerland, 27, 149.
1931 Arnold, A., Planta, 13, 529.
Kostychev, S., ibid., 13, 778.
1932 Chesnokov, V., and Bazyrina, K., Trav. inst. biol. Peterhof, 9, 58; Proc.
Leningrad Soc. Nat. Sci., 61, 279.

Schoder, A., Jahrb. wiss. Botan., 76, 441.
van der Paauw, F., Rec. trav. botan neerland., 29, 497.
Jaccard, P., and Jaag, 0., Ber. deut. botan. Ges., 50, 167.
Jaccard, P., and Jaag, 0., Botan. Centr., Beihefte, 50, [I] 150.
Harder, R., Filzer, P., and Lorenz, A., Jahrb. wiss. Botan., 75, 45.
Hiramatsu, K., Tlioku Imp. Univ. Science Repts., II, 7, 239.
Chesnokov, V., Grechikhina, 0., and Jermolayeva, I., Trav. inst. biol.
Peterhof, 9, 157; Proc. Leningrad Soc. Nat. Sci., 61, 377

1933 Schanderl, H., and Kaempfert, W., Planta, 18, 700.
Kursanov, A. L., Planta, 20, 535.
Harder, R., ibid., 20, 699.
Bosian, G., Z. Botan., 26, 209.

Hoover, W. H., Johnston, E. S., and Brackett, F. S., Smithsonian Inst.
Ppbl. Misc. Collections, 87, No. 16.

1934 Boysen-Jensen, P., Planta, 21, 368.
1935 Drautz, R., Jahrb. wiss. Botan., 82, 171.

von Guttenberg, H., and Buhr, H., Planta, 24, 163.
Singh, B. N., and Lai, K. N., Plant Physiol, 10, 245.
Stalfelt, M. G., Planta, 23, 715.
1936 Mitchell, J. W., Botan. Gaz., 98, 87.
1937 Monch, I., Jahrb. wiss. Botan., 85, 4.
Gessner, F., ibid., 85, 267.
van Hille, J. C, Proc. Acad. Sci. Amsterdam, 40, 793.

1938 Gessner, F., Jahrb. vnss. Botan., 86, 491.
Filzer, P., ibid., 86, 228.
Neuwohner, W., Planta, 28, 644.

Neubauer, H. F., ibid., 28, 738.
van Hille, J. C, Rec. trav. botan. neerland., 35, 680.
1939 Stocker, 0., Rehm, S., and Paetzold, I., Jahrb. wiss. Botan., 86, 556.
Waugh, J. G., Plant PhydoL, 14, 463.
Wassink, E. C, and Katz, E., Enzymologia, 6, 145.
Noddack, W., and Eichhoff, H. J., Z. phjsik. Cheni., A185, 222.
Meyer, B. S., Ayn. J. Botany, 26, 755.
1940 Brilliant, V. A., Sovet. Botan., 1940, No. 4, 28.

Pratt, R., and Fong, J., yl?n. /. Botany, 27, 431.
1942 Pratt, R., ibid., 29, 142.
Katz, E., Wassink, E. C, and Dorrestein, R., Enzymologia, 10, 269.
Steemann-Nielsen, E., Dansk Botanisk Arkiv, 11, No. 2.
1943 Pratt, R., Am. J. Botany, 30, 32.

Pratt, R., ibid., 30, 406.
Pratt, R., ibid., 30, 626.
1944 Spoehr, H. A., Smith, J. H. C, Strain, H. H., Milner, H. W., and Hardin,
G. Carnegie Inst. Washington Yearbook, 43, 67.
J.,
1945 Pratt, R., Oneto, J. F., and Pratt, J., Am. J. Botany, 32, 405.
Spoehr, H. A., Smith, J. H. C., Strain, H. H., Milner, H. W., and Hardin,
G. J., Carnegie Inst. Washington Yearbook, 44, 66.
1946 Spoehr, H. A., Smith, J. H. C, Strain, H. H., Milner, H. W., and Hardin,
Cx. J., ibid., 45, 101.

1947 Brilliant, V. A., Photosynthesis as Life Process of the Plant. Acad. Sci.
USSR, Leningrad, 1947.

1948 Scarth, G. W., Loewy, A.,and Shaw, M., Canadian J. Research, C26, 94.
1949 Bohning, R. M., Plant Physiology, 24, 222.
Chapter 27
CONCENTRATION FACTORS
In this chapter, we will describehow the rate of photosynthesis and the

yield of chlorophyll fluorescence depend on the concentration of the react-
ants. In the ordinary photosynthesis of green plants, the only reactant
the amount of which can be varied freely is the oxidant, carbon dioxide.
True, the activity of the reductant, water, also can be changed within certain
limits (c/. Vol. I, chapter 13, page 333); but the effect of such variations
is in the main an indirect one: Changes in hydration affect the colloidal
state of the protoplasm, which in turn influences all the activities of the
living cell. In bacterial photosynthesis ("photoreduction," cf. chapter 5,

Vol. I), where hydrogen, hydrogen sulfide, thiosulfaie or another inorganic or
organic reductant takes the place of water, its concentration can be varied
as easily as that of the oxidant, carbon dioxide. Thus, bacteria (and
"hydrogen adapted" algae, cf. chapter 6) open a new approach to the ki-
netic study of photosynthesis. Finally, the "Hill reaction" (chapter 6, page
63, and also chapter 35, in whole cells or in isolated chloroplast material,
permits one to measure the influence of concentration of substitute oxidants
(Fe+++, quinone, chromate, etc.) on the rate of liberation of oxygen.
We will also describe in this chapter the effect on photosynthesis and
fluorescence of varying amounts of additions such as catalyst poisons,
narcotics and inorganic ions. This section (part D) forms a quantitative
elaboration (and contains, inevitably, some repetition) of the qualitative
information presented in chapters 12 and 13 in Volume I.
A. Experimental Carbon Dioxide Curves*

1. Carbon Dioxide Molecules and Carbonic Acid Ions
In the "carbon dioxide curves," which will be discussed in this chapter,

the rate of photosynthesis is plotted as a function of the concentration of
carbon dioxide, while all the other kinetic conditions are assumed to be
constant. The concentration carbon dioxide molecules,
of free, neutral
[CO?], will be used as the independent variable, whether the experiments
* Bibliography, page 9G0.
886
CARBON DIOXIDE MOLECULES AND CARBONIC ACID IONS 887
were carried out with land plants, in an atmosphere containing gaseous

carbon dioxide, or with aquatic plants, in either acid or alkaline solutions,
despite the fact that in the last-named case carbonic acid was present
mainly in the form of carbonate and bicarbonate ions. This presentation
is chosen because the neutral molecular species CO2 easily enters and leaves
the cell, while ions appear to encounter a much greater difficulty in diffusing
through the cell membrane. Consequently, the intracellular concentra-
tions of all molecular species of carbonic acid, including the ions HCOs"
and CO3"", probably are determined mainly by the extracellular concentra-
tion of the species CO2 and largely independent of the concentration of the
carbonate ions (and thus also of the pH of the medium, since at a given
value of [CO2], variations of [HCO3-] and [COs^-] are uniquely correlated
with changes in the pH).
This simplification is convenient, but is likely to prove an oversimplifi-
cation. In chapter 8 (Vol. I, page 195), we described the controversy be-
tween Natansohn (1907, 1910), Wilmott (1921), Romell (1927) and James
(1928), on the one hand, and Angelstein (1911) and Arens (1930, 1933,
1936), on the other. Natansohn believed that only the concentration of
neutral carbon dioxide molecules in the medium is of importance for the
photosynthesis of aquatic plants the occasionally observed rate-enhancing
;
effect of bicarbonate ions (quoted by Angelstein as proof of their availabil-

ity for photosynthesis) was interpreted by Wilmott and Romell as a buf-
fering effect.(The dissociation of HCO3- ions into OH" and CO2 provides
ample replacement for the carbon dioxide molecules used up by photosyn-
thesis.) James found, in fact, that, if care is taken to avoid exhaustion of
carbon dioxide by strong circulation, the influence of bicarbonate ions on
the rate of photosynthesis tends to disappear. In many plants, excess
carbonates may even produce an inhibition attributable either to alkaline
reaction (c/. Vol. I, page 339) or to the damaging effect of the cations (as
indicated by the different influences of the bicarbonates of sodium and

potassium; cf. Vol. I, page 340, and p. 835). Experimental support of
Natansohn's concept was provided by the experiments of Osterhout and
Dorcas (1926), which the rate of penetration of carbonic acid into the
in
interior of giant Valonia cells was proved to be proportional to the external
concentration of the carbon dioxide molecules and independent of the
concentration of the anions of carbonic acid.
However, Arens (1930, 1933, 1936) observed that, in light, HCO3- ions
were taken up by the lower surface of leaves of aquatic plants (such as Elo-
dea), while COs^- or - ions were set free at the upper surface. He inter-
OH
preted this observation as proof that HCOs" ions actually penetrate into
cells and are used there for photosynthesis, either completely, according to
the equation:
:
888 CONCENTRATION FACTORR CHAP. 27
HCO3- > CO2 + OH-

or partially, according to the equation
2 HCO3- > CO32- + H2O + CO2
In Volume I (page 157), we said that Arens' results are in need of ex-
perimental verification, and that, if they prove to be correct, they may be
explained (a) by the diffusion of ions through the leaf without penetration
into the interior of cells, and (6) by cell wall penetration l:»y neutral salt
molecules, such as KHCO3.In connection with the latter possibility, it
would be important to obtain quantitative information on the rate of pene-
tration of bicarbonate as compared to that of free carbon dioxide conceiv- ;
ably, the observations of Arens could be explained even if the first rate is
only one hundredth or one thousandth of the second one, and thus negligible
from the point of view of the kinetics of photosynthesis.
By considering the penetration problem as a quantitative rather than
qualitative one, we can anticipate that the influence of the external con-
centrations [HCOa"] and [COs^"] on the carbonic acid system inside the
cell will depend on whether we deal with an approximate equilibrium (z. e.,
work in the dark, or in low light), or with a photostationary state in which

carbonic acid is rapidly consumed by photosynthesis. In the first case,
even a very slow penetration of salt molecules may result in considerable
changes in the composition of the cell fluids, while, in the second case, the
effect of such slow penetration may be completely negligible in comparison
with that of the much more rapid flow of CO2 molecules.
More recently, Steemann-Nielsen (1946) revived Angelstein's (1911)
argument. He studied the rate of photosynthesis as a function of the con-
centration [CO2] in the medium, using the two aquatic plants Myriophyl-
lum spicatum and Fontinalis antipyretica. In Fontinalis, the rates of oxygen
liberation found in alkaline solutions (pH 8.3, containing from 0.5 X
10-^
to 5 X 10-3 mole HCO3- per liter, together with from 0.5 X 10"^ to 5 X
10-^ mole CO2 per liter) were hardly different from those observed in acid
solutions with the same amount of CO2, but practically no HCO3- ions.
A significantly different result was obtained with MyriopMjllum: In this
plant, the yield of photosynthesis in alkaline bicarbonate solutions was
ten times higher than in acid solutions with the same content of CO2 mole-
cules! In some alkaline solutions the rate of oxygen liberation by Myrio-
phyllum was as much as one third of the rate found in acid solutions with
the same total concentration ([CO2] + [HCO3-]). This was interpreted
by Steemann-Nielsen as indication that Myriophyllum uses IICO3- ions
directly with about one third the efficiency with which it uses neutral CO?
molecules. The between the two species, Fontinalis and Myri-
difference
ophyllum, was tentatively related by Steemann-Nielsen to the fact that
CARBON DIOXIDE MOLECULES AND CARBONIC ACID IONS 889
Myriophyllum grew in a locality where the pH in summer was as high as

9-10 (corresponding to a ratio of 100 HCOs" to 1 CO2), while Fontinalis
was gathered in a locality where the water contained very Httle bicarbonate,
but as much as 30 X 10"^ mole per liter of free CO2 molecules (a remarkably
high figure, if one recalls that water equilibrated with the free atmosphere
contains only about 1 X 10 ~^ mole per liter CO2).
The behavior of the two species is illustrated by figures 27.1 A and B.
One can either accept these figures as evidence of direct participation of
E wu
E
Q.
O
c
0)
u
0)
Q.
o
3
O
o
Q.
.
890 CONCENTRATION FACTORS CHAP. 27
(1946) enlarged these observations by determining the carbon dioxide curves of photo-
synthesis in solutions containing different cations. He found that, with Myriophyllurn
spicatum, in acid solution, the presence of sodium or calcium (in the form of chlorides)
had no effect on the rate of photosynthesis. In alkaline solutions, on the other hand,
the rate was lowest in sodium bicarbonate, higher in potassium bicarbonates and still
higher in calcium bicarbonate. The highest rate could be obtained in a solution con-
taining K+, Na+, Ca2+, Cl~, and S04^~ ions in the same proportion as the water of the
was the natural habitat of the plants. In lake water, the rate was independent
lake that
~'
of between 8.5 and 10.5, while in potassium carbonate-bicarbonate buffer (10
pH
mole HCOs" per liter) the rate increased slowly between pH 8.4 and 10.5 and dropped
sharply to zero at pH 11
These results can be interpreted in terms of Steemann-Nielsen's concept of direct

participation of bicarbonate ions in photosynthesis (for example, by assuming different
rates of penetration of different neutral molecules, MeHCOs, through the cell membrane;
cf. Vol. I, page 197), but they may also be of a more indirect and complex origin. Ac-
cording to Pratt {cf. fig. 25. 1 ) the cation effects are largely irreversible, a complication
not considered by Steemann-Nielsen.
Tseng and Sweeney (1946) studied the red alga Gelidinium cartilagineum and found
that the rate of its photosynthesis was determined exclusively by the concentration of
free carbon dioxide molecules, [CO2], and not affected by the simultaneous presence of
a large number of bicarbonate ions, [HCOs"] > 10 [CO2].
Ruttner (1947) compared the limiting pH values established in water as the result
of prolonged photosynthesis of different aquatic plants. Elodea (canadensis or densa),
Photomageton, Myriophillum prismaium, Lemma trisuUa and several other aquatic
phanerogams continued to reduce carbon dioxide until the pH rose considerably beyond
pH 9; while several mosses (such as Fonlinalis antipyretica) ceased to assimilate carbon
dioxide when pH reached 9.0. At the latter pH, [CO2] = 0.4 IQ-^ mole/1.; this is X
the region in which the "carbon dioxide compensation point" was found previously
with land plants {cf. page 899). Ruttner suggested that the capacity of aquatic higher
plants to carry out net positive photosynthesis at [CO2] equilibrium values <^ 1 X
10~^
mole /I., if bicarbonate is present, indicates their capacity to utilize bicarbonate ions di-
rectly,and not merely as source of CO2 molecules in the medium. In a second paper,
Ruttner (1948) gave evidence supporting the assumption that the cessation of photo-
synthesis of Fontinalis at pH 9 is the result of low CO2 concentration (0.4 X 10 -'^ mole /I.,
corresponding to about 0.01 vol.%), and not of excess alkalinity. Earlier observations
of Shutov (1926), Bode (1926) and Dahm (1926), who found that many aquatic plants
can raise the pH of the medium to values as high as 11.8 (Spirogyra), can then be in-
terpreted as meanmg that these plants, too, can use bicarbonate ions directly as source
of carbon for photosynthesis (or, more exactly, as a vehicle to transport carbon dioxide
from the medium into the cells).
Osterlind (1948,1949) went even further than Steeman-Nielsen and Ruttner, and
asserted that certain plants use bicarbonate ions than carbon dioxide
more effectively
molecules. He noted that the alga Scenedesmus quadricauda did not grow at all at pH
5.5 (in a solution aerated with ordinary air). It reached a high rate of growth at about
pH 6.5, and increased it slowly up to pH 9. According to Osterlind, this increase is
not an effect of alkalinity, but a consequence of the presence of bicarbonate ions. He
based this conclusion on the observation that, in air containing 5% CO2, good growth
could be obtained even at pH 3-4. The maximum rate of growth was reached when
[HCO3-] exceeded 9 X 10-^ mole /I.; between 2 and 8 X lO^^ moIe/1., the rate was
proportional to [HCO3-]. Osterlind thought that, with the cell populations used, no
CARBON DIOXIDE CURVES 891
carbon dioxide exhaustion could occur even in the solutions which contained no bicar-
bonate ions. He also noted that, with 10 X 10 ^ mole/1, of HCOs" present, growth was
10 "« mole /I. of carbon dioxide; and
as much as twenty-five times faster than with 10 X
concluded that Scenedesmus quadricauda uses bicarbonate ions (for growth, and thus
presumably also for photosynthesis) twenty-five times more efficiently than free carbon
dioxide molecules. Later (1950'- 2) Osterlind found that Chlorclla pyrenoidosa does not
use bicarbonates; since he found no difference in the carbonic anhydrase content of
the two species, he suggested that their cell membranes arc different.
Pending further analysis concerning the role of carbonate ions (a ques-

tion which the above-described experiments have reopened), we will pro-
ceed on the old assumption that the rate of photosynthesis is primarily a
function of the concentration of the molecular species CO2 in the immediate
surroundings of the and that the main effect of the presence of HCO3-
cells,
ions is to prevent this concentration from depletion during photosynthesis.

Figures collected in Table 27.1, apart from those given for Myriophyl-
lum by Steemann-Nielsen, give no indication of a large, direct contribution
of carbonate ions to photosynthesis. We note, for example, that Emerson
and Green (1938) were able to achieve carbon dioxide saturation of photo-
synthesis in an acid phosphate buffer when the medium contained only
0.7 X 10 -^ mole/1. CO2. In experiments with carbonate buffers, in which
each carbon dioxide molecule was accompanied by 1000 bicarbonate ions
(and as many or more carbonate ions), saturation usually was observed
either at approximately the same or at an even higher value of fC02].
2. General Review of Carbon Dioxide Curves
necessity of carbon dioxide ("fixed air") for photosynthesis was

The
discovered by Senebier in 1782 (c/. Vol. I, chapter 2). The earliest quanti-
tative studies of the relation of the rate of photosynthesis to the concentra-
tion of carbon dioxide were made by Kreusler in 1885 and 1887, Brown and
Escombe 1902 and Treboux, and Pantanelli, both in 1903. Since these
in
observations showed an increase of the rate with increasing fC02] in the
region of low concentrations, and a decline at high concentrations, they were
interpreted on the basis of the then popular "optimum theory" (fig. 26.1),
untilBlackman and Smith suggested in 1911, that they can better be ex-
plained by the concept of "limiting factors." Blackman pointed out that
no evidence existed of a "minimum" [CO2] required for the beginning of
photosynthesis, or of a sharp "optimum"; instead of the latter, experi-
ments showed a wide range of [CO2] values over which the rate remained
approximately constant. As described in chapter 26, Blackman claimed
that correctly determined carbon dioxide curves must be broken lines of the
shape shown in figure 26.2; and many investigations have been carried
out with the expressed purpose of "proving" or "disproving" this "law."
I I
892 CONCENTRATION FACTORS CHAr 27
(NO
(NCOiO O
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fa 3 ^1 S\ S\ S\ SI
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o
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to
!/l
o .« *r +j
H
c « t. e _ CO lO CO lO lO lOiO
ooOiot>-cot--r-ot>-r>-t~ _
o O"*CRi0 0000C0>O0Ci0000 '^
H (N CO I— 1
— 1
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O
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o
< ^ 53 o3 03
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o I I
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o 03
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H S
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O
CARBON DIOXIDE CimVES 803
It hardly needs repeating that the problem must be treated, not from the
point of view of an apodictic "law," but on the basis of the general princi-
ples of reaction kinetics, and that these principles admit of "limiting fac-
tors" only as approximations, useful under certain extreme conditions.
Table 27.1 gives a summary of the most important experimental de-
terminations of the carbon dioxide curves of photosynthesis, since the time
of Blackman and Smith. As a general rule, these curves rise rapidly at
first, then more slowly and finally go over into "saturation plateaus."
At excessively high [CO2] values, the rate may decline again. Table 27.1
gives, in the last two columns, the concentrations found necessary to pro-
duce full carbon dioxide saturation and half saturation, respectively.
(When the approach to saturation is gradual, the second figure can often
be given with more precision than the first one.)
We
note that the observed saturating concentrations vary all the way
from 0.5 X 10 -s, to 400 X 10"^ mole/1. CO2. It will be shown in section 5
that the higher values are beyond doubt due to depletion of carbon dioxide
in the medium surrounding the plants, and consequent establishment of
large external concentration gradients. They can be strongly reduced by
accelerated circulation or buffering. The lower values, on the other hand,
may be determined either by diffusion resistance which is not affected by
buffering or stirring (e. g., that of the stomata, air channels, of adsorption
layers, cell walls and cytoplasm), or by intrinsic kinetic characteristics of
photosynthesis (such as the carboxylation equilibrium and the rate of car-
boxylation).
In the general discussion of the kinetic curves of photosynthesis in
chapter 26, three types of curve sets, P = f[Fi], with F2 as parameter, were
considered and designated as the first (or "Blackman") type, the second
(or "Bose") type, and the third type (see figs. 26.2, 26.3 and 26.4, respec-
tively). It was stated that curves type must arise when the
of {he first
parameter, F2, determines the maximum rate of a partial process that does
not depend on the independent variable, Fi, and therefore imposes a hori-
zontal "ceiling" on the curves P = without affecting the initial slope
f(Fi),
of these curves. Curves of the type are found when the parameter af-
third
fects only the initial slope of the light curves, for example, if it codetermines
the rate of a process that is also proportional to the independent variable,
Fi. In curve systems of the second type, the parameter affects both the
initial slope and the saturation level. Carbon dioxide curves of all three
types can be expected under appropriate conditions; theoretical examples
were given in chapter 26. So far, however, the only experimentally deter-
mined carbon dioxide curve sets have been obtained with light intensity as
parameter. Four sets of such curves, which appear comparatively reliable
as far as the measuring technique is concerned, are reproduced in figures
CARBON DIOXIDE CURVES 895
Fig. 27. 2A. Carbon .dioxide curves of Foniinalis aniipyretica at various light
intensities (in lux) (after Harder 1921). Abscissa, [CO2] in (mole/1.) X 10^
o Intensity 6.2 (20°C)

+ Intensity 6.2 (I2''C )
A Intensity 20(20°C )
^ 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150

COz, 0.001 vol.%
Fig. 27.2B. Carbon dioxide curves of Hormidium flaccidum at two light in-
tensities (in relative units), and two temperatures (after van der Honert 1930).
0.01 volume per cent COo corresponds at 20 °C. to 3.37 X 10 -« mole/1, (c/. Vol. I, p.
174).
PC02>'°''°°'^
0.2 0.3 0,4 0.5

CO
CARBOX DIOXIDE CURVES 897
10"^ mole/1, and then abruptly became horizontal (fig. 27. 2C). In this
figure the maximum yield corresponds to one volume oxygen per volume of
cells each three minutes. The rate values were obtained by admitting a
known amount of carbon dioxide into a Warburg vessel, shaking vigor-
ously and measuring the pressure changes at short intervals until all carbon
dioxide was used up.
200
6 31 klux
.74 klux
0.407
b8-8 — 8-
klux
_i_
5 10 15 20 25 30
[C02],in 10'^ mole/ 1.
Fig. 27.4. Carbon dioxide curves of Cabomba caroliniana

(after Smith 1938).
Figures 27.3 and 27.4, obtained with wheat and with the water plant
Cabomba, respectively^ show a more gradual approach to saturation, but
they, too, indicate a coincidence of all curves at low [CO2] values, which is
characteristic of the Blackman type. Harder's Fontinalis curves (fig.
27. 2A),on the other hand, are of a pronounced "Bose type": Curves
corresponding to different light intensities diverge from the beginning, and
remain an approximately constant ratio; saturation is approached very

in
gradually, and is not quite reached at 32 X 10 ~^ mole/1., even on the curve
that corresponds to an illumination of only 2000 lux. Harder's curves
indicate that, in his plants, the rate of oxygen liberation did not become
limited entirely by carbon dioxide supply even at the lowest used bicar-
bonate concentrations, and did not become independent of [CO2] even at
the highest used concentrations. The range studied, 0.03 to 0.3% KHCO3,
i. e., from 4 X IQ-^ to 40 X
10-^ mole/1. CO2 (c/. Vol. I, page 178), was,
however, a rather narrow one. The curves in figs. 27.2-4 clearly tend to
coincide only below 1 X 10 ~^ mole/1.
In the theoretical discussion later in this chapter, we will see that carbon
dioxide curve? that diverge from the origin can be expected if the carbon
dioxide-acceptor complex, ACO2, is not fully saturated with carbon di-
oxide, at low [CO2] values, even in the equilibrium state; while carbon
dioxide curves that coincide at low [CO2] values can be predicted if the
carbon dioxide dependence of photosynthesis is due entirely to the limita-
tion of the rate of processes by which carbon dioxide is made available for
photosynthesis (such as liberation of CO2 from HCOa", diffusion, and car-
boxylation of an "acceptor").
Table 27.1 shows that, with increasing light intensity, the "half satura-
tion point," which we will designate by 1/JCO2], generally shifts toward
the higher concentrations; so that, in very intense light, it may fall con-
siderably beyond 10 X 10 ~^ mole/1. This fact can be significant, indi-
cating certain kinetic conditions (as will be shown later in this chapter,
see p. 934 ff); often, however, it merely means increasing depletion of
carbon dioxide in the neighborhood of the cells when photosynthesis pro-
ceeds at a faster rate.
3. Carbon Dioxide Compensation Point
For each light intensity, there must exist a carbon dioxide concentration
at which photosynthesis just compensates respiration and the net gas ex-
change is zero, and below which respiration exceeds photosynthesis. This
"carbon dioxide compensation point" has not been studied in the same sys-
tematic way as was the "light compensation point" (c/. Table 28. Ill);
Miller and Burr (1935) first devoted an investigation to it. In their
experiments, a large variety of potted plants were enclosed in vessels filled
with gas mixtures of different composition and illuminated with white
light of about 20,000 lux, until all observable gas exchange stopped, i. e.,
until the carbon dioxide concentration had declined to the compensation
point. It was found that this occurred —
at temperatures from 5° to 35°
C.— when the carbon dioxide content was down to about 0.01%. At low
CARBON DIOXIDE COMPENSATION POINT 899
temperatures, this gas composition remained unchanged for several hours;

at 35-37° C, after a short period of constancy, the carbon dioxide pressure
—
began to increase again probably because photosynthesis suffered slow
thermal inhibition (c/. chapter 31), while respiration remained constant.
Thomas, Hendricks and Hill (1944) found that in beet plants, at 15° C,
photosynthesis compensated respiration at fC02] 0.003% about one — —
third of the value found by Miller and Burr. Gabrielsen (1949) found a
value of 0.009 vol.% for the CO2 compensation point of Sambucus leaves
at 10 klux.
In submerged plants, one has to distinguish between the compensation
point at constant pH and the steady state reached after prolonged photo-
synthesis with limited carbon dioxide supply: In the latter case, both
[CO2] and pH change with time (OH" ions being left behind when CO2 is
withdrawn from HCO3-), and the final steady state may be determined by
either one or both of these factors.We have referred above to the experi-
ments of Dahm Shutov (1926) and Ruttner (1947, 1948), which
(1926),
were interpreted by Ruttner (1948) and Osterlind (1948, 1949) as indicating
that some aquatic phanerogams and algae can use bicarbonate ions so
efficiently that the presence of a minimum concentration of free CO2
molecules not needed to maintain their photosynthesis; these plants

is
are able to continue -net synthesis even after [CO2] has been reduced to
10-^ mole/1, or less, and pH had risen above 10 or 11. (The pH of the
sap inside the cells remains approximately neutral.) Aquatic mosses, such
as FontinaJis, on the other hand, cease to liberate 0? when [CO2] is
reduced to some such value as 0.4 X 10"^ mole/1. (Ruttner 1948). This
corresponds to 0.01 vol. %
CO2 in the atmosphere, and indicates a compen-
sation point similar to that found with land plants. In the steady state
reached by photosynthesis of aquatic plants of this type, the reaction of
the medium is below, or about equal to pH 9.
A rather striking observation of Miller and Burr was that the carbon
dioxide compensation point did not depend on temperature. This contrasts
with the strong dependence on temperature of the light compensation point
(c/. page 984). The reason for this difference is that temperature has a
strong influence on respiration, as well as on photosynthesis in strong light,
but only a weak effect (or none at all) on photosynthesis in light of low
intensity (c/. chapters 29 and 31). In the measurement of the "light com-
pensation point," photosynthesis is in the "light-limited" and therefore
temperature-independent state; while in the measurement of the carbon

dioxide compensation point, "carbon dioxide limited" state and
it is in the
therefore depends on temperature. However, the exact coincidence of

the temperature coefficients of respiration and photosynthesis, implied in
the results of Miller and Burr, is unlikely to be more than an accident.
We assume that the carbon dioxide curves of photosynthesis, if properly

corrected for respiration, continue smoothly below the compensation point
and reach zero when the carbon dioxide concentration is zero. However,
their exact determination in the region of very low carbon dioxide concen-
trations is difficult because of the production of carbon dioxide by respira-
tion. It is difficult to remove this carbon dioxide completely (e. </., by
an alkaline absorber) before some of it is utilized for photosynthesis, since
,
this may occur even before the carbon dioxide has left the interior of the
cells (c/. Vol. I, chapter 19, page 529). Some intermediates of respiration,
such as certain carboxylic acids, may perhaps be utilized for photosyn-
thesis without conversion to free carbon dioxide. One would then obtain
small positive values of "true" photosynthesis (i. e., of the difference be-
tween the gas exchange in light and in the dark), even when the concen-
tration of carbon dioxide is zero, not only in the medium, but also inside
the cell.
Experimental investigation of the relation between photosynthesis and

respiration at low [CO2] values is further complicated by the observation
that, if carbon dioxide is removed very effectively, photoxidation is apt to
occur upon exposure to light, and oxygen consumption becomes larger in
light than in the dark (instead of being decreased in consequence of re-
assimilation of respiration products).
One is thus caught on the horns of a dilemma: (a) either the respira-
tory carbon dioxide is not removed effectively enough — in which case il-
lumination produces an apparent reduction in the volume of respiration,

and it appears as if photosynthesis can proceed at a positive rate even at
[CO2] — (fig. 28.5 gives an extreme example of this kind); (h) or the
removal of carbon dioxide is fully effective —then, photoxidation sets in,
and the rate of photosynthesis appears to become negative at [CO2] = 0.
In chapter 19 (page 528) was noted that Noack (1925, 1926) had o))-
it
served mainly the first phenomenon (i. e., an apparent "light inhibition of
respiration" in a CO2 free atmosphere); whereas van der Paauw (1932)
had discovered, and Franck and French (1941) further explored, the second
effect — the photoxidationin carbon dioxide starved leaves.
Photoxidation can be prevented by using low light intensity and short
exposuies. Whether it is possible to avoid the reassimilation of a part of
respiratory carbon dioxide (or of its precursors) is a controversial matter.
Gabrielsen (1949) noted that in the comparatively thick sun leaves of
Samhucus, in streaming, carbon dioxide free air, as much as 56% of respira-
tory carbon dioxide were reassimilated ; but that in the thinner shade
leaves, reassimilation was negligible. Reassimilation could be reduced by
lowering the temperature (e. g., to 5° C), and by increasing the rate of flow
of the gas, e. g., to 33 cc./m.^ min. Gabrielsen concluded from these ob-
CARBON DIOXIDE FERTILIZATION 001
servations that there is no evidence of photochemical reduction of precur-

sors of free carbon dioxide formed in respiration (since the lattci- would
manifest a general reduction of oxygen consumption in light).
itself in
Warburg, Burk and co-workers (1949) reached the same conclusion in

experiments with strongly stirred, dense ChloreUn pyrenoidosa suspensions:
With alkali present in the side arm of the reaction vessel, illumination with
Aveak red light (lielow compensation) had practically no
on the con- effect
sumi)tion of oxygen. This showed that all the carbon dioxide produced by
respiration was conveyed to the alkali and absorbed there (and none re-
assimilated in light), and that no intermediate products of respiration were

used up as substitute oxidants in the photosynthetic process in the absence
of the normal oxidant, external carbon dioxide.
We \\\\\ have to return to these observations in chapter 29, because of
their significance for the calculation of the quantum yield of photosynthesis
in weak We will note there that the results of Warburg et at. may
light.
have been associated with the intermittency of illumination, caused in their

experiments by the rapid stirring of dense cell suspensions. Most of the
respiratory carbon dioxide was produced while the cells were in the shade and
could escape into the medium before entering the small illuminated zone.
Supporting Kostj^chev's concept of indirect, physiological regulation of photosynthe-

sis (cf.chapter 25), Chesnokov and Bazyrina (1932) concluded, from experiments on
higher plants, that the rate of photosynthesis is not a smooth function of the external
carbon dioxide concentration at all. They asserted that photosynthesis drops to zero
when the external carbon dioxide concentration declines below 0.2 X 10 ~^ M, while above
1 X 10-5 ji/ the rate is not affected by changes in [COj]. They considered this be-
havior a proof of the admirable adaptation of plants to natural conditions a "trigger —
action," which puts the photos3Tithetic mechanism to work when conditions are "nor-
mal" and stops it completely when the conditions become unfavorable. A direct in-
fluence of external carbon dioxide concentration on the reaction rate, subordinated to
the law of mass an "all or nothing" response.
action, could not, they argued, produce such
However, the alleged discontinuity of the carbon dioxide curve, and the consequent
assumption of the existence of a "carbon dioxide threshold" of photosynthesis, is not
confirmed by kinetic investigations under well-controlled laboratory conditions (e. g.,
by the measurements presented in figs. 27.2A-27.4).
4. Carbon Dioxide Fertilization and Inhibition

Practically carbon dioxide curves show that neither the normal car-
all
~^
bon dioxide concentration of the air (0.03%, or approximately 1 X 10
M) nor the content of this gas in water equilibrated with the free atmos-
phere is sufficient for complete saturation of photosynthesis in moderate
or strong light — at least, without exceedingl}^ strong stirring. The curves
indicate that it should be possible to improve, perhaps by as much as 50-
100%, the yield of photosynthesis under natural conditions, by means
of
"carbon dioxide fertilization," and one may expect that this will lead to a
proportionate increase in crop. Experiments tend to confirm this con-

clusion.
Among the earliest attempts at carbon dioxide fertilization were those of Demoussy
(1904) ; the first practical successes were achieved by Klein and Reinau (1914). Among
the more recent investigations, those of Lundegardh (1924), Rippel (1926), White
(1930), Harder, Keppler and Reuss (1931), Johnston (1935), Richter (1938) and Katun-
sky (1939) may be quoted. In numerous experiments, carbon dioxide-fertilized cultures
produced crops 50-100% larger than control cultures grown in rooms with the normal
concentration of this gas. Carbon dioxide fertilization has found some practical applica-
tion (c/., for example, Reinau 1927) in the suburban greenhouse cultivation of fruits and
vegetables, since greenhouses can be "fertilized" comparatively easily and inexpensively
by compressed carbon dioxide from cylinders. The possibility of a similar fertilization
on a large scale in open fields depends on the availability of cheap combustion gases
rich in carbon dioxide, but free of sulfur dioxide and other plant-damaging components
(cf. Katunsky 1939).
Thomas and Hill (1949) made improved measurements of the rate of photosynthe-
sis of tomatoes, sugar beet and alfalfa under field conditions, and found continuous in-
—
crease in rate even at 0.3 or 0.4% CO2 except in the case of a sulfur-deficient beet
culture, which apparently was unable to use an increased carbon dioxide supply. The
maximum fertilization effects observed in these experiments were rate increases by a
factor of about three.
Possibility of fertilization by bicarbonates plays an important role in speculations
on large-scale culturing of unicellular algae as source of fuel or food, for man, animals, or
protein and fat-producing microorganisms, such as yeast.
It was mentioned above (see page 901) that Chesnokov and Bazyrina (1932)
denied that external carbon dioxide concentration has a direct effect on the rate of photo-
synthesis at all. Bazyrina and Chesnokov (1930) sought a different explanation of the
phenomenon of CO2 fertilization, and thought they found it in the stimulating action of
carbon dioxide on plant growth. They denied that field crops are primarily determined
by the intensity of photosynthesis, and pointed out that accelerated photosynthesis
sometimes causes an unbalanced or premature development and thus diminishes rather
than increases the crop. Granted that the size of the crop depends on many factors, it
is certain that photosynthesis is one of them, if not the main one; and it is
hardly
a coincidence that not only the possibility of crop increase by carbon dioxide fertiliza-
tion, but also its approximate maximum extent (50-100%) can be anticipated from the
shape of the carbon dioxide curves obtained under controlled laboratory conditions.
The question of how strongly the actual concentration of carbon dioxide

surrounding vegetation deviates from the average (0.03%) has been much dis-
cussed, and widely divergent opinions have been expressed on this sub-
ject. Undoubtedly, carbon dioxide concentration near the ground in dense
vegetation can rise considerably above the average, particularly at the end
of night. However, extreme figures such as [CO2] > 1%, given by some
investigators, are unlikely. We
quote two examples of more reliable
will
determinations: Verduin and Loomis (1944) found that, in a maize field,

the concentration of carbon dioxide 100 cm. above ground, was 0.055-
0.080% at night, and rapidly declined to 0.045% in the morning. Fuller
(1948) found that CO2 concentration near the ground (0-1 cm.) reached
EXTERNAL SUPPLY AND EXHAUSTION EFFECTS 903
(at 1P.M., in June), 0.13% in a forest, 0.10% in grassland, and 0.18% in a

river bottom. The concentration declined steeply with height above
ground in all these habitats, dropping to near average (~0.04%) 8 or 10
cm. above ground.
The decline of photosynthesis at excessively high concentrations of carbon
dioxide 10 volumes per cent CO2 or more, corresponding to over 300
{e. g.,
X 10-^ M), which, before Blackman, was considered a confirmation of the

"optimum theory," was reinterpreted by Blackman as an inhibition effect,
alien to the intrinsic kinetic mechanism of photosynthesis. It was dis-
cussed as such in chapter 13 (Vol. I) which dealt with various inhibitors
and stimulants. Referring the reader to this chapter, we merely repeat
here references given there to the work of de Saussure (1804) (who dis-
covered the effect), Boussaingault (1865), Bohm (1873), Ewart (1896),
Chapin (1902), Pantanelli (1903), Jaccard and Jaag (1932) and Livingston
and Franck (1940). A recent study by Ballard (1941) with leaves of
Ligustrum can be added to the list. It showed that, at 17° C, inhibition
occurred (at 35,000 lux) at [CO.] = 2%, while at low temperatures (6° C.)
no inhibition was noticeable up to 5%. We recall that Chapman, Cook
and Thompson (1924) found that high carbon dioxide concentration induces
closure of the stomata; it was therefore suggested in chapter 13 that sto-
mata may account for some of the observed carbon dioxide inhibition ef-
fects. Other phenomena, which also may contribute to the inhibiting
influence of excess carbon dioxide, are its adsorption on catalytic surfaces
("narcotization"), and possibly also acidification of the cell fluids (shift
of intercellular buffer equilibria).
That the not the only reason for carbon dioxide
closure of stomata is
inhibition is illustrated by the observation of Osterlind (1949) that it also

occurs with algae such as Scenedesmus quadricauda. An inhibition of the
growth of this alga became noticeable at 2 X 10"^ mole/1., and reached
50%, at 10 X 10-3 mole/1. CO2.
5. External Supply and Exhaustion Effects
In commenting on Table 27.1, we noted wide variations in the numerical

values of the saturating carbon dioxide concentration' and suggested that
these variations may be due largely to the exhaustion of carbon dioxide in
the immediate neighborhood of the plants. We will now consider this as-
pect of the problem more closely.
The experimental One group,
results fall roughly into three classes.
which includes the results of Blackman and Smithand Singh and
(1911)
Kumar (1935), the somewhat less extreme data of James (1928) and the
figures given by Steemann-Nielsen (1946) for Fontinalis, and by Wassink
and co-workers (1941-1942) for purple bacteria, is characterized by con-

tinued increase of the rate of photosynthesis with increasing carbon dioxide
concentration until the latter has reached 50, 80, 200 (Singh and Kumar)
or even 400 X
10""^ mole/1. (Blackman and Smith) the last value corre-
;
sponds to 12% carbon dioxide in the air! Earlier measurements of Kreusler
(1885, 1887) and Brown and Escombe (1902), not included in the table, fall
into the same category.
An intermediate group of results, including Harder's (1921) and Smith's
(1937, 1938) on higher aquatic and Emerson and Green's (1934)
plants,
on Gigartina, place carbon dioxide saturation at 20-30 X 10"^ mole/1. CO2.

Finally, in several careful investigations, the rise of photosynthesis with
increasing carbon dioxide concentration was found to cease as early as be-
tween 0.5 and 5 X 10"^ mole/1. CO2 (Hoover and co-workers 1933, and
Singh and Lai 1935, higher plants; van der Honert 1930 and van der
Paauw 1932, Hormidium; Emerson and Green 1938, Chlorella; and
Barker 1935, diatoms). It will be noted that results of this low order of
magnitude have been obtained both with land plants in rapidly circulating
gas, and with algae in well-stirred acid or alkaline solutions.
There is little doubt that most if not all results of the first type were due
to insufficient circulation and consequent depletion of carbon dioxide in
the medium surrounding the plants. It is by no means certain that con-
centration gradients in the external medium did not affect significantly
also the results in group 2, or even in group 3. And, in addition to gradi-
ents in the external medium (which can be reduced by intense circulation),

we also must consider those in the stomata, air channels, cell walls and
cytoplasm.
of rapid circulation can be understood by considering
The importance
that green such as Chlorella, can consume, in strong light, up to one
cells,
half their own volume in carbon dioxide each minute. In cell suspensions,
the volume of the cells usually is from 0.1 to 1% of the volume of the
medium. Consequently, the suspension as a whole will use up its own
volume in carbon dioxide in from 200 to 2000 min. In other words, the
10"*
rate of consumption of carbon dioxide will be from 2 X 10 "Ho 2 X
mole CO2/I. min. Consequently, if the concentration of carbon dioxide
in the medium is a; X 10"^ ilf, it will be all used up in from 0.05 x to 0.5
X minute, or from 3 a; to 30 x second. Consulting Table 8.II, we note that
in water equilibrated, at 25° C, with an atmosphere containing 0.01%
CO2, X = 0.4; 0.1% CO2, a: = 4.1; 1% CO2, x - 41, and so on. Conse-
quently, a cell suspension in an acid medium that contains no significant
amounts of HCO3- ions, containing 0.1 to 1% cells by volume, will con-
sume all its carbon dioxide in from 1.2 to 12 seconds, if it has been equili-
brated with air containing 0.01% CO2; in from 12 to 120 seconds, if the
CARBON DIOXIDE EXHAUSTION EFFECTS 905
atmosphere contained 0.1% CO2, and so on. Bicarbonate solutions con-

tain, for each GO2 molecule, about 100 HCOs" ions; they should therefore
last one hundred times longer than acid solutions with the same value of
[CO2]. Finally, 0.1 M
carbonate-bicarbonate buffers containing from 2 X .
10^ (buffer No. 330 (buffer No. 11) carbonate and bicarbonate ions
1) to
for each CO2 molecule provide sufficient reserves to maintain full photosyn-
thesis, in suspensions containing 0.1 to 1% cells by volume, for from 500
to 5000 minutes, or S to 80 hours.
These figures lead to several conclusions. First, measurements of the
rate of photosynthesis at low carbon dioxide concentrations (e. g., less than
M
1% CO2 in the air, or 30 X 10 -^ in solution), if they are to last for more
than a few seconds, require an ample supply of carbon dioxide, either in
situ, in the form of carbonate and bicarbonate ions, or from outside, in
the
form of large amounts of circulating liquid or gas, which must be kept well
supplied with fresh carbon dioxide to replace losses. Second, whenever
leaves or multicellular algae are used, very intense stirring or circulation is
required to prevent the establishment of a carbon dioxide concentration
gradient around the plants. The required stirring depends on the ratio of
surface to volume. This is well illustrated by the following example:
Gessner (1938) measured the oxygen liberation by two varieties of Proser-
—
pinaca palustris one with large leaves and one with finely divided, feather-
like leaves. In stagnant water, the first variety produced much less oxygen
than the second one; stirring improved strongly the efficiency of the large-
leafed, but did not affect the oxygen production by the feather-leafed
variety. In other words, in the absence of circulation, external carbon
dioxide supply must have been the rate-limiting factor for the large-leafed,
but not for the feather-leafed, variety.
It seems that, with multicellular objects, even the provision of a
strongly stirred bicarbonate-buffered medium does not always guarantee
the absence of carbon dioxide exhaustion effects. Wassink (1946) found,
for example, that the photosynthesis of 5 mm. discs cut out of leaves, sus-
pended in carbonate buffer No. 9 (7.9 X
10 -^ mole/1. CO2) and shaken in a
Warburg apparatus, was largely "carbon dioxide limited." The

still
equilibrium concentration of carbon dioxide in the atmosphere above this

buffer is 0.25% (cf. Tables 8.V and 8.II). By increasing the initial carbon
dioxide content in the air space to 2% in some cases, and to as much as 9%
in others, Wassink was able to obtain carbon dioxide saturation. Without
exact knowledge of the dimensions of the apparatus, it is difficult to esti-
mate the final carbon dioxide concentration and the pH of the solutions
treated in this way.
Closure of the stomata in punched leaf discs may have been one of the
reasons for apparent extreme carbon dioxide requirements observed in
these experiments. From this point of view, and from the point of view
of favorable ratio of surface to volume, unicellular algae offer much better
conditions. In brief experiments, or in weak light, they can be used in
.acid solutions previously equilibrated with carbon dioxide of sufficiently
high partial pressure (>1%; it was calculated above that a suspension
containing 1 volume per cent of cells will use, in saturating light, all
the carbon dioxide contained in water equilibrated with 1% CO2, in 1.5
minutes). If stronger illumination or longer duration of experiments is
desired, acid solutions can be used only if the carbon dioxide content is
continuously renewed, by stirring with a gas the carbon dioxide con-

e. g.,
tent of which is maintained by contact with an alkaline carbonate buffer.

More efficient should be the provision of carbon dioxide reserves in
situ by using carbonate buffers directly as suspension media, as first sug-
gested by Warburg. However, a certain difficulty arises from their un-
physiological and variable alkalinity. In progressing from M/IO buffer
No. 1 (0.5 X lO-'^ mole/1. CO2) to M/10 buffer No. 11 (29 X 10"^ mole/1.
CO2), we find the pH declining from 11 to 8.5. Since all living cells are
more or less sensitive to excess alkalinity (even if Chlorella appears to be
remarkably resistant to it), this drop of pH could cause continued increase
of the rate of photosynthesis in a range where this rate is intrinsically in-
dependent of carbon dioxide concentration. This may explain, for ex-
ample, the difference between the carbon dioxide curve of Chlorella as de-
termined by Warburg (1919) in carbonate buffers, and the same curve ob-
tained by Emerson and Green (1938) in a phosphate buffer. The first
one continues to increase up to and beyond 9 X 10^^ mole/1. CO2, while
the second one is perfectly flat above 0.7 X 10"^ mole/1. CO2.
On the other hand, observations of Ruttner (1947, 1948) and others on
the maximum pH reached in non-renewed media after prolonged photosyn-
thesis by aquatic plants (c/. above page 890), tend to discount the damaging
effect of alkalinity on algae and submerged phanerogams (as contrasted
to water mosses), by indicating the continuation of photosynthesis up to
pH 11 or 12; pH measurements on cell sap showed it to maintain its ap-
proximately neutral reaction even in such highly alkaline media.
(For other possible explanations of the difference between the CO2 curves
of Chlorella as observed by Warburg, and by Emerson and Green, see
page 908.)
Another pertinent question is whether the rate of conversion of HCOa"
ions into CO2 molecules always is high enough to provide effective replenish-
ment of used-up carbon dioxide. In chapter 8 (cf. Vol. I, page 175) we dis-
cussed the finite rate of hydration and dehydration of carbon dioxide, and
estimated that, in acid solution at room temperature, an H2CO3 molecule
lives ca. 0.1 sec. before dissociating —the monomolecular rate constant
: :
of dehydration being about 10 sec.-^ at 18° C; Table 8.III). The

(c/.
rate of dehydration of HCOa" ions was not given but we can esti-
there,
mate it from the rate of addition of OH" to CO2, determined experimen-
tally by Brinkman, Margaria and Roughton (1933)
CO2 + OH- , HCO3-

k'
k = 2.05 X 103 (18° C.)
To obtain k', first calculate the equilibrium constant of the above reaction
from the known constants of dissociation of water (1.04 X 10~"), ionic
dissociation of H2CO3 into H+ and HCO3- (1.8 X 10""), and hydration of
CO2 (2.2 X 10-^), and obtain:

K = k/k' = 4.4 X 10'
This, together with the above value for k, gives (for 18° C.)
A;' = (2.05 X 103)7(4.4 X 10') = 0.47 X 10"*
This indicates that an HCOa" ion lives, on the average, at 18° C, as long
as 2.7 X 10^ sec. before being dissociated into OH ~ and GO2. A bicarbonate
buffer containing y mole HCOs^/l. can therefore supply a maximum of 4.7
X 10 ~^ y mole C02/l./sec. by this dehydration process. At pH < 10,
dehydration via H2CO3 must be added; at pH 9 it can double the rate
of conversion of HCOs" to CO2 (assuming the association of HCOs" and
H+ to H2CO3 to be practically instantaneous). A solution containing
y = 0.02 mole/1. HCO3- (Warburg's il//10 buffer No. 2, pH ^ 10.7)
is thus able to supply a maximum of 9 X 10"'' mole CO2/I. sec. The cor-
responding figure for buffer No. 9 (0.085 HCO3-, pH 9.4) is 5 X M ^
10 ~^ mole CO2/I. sec. Comparing these figures with the above-estimated
maximum rates of photosynthesis in strong light (from 2 X 10~Ho 2 X
10-^ mole CO2/I. min., or from 3.3 X 10 ^^ to 3.3 X 10 "^ mole CO2/I. sec.
for suspensions containing from 0.1% to 1% cells by volume), we note that
the maximum supply exceeds maximum consumption in the 0.1% suspen-
sion by a factor of about three in buffer No. 2 and by a factor of about fifteen
in buffer No. 9. In 1% suspension, the supply is quite insufficient in buf-
fer No. 2 and barely sufficient in buffer No. 9. Considering the roughness
of the calculation (e. g., the use of concentrations instead of activities), the
margin is by no means secure even in the dilute suspension. Assuming the
calculation to be exact, a supply process with a maximum rate equal to
only 3 times the noninhibited rate of reaction bound to cause a marked is
inhibition (c/. chapter 26). It is therefore an open question whether the

limited rate of reproduction of CO2 molecules from HCOs^ ions can play a
role in the determination of the rate of photosynthesis of dilute suspensions
in strong light, at least in the more alkaline carbonate buffers. This
•
"bottleneck" may well have contributed, c. gr., to the decline in rate ob-
served by Warburg (1919) in Chlorella at [CO2] < 9 X 10"^ M. (As
mentioned before, Emerson and Green have noted no such decline until
[CO2] was down to 0.7 X 10~* M, and have suggested that damage caused
by increased alkalinity of the lower carbonate buffers could provide an
explanation of Warburg's results.)
Carbon dioxide exhaustion effects are not restricted to experiments in
liquid media, but affect alsomeasurements made with land plants in a car-
bon dioxide atmosphere, if it is stationary (c/. Lundegardh 1921), or in-
sufficiently agitated (Kreusler 1885,1887; Singh and Kumar 1935); this
was demonstrated by Kostychev ef aZ. (1927) and Chesnokov and Bazyrina
(1932). Here again, not only the rate of gas circulation, but also the size
and shape of the plants may be of importance and the opening of the
stomata constitutes an additional complication.
To sum up it seems safe to assume that, whenever the rate of photo-
synthesis was found to continue its increase with the external concentra-
tion of carbon dioxide much above [CO2] = 10 X 10 "^ M, the reason was
slow outside supply of carbon dioxide to the photosynthesizing cells, and
consequent exhaustion of the reduction substrate. Experiments in vigor-
ously stirred solutions, or in rapidly circulating gas mixtures, regularly
showed the photosynthetic apparatus to become saturated with carbon
dioxide at concentrations not much higher, or even lower, than 1 X 10 ~^
M. Even in experiments of this type,one cannot be certain whether all
diffusion effectshave been eliminated, particularly in higher plants, where
the diffusion resistance of the stomata, epidermis and air channels cannot
be destroyed by stirring or gas circulation. The diffusion resistance of the
cell walls or protoplasmic layers also remains unaffected by all me-
chanical means (although it may perhaps be changed by chemical agents).

Another source of distortion of the carbon dioxide curves of photosyn-
thesis was noted by Howies (unpublished) and Whittingham (1949) in
Brigg's laboratory. They observed that the photosynthesis of Chlorella
in carbonate buffers with low [CO2] values was time-dependent, if the cells
had been transferred into the CO2 deficient medium from a culture medium
of higher concentration (such as 4% CO2). The initial rate was low; it
increased by a factor of 3 in the course of two or three hours, and then be-
came constant. If the cells were cultured in air (0.03% CO2), the rate
was high and constant from the beginning. Obviously then, with cells
"incubated" at high [CO2], the shape of the carbon dioxide curve will de-
pend on the duration of the measurement.
If the carbon dioxide curve of Chlorella is determined at low [COu]
values, with cells "adapted" to low carbon dioxide concentration, the value
of VJCO2] is as low as 0.5 to 1.0 X 10-« mole/1.
The initial inhibition of photosynthesis in low [CO2], shown by cells
previously exposed to high [CO2] values, could be related to the photoxida-

tion phenomena observed in CO2 starved plants (c/. Vol. I, chapter 19).
Using Franck's picture, it can be suggested that CO2 satiated cells, placed
in CO2 deficient medium and exposed to light, develop a large quantity of a
"narcotic" (perhaps, because they were full of metabolites), which settles
on chlorophyll and holds photosynthesis down. The autocatalytic removal

of this inhibition seems to require 2-3 hours (as against a few minutes in
ordinary induction, cf. chapter 33). That such cells in fact are inhibited
is confirmed by the observation that if, after brief exposure to light in low
[CO2], they are brought back into a medium of high [CO2] (such as buffer
No. they show a reduced rate of photosynthesis in this medium as well.
9),
Since these experiments were carried out in carbonate buffers, the ob-
served effects can be attributed either to changes in [CO2], or to those in
If all carbon dioxide activity gradients between the outside medium

and the site of photosynthesis could be avoided, we would still anticipate,
on theoretical grounds, that carbon dioxide concentration will retain an
influence on the rate of photosynthesis: first, because of dissociation,
under low partial pressure of carbon dioxide, of the carbon dioxide-acceptor
compound that we assume is formed as an intermediate in photosynthesis
(cf. chapter 8) and second, because of the dependence of the rate of forma-
;
tion of this compound ("carboxylation") on the factor [CO2]. These two

relationships will be discussed theoretically in sections 7b and c but there ;
can be no certainty, until much more precise measurements have been

carried out, that any of the observed carbon dioxide curves actually reflect
one or both of these intrinsic kinetic relationships, rather than the more
incidental diffusion phenomena. As long as a [CO2] effect can be made to
disappear by improved stirring, it reveals itself as due to external diffusion;
but, when no further improvement in rate can be achieved in this way, this
does not mean that the remaining [CO2] effect is not caused by diffusion in
those parts of the gas path were external stirring can do no good.
In estimating the supply of carbon dioxiile to plants under natural conditions, the
llir rools nuist not be overlooked.
possibility of caibon dioxide siipplij through, It was
mentioned in chapter 2 (N'ol. I) that the doclrine of the aerial nijurishment of plants was
the second accomplishment of the discoverer or, more exactly, co-discoverer of ])hot()-
synthesis, Ingen-Housz. Since the time of Liebig, this doctrine has become the basis
of the science of plant nutrition. However, under certain conditions, Senebier's concept
that carbon dioxide can be supplied by the soil water to the roots and thence to the
leaves, scorned l)\' Tngcn-TTousz, may be correct. This may affect, field determinations
of the rate of pliotosynthcsis, basedon measui'ements of the carbon dioxide consumption
from the air, and may by other methods, if the ob-
also influence the results obtained
served rates are considered in relation to the external carbon dioxide concentration. For
recent discussions of this question, we Bergamaschi (1929), Livingston and Beall

refer to
(1934), Suessenguth (1937), Overkott (1938, 1939) and Hartel (1938). The experiments
of the two last-named authors, in particular, have confirmed unambiguously that a cer-
tain amount of carbon dioxide can be conveyed from roots to leaves by convection (and
to a smaller extent by diffusion), and that this supply can be utilized by leaves for the
synthesis of carbohydrates. It was even suggested that this "invisible" CO2 supply,
brought about by increased transpiration during the hot hours of the day, may be
the cause of the decline of the carbon dioxide absorption from the air, which is often ob-
served at midday (c/. page 873). Whether this is a valid hypothesis cannot be judged
without quantitative investigations; but in any case, it cannot explain all the aspects
of the so-called "midday depression," (o) because these also include a decline of oxygen
liberation, (6) because they have been observed not only in the higher land plants but
also in aquatics.
6. Role of the Stomata
It was stated above that, in experiments with the leaves of the higher
land plants, a special problem is posed by carbon dioxide passage through
Diagram of a section through stoma and substomatal cavity of a

Fig. 27.5.
leaf toshow direction of diffusion of gases in photosynthesis (after Robbing and
Rickett). Arrows with black balls represent carbon dio.xide; those with tri-
angles, oxygen.
the stomata and air channels, through which it has to flow in order to reach
the photosj^nthesizing cells of the palisade tissue and of the spongy paren-
chyma.
A controversy as to whether the carbon dioxide enters the leaf only through the
stomata or also through the cuticle was decided by Blackman (1895). He proved, by
ROLE OF THE STOMATA 911
experiments with paraffined leaves, that gas exchange takes place almost exclusively
through the stomata, in the way indicated in figure 27.5. Only under very high pres-
sure of carbon dioxide did Blackman observe a slight penetration of the gas through the
cuticle. According to St&lfclt (1935), in the free atmosphere, carbon dioxide pene-
trates the cuticle at a rate of only between 3 and 6 X 10~^ mole/cm. ^ hr.; the gas flow
through the stomata may be as much hundred times faster, i. e., of the order of
as one
5 X 10"'' mole/cm. 2 hr., despite the fact that their openings occupy only about 0.1%
of the total leaf surface. Freeland (1946) found more recently that, in some leaves, the
relative rate of passage of carbon dioxide under pressure through the lower and the up-
per surface is so low as to suggest predominance of diffusion through the epidermis over
passage through the stomata. The thickness of the epidermis may be an important
factor in the determination of the relative role of stomata and epidermis as routes for
the entry of carbon dioxide into the leaf.
Ferns and other lower land plants possess no stomata, and therefore must receive
all their carbon dioxide supply through the epidermis. Stomata also are absent in
—
aquatic plants and algae, where their main function regulation of evaporation is not —
required.
Between 10,000 and 30,000 stomata are present on each square centi-
meter of the on both sides or on the lower side only.
leaf surface, either
They are elongated slits, usually from 10 to 15 ix long, flanked by two "guard
cells" {cf. figs. 27.5 and 27.6), which are capable of changing shape so as to
effect the opening or closing of the slit {cf. fig. 27.7).
Fig. 27.6. A portion of the lower epidermis of a geranium leaf (after

Robbins and Rickett).
This mechanism is brought into operation by shifts in the sugar-starch

equilibrium, which increase the turgor when the slits are to be opened, and
decrease it when they must be closed.
The problem of the diffusion resistance of stomata has been considered
from two points of view: First, it was asked: Is it possible for a diffusion
flow of up to 10-5 mole (0.24 cc.) COa/hr. (c/. chapter 28, Table 28.5) to
pass through the stomata on 1 cm.- of the leaf surface, when the total open
area is less than 1 mm.^ and the concentration drop is not more (and often
less) than 1 X 10~'^ mole/1, (which is the normal C(\ concentration in the
open air)? Granted a remarkably low diffusion

The second question was :
resistance of the stomata, is this resistance nevertheless an important

"limiting factor" in photosynthesis of higher plants, particularly at low
carbon dioxide concentrations?
To understand why the first question had to be asked, suffice it to recall
the experiment of Brown and Escombe (1900), who showed that a leaf
takes up carbon dioxide from quiet air almost as rapidly as an eciually large
surface of an alkali solution! It Avas soon found that this unexpectedly
high rate of diffusion has nothing to do with the physiological properties of

the leaf but a general property of multiperf orate septa, i.e., barriers con-
is
taining many small openings. Model experiments on transpiration showed

{cf. Sierp and Seybold 1929, 1930) that the rate of evaporation from a ves-
Fig. 27.7. Stoma of Hellehorus sp. in transverse section. Darker lines show shape
assumed by guard cells open; lighter lines when stoma is closed (from
when stoma is
Strassburger et al, after Schwendener). In closed state, vacuole (shaded area) con-
tracts because of water loss caused by decreased turgor (produced by polymerization
of sugars).
sel,covered with a septum, can be as high as three fourths of that from an

equally large open vessel— even if the aggregate area of the holes is less
than 1% of the total surface of the liquid! The theoretical solution of this
apparent paradox was given (for the case of evaporation) as early as 1881
by the Austrian physicist Stefan. He used, for this purpose, the formal
similarity of the equations describing the diffusion flow of matter from
an extended surface and from a point source, with the equations de-
scribing the hues of force in the electrostatic field in front of an extended
conducting surface, and around a small conductor. In this formal
analogy, the diffusion flow corresponds to the electrostatic capacity of
the conductor; and it is known that the capacity of a large flat condenser
is determined by the area of its plates, while the capacity of a
small spherical
conductor is determined by its radius. In the same way, the amount of
DIFFUSION THROUGH SEFTA 913
evaporation from an extended surface is proportional to its area, while the

amount of evaporation from a small sphere is proportional to its radius;
the same applies to the comparison of diffusion across an extended plane
(the case usually considered in the derivation of diffusion equations) with
the diffusion through a small hole. Diffusion through a multiperforated
septum can be treated in the same way as that through a single hole as long
as the distance between the holes is large enough (compared with the radius
of the holes) for the half-spherical surfaces of equal concentration (and the
radial lines of flow, which are normal to these surfaces) to be established
around each hole without marked interference by the neighboring holes.
This principle was first applied to the penetration of carbon dioxide
through the stomata by Brown and Escombe (1900) upon advice of the
physicist Larmor. Renner (1910, 1911), Brown (1918), Freeman (1920),
Sierp and Noack (1921), Sierp and Seybold (1927, 1928, 1929), Huber
(1928) continued the study, being, however, mainly concerned with the
trans-piraiion of plants. As a typical result, we reproduce a table from
the paper by Sierp and Seybold (1929). Table 27.11 shows the rates of
evaporation of water through septa with different numbers of holes but a
constant total open area. The next-to-last row shows the flow-retarding
effect of an inadequate distance between the holes. The table indicates
that a maximum rate of diffusion is reached asymptotically when the holes
are reduced to 20-10 n in diameter. Although the aggregate area of the

holes (3.14 mm.2) is less than 1% of the total area of the vessel (-400 mm.^),
the evaporation rate through the septum with holes 10 n in diameter is as
high as 70% of that from the open vessel. These figures indicate that the
dimensions of the stomata (5-15 n) may be appropriate to ensure the de-
sired rate of gas exchange through the smallest possible number of openings.
Table 27.11
Evaporation through Septa (After Sierp and Seybold 1929)
interference of the openings. He calculated that interference should be

inversely proportional to the square of the distance between pores, d:
logQ/Qi = -k/d^
where Qi is the diffusion rate at d = oo This equation agrees well with

.
experimental results of Verduin (1949) and Weishaupt (1935). At a given

ratio of pore diameter and pore distance, the interference must be stronger
the smaller the pores. The stomata are so small that the diffusion through
each of them is
—
reduced significantly by interference ^sometimes by > 50%
of the theoretical value for an isolated opening of the same size. As
stomata close gradually, interference weakens; and the diffusion rate
therefore declines slower than proportionally to the open area.
These experiments and their theoretical interpretation explain how the
tiny stomata can allow a large volume of carbon dioxide to diffuse into the
leaf, thus permitting a high rate of photosynthesis. We now turn to the
second question; stomata impose a significant
does the resistance of the
limit on the carbon dioxide supply and, with it, on the rate of photosynthe-
sis? Closed stomata undoubtedly must curtail photosynthesis drastically
(restricting it to the utilization of the carbon dioxide that can reach the
chloroplasts by diffusion through the cuticle, or is produced in the leaf by
respiration). The question is: How far must the stomata be open to
cease exercising a restrictive influence on photosynthesis? May this
restriction be significant even when slits are fully open? Are they the
bottlenecks responsible for the "Blackman features" of many carbon
dioxide curves? be recalled that, in the preceding chapter, it was
It will
shown that the restrictive influence of a reaction step generally becomes felt
long before the rate of the over-all process closely approaches the "ceiling"
imposed on it by this step. Therefore, the resistance of the stomata may
affect the shape of the carbon dioxide curves even when the rate of photo-
synthesis is not more than one half or one quarter of the maximum possible
flow of carbon dioxide through the stomata.
For an experimental study of the influence of stomata on photosynthesis,
one must measure the rate of photosynthesis under constant external con-
ditions, but with varying apertures of the stomata. Unfortunately, treat-
ments used to enforce partial closure of the stomata (such as incubation in
darkness or in dry air) may also directly affect the efficiency of photosyn-
thesis, so that caution is required in the interpretation of the results. In
order to arrive at reliable conclusions, the width of the stomata and the
rate of photosynthesis must be determined with the same leafâ condition
that has not always been fulfilled.
The relation between stomatal openings and the rate of photosynthesis

ROLE OF THE STOMATA 915
has been the subject of study by several investigators, among them Iljin
(1923), Geiger (1927), Maskell (1928), Johansson and Stalfelt (1928),
Kostvchev, Bazyrina and Chesnokov (1928), Boysen-Jensen (1932),
Schoder (1932), Stalfelt (1935), Newton (1936), Heath (1939) and Heath
and Penman (1941).
Of these, Kostychev, Bazyrina and Chesnokov (1928), and Schoder
(1932) could find no correlation between the two magnitudes. All other
observers concluded that, under certain conditions, a clear-cut relationship
can be noted between them.

Thus, Maskell (1928) found a parallelism between the diurnal and seas-
onal course of stomatal apertures (as measured by a porometer) and the
16
—
one quarter open (2 ju). This difference is understandable since at 8000

lux the maximum only 8 mg. COo/cm.- hr., while
rate of photosynthesis is
in 26,000 lux, it rises to > 20 mg. C02/cm.2 hr. Stalfelt concluded that
stomatal openings easily may limit the carbon dioxide supply in ordinary
air, and therefore also the rate of photosynthesis under natural conditions,
particularly in strong light. Like Maskell, she supported this view by cal-
culations of the rate of diffusion through the stomata, based on equations
of Brown and Escombe (1900). These calculations confirmed that the
maximum rate of carbon dioxide flow from ordinary air through wide open
stomata is of the same order of magnitude as the maximum rate of photo-
synthesis.
These results, while clearly showing the possible "bottleneck" role of
the stomata, do not that other parts of the path between atmosphere
mean
and chloroplasts do not contribute commensurable or even greater —
terms to the total diffusion resistance.
In the face of these results, one must disagree with Renner (1910), who thought that
the resistance of the stomata represents only a negligible fraction of the total diffusion re-
sistance on the carbon dioxide path from the atmosphere to the chloroplasts, as well as
with Schroeder (1924), who attempted to prove that the diffusion resistance of the air
channels is the rate-limiting influence in the photosynthesis of the higher plants, and in
this proof altogetheromitted the resistance of the stomata.

Romell (1927) pointed out that Schroeder neglected, not only the flow resistance
of the stomata, but also that of the gas-liquid interface, and of the liquid phase between
the cell wall and the chloroplasts. Romell calculated that the gradient of the carbon
dioxide concentration in the air channels must be smaller than in the protoplasm (be-
tween cell wall and chloroplast), and that both these gradients should be negligible in
comparison with the drop of concentration at the phase boundary, caused by the rela-
tively small accommodation coefficient of carbon dioxide on water (as calculated from
Bohr's measurements of the velocity of escape of carbon dioxide from aqueous solution).
The maximum theoretical rate of diffusion, calculated by Romell by taking all these
factors into account, proved to be considerably lower than the maximum rate of photo-
synthesis that the leaves actually can reach in open air. One is thus led to assume (c/.
van der Honert 1930) that the accommodation coefficient of carbon dioxide is larger on
the cell wall than on a water-air interface.
7. Interpretation of Carbon Dioxide Curves
The preceding pages show that reliable experimental material for analyt-
ical interpretation of the carbon dioxide curves of photosynthesis is hardly
available at present, not be easy to obtain. We have stated that
and will
at least two intrinsic kinetic factors could make the rate of photosynthesis
a function of the external carbon dioxide pressure: the probable reversi-
bility of the primary carbon dioxide absorption (carboxylation) step, and
a finite rate of carboxylation. The difficulty is to recognize the workings
of these "intrinsic" or chemical factors behind the more incidental, physi-
INTERPRETATION OF CARBON DIOXIDE CURVES 917
cal flow plienomena outside and inside the plant. We cannot be sure at
present whether any of the observed carbon dioxide curves reflect reason-
ably well the effect of carboxylation equilibrium (or of the rate of carboxy-
lation), or whether practically all carbon dioxide dependence of photosyn-
thesis, known so far, is due to diffusion phenomena, with possible addi-
tional distortions by the time effects noted on page 908.
Despite this unsatisfactory state of our experimental knowledge, we
willgo through with some kinetic derivations leading to general equations
for the shape of carbon dioxide curves, as affected by the several factors of
slow diffusion, limited rate of carboxylation, reversibility of carboxylation
and limited supply of light energy. We
thus obtain a kind of skeleton
will
analytical theory of the carbon dioxide curves, which could prove useful
for devising and interpreting future kinetic measurements if only inves- —
tigators of the kinetics of photosynthesis would change their present habit
of considering only their own limited data, and ignoring all but their own
ad hoc derived equations.
(a) Carboxylation Equilibrium
Two steps in photosynthesis, the rate of which depends directly on the

factor [CO2] are: First, diffusion of carbon dioxide from the medium to
the reaction site, and second, the first chemical reaction of carbon dioxide.
In chapter 8 (Vol. I), we decided that this reaction is a nonphotochemical,
catalytic carboxylation. We usually described it by the formula CO2 -^
{CO2}, but since the concentration of the "carbon dioxide acceptor" (until
now symbolized by braces) enters explicitly into many of the following ki-
netic equations, we will from now on designate it as A, and the product of
carboxylation as ACO2. (Franck and Herzfeld, 1941 used the more specific ,
symbols RH for acceptor and RCOOH for the product.)

The two [CO.j]-dependent steps can then Ijc written as follows:
Ki
^
(27.1) CQ2 . (CO,)a
(where (C02)a refers to carbon dioxide in the immediate neighborhood of

the acceptor, and k^ is a diffusion constant) and:
(27.2) (C02)a +A , '^ ^ ACQ., ( > reduction)

a
The ACO2 may be either a direct photochemical process (as

reduction of
assumed by Franck and Herzfeld; cf. scheme 7.VA), or a nonphotochemi-
cal reaction with an intermediate, as postulated in many other schemes
in chapters 7 and 9. Even in the latter case, the rate of reduction is hkely
to be a function of light intensity, because the partner with which the com-
pound ACO2 reacts must be a —direct or indirect—product of the primary

photochemical process.
Two different premises can be used in the kinetic analysis of the effect
of carboxylation. One alternative (indicated by arrows in equation 27.1,
is to assume that the carboxylation is markedly reversible, i. e., that k^ is
of the same order of magnitude as A-a[C02]a. In this case, the association
of the acceptor A is not complete even without any

with carbon dioxide
dislocation of the equilibrium by the consumption of ACO2 in light. The
other alternative, preferred by Frank and Herzfeld, is to assume that the
carboxylation equilibrium lies entirely on the side of association (meaning
A-a[C02] » A-a), so that in the dark practically all acceptor is "saturated"
by carbon dioxide molecules (at all practically significant partial pressures
of carbon dioxide), unless the carbon dioxide molecules are displaced by

other association partners, such as reduction intermediates, narcotics,
etc. Free molecules A occur in this picture only during (or immediately
after) intense photosynthesis, when reduction of ACO2 is (or has been) too
rapid for the recarboxylation to keep step with it.

As described in chapter 8 (vol. I) the known equilibria of carboxylation
in vitro correspond to practically complete dissociation. Only few cases
are known which the carboxyl group is thermodynamically stable with
in
respect to decarboxylation (at least, under sufficiently high carbon dioxide
pressures). The "saturation" of photosynthesis with carbon dioxide, which
occurs under partial pressures as low as 0.1%, indicates that conditions may
be different here, perhaps in consequence of a coupling of carboxylation
with another reaction, such as degradation of a "high energy phosphate,"
or an "endergonic" oxidation-reduction (c/. Vol. I, page 201). However,
there is no experimental or theoretical reason except convenience —
in ana-
lytical formulation— to postulate that in photosynthesis the carboxylation

equilibrium completely on the side of synthesis, even at the lowest practi-
lies
cally significant carbon dioxide pressures. We

will therefore begin our
analysis by assuming that the degree of saturation of the acceptor with car-
bon dioxide does depend on the external concentration of carbon dioxide.

If one molecule of carbon dioxide is taken up by one molecule of ac-
ceptor, the carboxylation equilibrium is determined by the equation:
(27.3) [ACO2] = (A'aAo[C02]a)/(l + iâ[C02]a)
where ^0 is the total available concentration of the acceptor:
(27.4) Ao = [A] + [ACO2]
and iCa is the equilibrium constant of carboxylation:
(27.5) Ka[C02]a[A] = [ACO2]

: : — :
If the carboxylation mechanism is as simple as postulated in (27.1), the

equilibrium constant Kg, is equal to the ratio of the two rate constants ka
and h\.
In equation (27.4) it is assumed that the acceptor, A, is either free or
occupied by CO2. This may not be the complete description for two rea-
sons: In the first place, the first reduction product of ACO2, designated by
us as AHCO2, may require time for its dissociation into and HCO2; part A
of the acceptor is then "blockaded," during photosynthesis, by this reduction
product {cf. section / below). In the second place, the photochemical re-
duction may have to be repeated several times, e. g.
(27.5A) ACO2 —^^ AHCO2 —— > AH2CO2 ^ AH3CO2 ^

AH4CO2 > A + H2O + {CHoO!
before the reduction product can separate itself from the carrier A (as
in the Franck-Herzfeld mechanism discussed in section d below.) If
AHCO2 is assumed to be the only product of photochemical reduction, the

completion of its reduction to AH4CO2 i. e., to the carbohydrate level-
must be ascribed to dismutations
4 AHCO2 > AH4CO2 + 3 ACO2
cj. Vol. I, p. 158.

The simplest assumption that can be made in the interpretation of the
carbon dioxide curves of photosynthesis is that they are, at least basically,

saturation curves of the acceptor A. This means that one assumes (a)
that equilibrium (27.3) is not strongly dislocated during photosynthesis,
at least under moderate conditions, and (6) that the rate of photosynthesis
isgiven by the rate of reduction of the compound ACO2, and the latter is
proportional to the concentration [ACO2]:
(27.6) P = nkf X [ACO2]
where the constant k* depends on the intensity of illumination (as indi-

cated by the asterisk). We will deal with the possible limitations of as-
sumption h later (see, e. g., section e). The condition for the correctness of
a (i. e., for the maintenance of equilibrium 27.3 in light) is (cf. formula
27.2)
(27.7) k* <K
That this condition is not always satisfied is demonstrated by the "pick
up" phenomena, described in chapter 8 (Vol. I). These observations show
that in very intense light (i. e., when k* is very large) or in the presence of
certain poisons (when k'a is very small) the acceptor A becomes "denuded"
of carbon dioxide and afterward "picks it up" in the dark.
: :
The factor n in (27.5) is either 1 or a fraction of 1, depending on how

many molecules CO2, at best, can be reduced to the carbohydrate level,
{CH2O for each molecule CO2 which undergoes the first reduction step
}
,
(to If the mechanism of reduction involves only one photo-

AHCO2).
chemical step (ACO2 -^ AHCO2), followed by a two-step dismutation:
4hv
4 AHCO, > 3 ACO2 + HoO + (CH2OI +A
then /; is 1/4. If an energy dismutation step of the tj^pe discussed in
chapter 9 (page 264) also is involved, n is reduced to 1/8. If, on the other
hand, CO2 is reduced to the H4CO2 level in a straight series of photochemi-
cal reduction steps:
AGO.
^"'
) AHCO, -^ -^ -^ ATI4CO, > A + H,0 + CH,0
then 71 is equal to 1. (As a compensation, the constant /;;* can be equal in
the dismuation model to the number of the absorbed light quanta, but
must be l/n times smaller in the straight reduction model, where 1/n
quanta are needed to carry a single CO2 molecule through all four reduc-
tion steps.)
Assuming that the conditions (27.6) and (27.7) are satisfied, we can
insert into (27.6) the equilibrium value (27.3) and obtain, for the carbon
dioxide curves of photosynthesis, the equation:
(27.8) P = 7lkt KaAo[C02]a/(l + iâ[C02]a)
For various values of k* (e. g., for various light intensities, 7), equation
(27.8) represents a family of curves of the "Bose type." These curves are
hyperbolae
(27.9) P/(Pxnax. - P) = K,[C02]
At the saturating concentrations of carbon dioxide, P approaches asympto-

tically the maximum rate:
(27.10) Pmax. = nk* Ao
All carbon dioxide curves separate from the beginning, their initial slopes
being
(27.11) {dP/d[C02].)a = nk*,K,Ao
They all reach "half saturation" at the same carbon dioxide concentration:
(27.12) l/JC02]a = 1/iâ
The empirical carbon dioxide curves deviate more or less widely from this
simple type: Even the "Bose type" curves, shown in figure 27. 2A, do not
all reach half saturation at the same value of [CO2]. We can attempt to
consider the curves (27.8), determined exclusively by static conditions,
—
CAKBOK DIOXIDE DIFFUSION 921
as the "primary" carbon dioxide curves, and treat the empirical curves as
if they were basically such curves, deformed by superimposed kinetic in-
fluences. In the region of low carbon dioxide concentrations (or in the
presence of poisons such as hydrogen cyanide), these additional influences
comprise supply reactions of limited efficiency slow carbon dioxide dif-
fusion and slow carhoxylation. These two processes of limited, but [CO2]-
proportional, maximum rate tend to impose a slanting "roof" on the
P — /[CO2] curves and thus to convert Bose's several divergent hyperbolae
into Blackman's single and almost straight line. In the region of high CO2
concentrations, the additional kinetic influences must be due to [CO2]-
independent factors, such as catalyst deficiencies, which tend to impose a
horizontal "ceiling" on the over-all rate, i. e., to produce "carbon dioxide
saturation" of photosjiithesis even before the acceptor A has become sat-
urated with carbon dioxide.
(6) Diffusion Factors
When and carboxylation are slow processes (more exactly,

diffusion
when their maximum rate under the given
conditions is not rapid compared
with the actual rate of photosjoithesis, P) the concentration [C02]a of the
carbon dioxide molecules in the immediate neighborhood of the acceptor
may decline during photosynthesis considerably below the concentration of
the same species in the medium, [CO2]; while the concentration of the
carboxylated acceptor, [ACO2], may decline markedly below the value
corresponding to the thermodynamic equilibrium, as determined by equa-
tion (27.3). The stationary concentrations, [C02]a and [ACO2], estab-
lished under such conditions, can be calculated by the application of the
law of mass action to the reactions (27.1) and (27.2):
(27.13) [CO^la = (fcdlCOo] + A-;[AC02])/(A:aAo - A^JACO,] + k,)
and:
(27.14) [ACO2] = (A-a[COo]aAo)/(A;* +K + k^COÛ)
Combined, these two equations give a quadratic equation for [ACO2]

(and thus also for P) as a function of [CO2]. Its one physically significant
solution is:
(27.15) [AGO,] = — — + Kkd + — +

k^k* Ao
-
k*kd
'
A;,fc4COo]
'
kJcÂo + <A;j + kfk,i + k^kj[C02]\ AoAvlCOo]
2 kfk^ J k*
Inserting this value of [ACO2] into (27.6), we obtain:
(27.16) - =
2^;;
^(k^k* Ao + klkd + k*kd + kJ<:d[C02]\ _ Aofcd[C02]A;*

^\ 2K /
This equation represents, for different vakies of the parameter A-* {i. e.,
for different light intensities), a set of hyperbolae. Similarly to the "pri-

mary" carbon dioxide curves (27.8), these hyperbolae approach the satura-
tion values (27.10), but, in contrast to the primary curves, they do not all
reach half saturation simultaneously. The expression for the half-saturat-

ing external carbon dioxide concentration can be derived from (27.15) by
assuming [ACO2] ^ 3^4o, and is as follows:
(27.17) .,.1C0,, . i-
[1 + *;
&A^^']
This equation shows that, because of delayed diffusion and carboxylation,
the half-saturation point advances with increasing light intensity toward
higher carbon dioxide concentrations —a behavior actually shown by most
if not all of the experimental carbon dioxide curves (c/. figs. 27. 2A, 27. 2B
and 27.3).
The initial slopes of the curves (27.16) are not proportional to k*, as
were the slopes of the primary curves according to equation (27.11), but
are given by the equation:
(27.18) (rfP/d[C02])o = nkMkdk* /{kÂokt + Kkd + kdk*)
This equation shows that carbon dioxide curves for all k^ values are con-
fined within an angle formed by the axis of the abscissae and a slanting
straight line ("roof"):
(27.19) Pur.. = [CO:^]

^^l^'l\^
and, of course, also under the horizontal ceiling (27.10). Equations

(27.16), (27.17), (27.18), and (27.19) represent the combined effects of slow
diffusion and slow carboxylation.
If only one of these factors is operative,
the equations can be simplified. Pure diffusion limitation can be assumed

if the maximum diffusion supply is much smaller than the maximum rate of
carboxylation, while pure carboxylation limitation must prevail under the

reverse condition. In the first case:
(27.20) kd<^kM
: : .
and equation (27.16) is reduced to:
(27.21)
P =
-
n
—
2i:aAoA;* +k.i
2^^
+ K^kACO,]
'kJ^Jc* -\-kd + KJzd[C02]\ _ AoA;rfA;*[C02]

,'
2^ /
(which contains, as expected, only the equilibrium constant, K^, instead of

the velocity constants k^ and Ic'j. The half-saturating concentration is:
1 / K,A,k*\
(27.22)
and the initial slope:
(27.23) (dP/d[C02])<> = nKMk*kd/{K^k^k* + kd)
The limiting slanting line (roof") is:
(27.24) Piim. = nkd[COi]
—an expression obviously representing the maximum possible supply of

carbon dioxide by diffusion (multiplied by n)
The initial slope of the particular carbon dioxide curve, which, without
the diffusion limitation, would have started with the slope equal to that of
the limiting line (equation 27.24), i. e., according to equation 27.11, the
curve corresponding to /r* = ka/KÂo, is reduced, by slow diffusion, to
one half its former value
(27.25) {dP/d[C02])o = nkd/2
More generally, a primary curve wdth an initial slope aka is reduced by the
diffusion limitation to an initial slope of
(27-26)
{dm]). = (^) "'^
Thus, primary curves with initial slopes between 10 and 100 ka, will
be confined, in consequence of slow diffusion, between ^%\ka and ^^%Q\kd,
i. e., their initial parts will practically coincide with the
limiting straight
line,and thus present a picture of the "Blackman type." On the other
hand, primary curves that would have exceeded the limiting rate only by
factor of the order of unity, as well as those that would have merely ap-
proached, but never exceeded, this limit, will retain their individuality and
nonlinear shape, and will show a gradual transition from the "Blackman
type" to the "Bose type." A certain depressing effect of diffusion will be
felt even in a curve the original slope of which was as low as 0. 1 ka- (The
:
slope of this curve will be reduced by 10%.) This example of the "advance
effect" exercised by a "limiting factor" according to the general laws of
reaction kinetics has already been quoted in chapter 26.
(c) Slow Carboxylation
This case is contained in the aboA'c-derived general equations (27.16-

27.19), if one makes the assumption:
(27.27) kÂo<^kj
This implies another inequality

(27.28) /crf [CO2 ]»/(-.' [ACOol
(since A-,.-lo[CO,] > AvUiCO,], > A-JA]lC"().], = /.{[AC'O,] + A*, the last
equation being the steady state condition for the complex ACO2).
Conditions (27.27 and 27.28) reduce (27.13) to:
(27.29) [COaJa = [CO2]
as it should be when the diffusion supply is ample. Consequently, (27.14)

and (27.15) are replaced by:
(27.30) [ACO2] = A-.,[C02]Ao/(A:: + k* + A:, [CO-,])
and (27.16), by the much simpler equation:
(27.31) P = nkM[CO,]k*/ik: + k* + kîCO,])
This equation can be written as:

(27.32) P/(Pn>ax. - P) = k,[CO,]/{k: + kt)
These hyperbolae reach half saturation at:
(27.33) ./JCOd =
^ (1 + I)
(as could be derived also directly from equation 27. 17) . Their initial slopes
are (c/. 27.18):
(27.34) {dP/d[C02])a = nk^k^*/{K + k*)
All curves (27.31) are confined under the "roof":
(27.35) Pli:n. = nfcaAo[C02]
an equation that can also be derived from (27.19), and obviously represents
the maximum possible rate of carboxylation. By reasoning similar to that
employed just above, it can be sho^vn that a "primary" carbon dioxide
curve with an initial slope akÂîn will have its slope reduced, in consequence
of slow carboxylation, to a/ {a + l)/Cai4on. In other words, in this case, too,
the influence of the Hmiting process is felt long before the rate approaches
the limit.
Carboxylation was treated so far as a one-step reaction, with a rate
proportional to the concentration [C02]a- I* is known, however (c/. Vol.
I, page 203), that carboxylation in photosynthesis is cyanide-sensitive,
and thus undoubtedly a catalytic reaction. It must therefore consist of

several steps, such as the formation of a substrate-catalyst complex, and
the transformation of this complex. We have no information as to the
precise nature of these steps, but it can be assumed that at very low [CO2]
values, the rate-determining step will be one with a rate proportional to
[CO2], e. g., reaction (27.36). Under these conditions, the above-given
derivations will be valid for the catalyzed as well as for direct carboxyla-
tion. At the higher [CO2] values, however, other steps or the carboxyla-
tion process may become rate-determining — steps limited in their maxunum
efficiency by the available amount of the relevant catalyst (carboxylase).
A [C02]-independent "ceiling" will thus be imposed on the rate of the over-

all process, which will be determined by the product of the amount of the
catalyst available and the average time a catalyst molecule requires to

complete the desired transformation.
The specific form of the corresponding kinetic equations will depend
on the postulated mechanism of the catalytic action, and, as stated above,
we have at present no reasons to favor any one mechanism among the sev-
eral compatible with our general knowledge of the mechanism of enzymatic
processes.
As an illustration, we will go through a calculation based on the simple mechanism:
K
(27.36) COo + Ea .
Ej, CO2
*.'
(using Ea, as in Volume I, as symbol for the carboxylase).
(27.37) Ea -CO, + A :î=^ Ea + ACO2
(For the sake of simplicity, we neglect diffusion and use [CO2] where [C02]a should be
used.) The equilibrium constants K, and K,^ are subject to the condition:
(27.38) ifeA'ea ( = p >< ^) = K.
We assume that equilibrium (27.36) is established practically instantaneously, and

remains undisturbed during photosynthesis, but that equilibrium (27.37) is less rapidly
attained, and can therefore be displaced in light.* Designating by El the total avail-
is a slow reaction would lead to more com-

* The assumption that (27.36), too,
plicated quadratic equations for [ACO2] and P, similar to those obtained in section b
for the combined effects of slow diffusion and carboxylation.
able quantity of the enzyme Ea we can derive the following equations for the steady
state:
k*K k* \
/([l + KACO2] + ^' [CO2] + yô)
(27.40) P = n/c*KaAo[C02] /( 1 + K.iCO^] + p^ [CO2] + ^ )
The term proportional to A;;.[C02] in the denominator imposes on these carbon di-
oxide curves an "absolute ceiling" (i. e., a maximum rate independent of both [CO2] and
A;*, and thus of 7):
(27.41) PStJ: = nAêaEÂo
(The lower index refers to [CO2], the This obviously is n times the maximum
upper to I.)
mechanism (27.36 and 27.37).)
possible rate of carboxylation according to the
For relative saturation, as a function of [CO2], we obtain:
[C02
(27.42) P/(Pmax. -P) = '^.iiEt^L.
1 + {k*IK.^l)
and for half saturating carbon dioxide concentration:
(27.43)
which reduces
„JC0,1 =
itself to:
i (i|, + 1)/ (1 +
^J
(27.44) ,/JC02] = UK,
for high A;* values (strong light), and to:
(27.45) ,/JC02] = 1/ifa
for low kr values(weak light).

Depending on whether K& < Ke, or vice versa, 1/JCO2] will shift upwards or down-
wards with increasing light intensity. The initial slope of the curves (27.40) is:
(07 An / rfP \ nktKÂo

^"^'^^^
\d[C02])o 1 - AfAe'aE^
The reduction in rate caused by Ea deficiency is, by comparison of (27.40) with (27.8):
K ^^ A'e[C02] + 1
(27-46a) ^ = VV+E!E^r:[C02] + l.
For saturating [CO2] values:
=
(27.46b) /3
V (' + sL)
and is thus equal to 3^ for a light intensity k* — Âêa, which without enzyme deficiency
would have given the saturation value (equation 27.41). Generally, the saturation
level is reduced from nakeaE°AAo to
the limiting process becomes felt

fe)
{ )
when the nonlimiting

A-ea^Âô.
rate
Thus, here again, the
is still far
effect of
below the imposed

"ceiling."
(d) Nondissociahle ACO2 Corn-pound. The Franck-Herzfeld Theory
So far, we have considered the carbon dioxide curves as, basically,

ACO2 saturation isothermals, merely distorted by slow diffusion, slow
carboxylation and limited quantity of the carboxylase Ea. An alternative
was mentioned several times before. The carboxylation equilibrium may
lie practically completely on the side of association, and the effect of the
factor [CO2] on the rate of photosynthesis may be due entirel}^ to kinetic
phenomena, such as the limited rates and carboxylation. The

of diffusion
corresponding kinetic equations can easily be derived from the more general
formulae given in sections h and c by putting fc^ = 0, i. e., assuming that
the rate of decarboxylation is negligible. For example, if the carbon di-
oxide limitation comes exclusively from slow carboxylation (while the sup-
ply of carbon dioxide by diffusion is ample), we can start directly with equa-
tion (27.31); omitting the k^^ term in the denominator of this equation,
we obtain:
(27.47) P = nKAo[C02]k* /(K + â[COo])
(If k* «: ks[C02], this equation is reduced to P = nA;*Ao as expected.)

This equation, too, represents hyperbolic carbon dioxide curves:
(27.48) P/(Pmax. - P) = K[C02]/k*
with half saturation at:

(27.49) ./JCOa] = A;*Aa
and the initial slope:
(27.50) {dP/d[C02])o = nkM

It will be noted that, in this case, all carbon dioxide curves make the same
angle with the [CO2] axis, independently of A:^ (i. e., of the light intensity).
Half saturation, on the other hand, occurs at [CO2] values that are propor-
tional to k* (i. e., increase with increasing light intensity).
The same method can be extended, to account also for saturation ef-
fects caused by limited amount of the carboxylating catalyst âi e. g.,
by assuming fc^ = in formula (27.37). Equation (27.40) is reduced, by
this assumption, to:
(27.51) P = n2fefceaA:?AoE° [C02]/(KefceaEi[C02] + Kek* [CO2] + k*)
The "absolute ceiling" of this family of hyperbolae, approached when both

: : :
k* and [CO2] are the same as in formula (27.41), i. e., equal to n

high, is
times the maximum rate of formation of ACO2 by reaction (27.37).

The equation of the individual carbon dioxide hyperbolae is
(27.52) P/(Pn,ax. - P) = {keJil + fc*)K%[C02]A*
They reach half saturation at
(27.53) ,/JC02] = k*/K,(k,,El + fc*)
and have the following initial slope (independent of k*, and thus of light
intensity)
(27.54) {dP/d[C02])o = nKeke.AÊl
The assumption of a stable ACO2 compound was used by Franck and

Herzfeld (1941) in their detailed kinetic theory of photosynthesis— the
most elaborate to be found in the Uterature. The rather complex chemical
mechanism on which the kinetic analysis was based was illustrated by
scheme 7.VA in Volume I.
would have been inconvenient for Franck and Herzfeld to use a different postu-
It
ACO2, because they assumed that the carrier is associated
late concerning the stability of
not only with carbon dioxide molecules, but also with seven reduction intermediates.
To consider the equilibrium ACO2 Â+ CO2 as reversible, while postulating a firm
attachment to the same carrier of the reduction intermediates, would have meant added
mathematical complications; while to assume a different reversible association equilib-
rium for each intermediate (as was once suggested in chapter 9) would have been still
more cumbersome. Thus, Franck and Herzfeld chose the simplest way when they as-
sumed the complexes of A with CO2 as well as with all seven intermediates to be practi-
cally undissociable (except by light —
a complication discussed in Vol. I, p. 167, and in
chapter 29).
Because of the assumption of an undissociable complex, the derivation of Franck
and Herzfeld contains no equivalent of section a. Since they did not take into account
it also contains no equivalent of section b.
the effects of slow diffusion, The aspects of
the carbon dioxide supply problem that Franck and Herzfeld did consider were the
phenomena treated in section c, i. e., slow carboxylation, caused either by low carbon
dioxide concentration, or by carboxylase deficiency.
Their treatment of these two effects was somewhat different from that given in sec-
tion c because they postulated a different mechanism of catalysis. Instead of the reac-
tion sequence (27.36, 37), Franck and Herzfeld assumed the following three reactions:
K
(27.55a) CO2 +A , A CO2
•
(formation of a "loose" complex)
(27.55b) ACO2 + Ea > AGO, + EI

(catalyzed formation of a "stable" complex and inactivation of the catalyst)
k'
(27.55c) E; ^—^ Ea
(reactivation of the catalyst)

—
FRANOK-HERZFELD THEORY 929
Franck and Herzfeld assumed equilibrium (27.55a) to be established practically
instantaneously, whereas reaction (27.55b) was assumed to have a finite velocity, and
thus to be capable of becoming a "bottleneck" of photosynthesis. This occurs when
either the substrate concentration [A-C02], or the enzyme concentration [Ea] is low,
or, more generally, when the product of the two concentrations is small. According to
(27.55), the concentration of the "loose" complex is:
(27.56) [A-COs] = K [AJICO^]
(For the sake of consistency cf. equation 27.5- we use as equilibrium constant the in-
verse of Franck and Herzfcld's K). The rate of the bottleneck reaction (27.55b) is:
(27.57) {dlAC02]/dl) = ^•a[A•C02][E] = k^K[A][C02][EA]
We assume— as we did in all our derivations so far— that no kinetic factors other than
those connected with the supply of carbon dioxide affect the rate of photosynthesis.
Under these conditions, the equations of the carbon dioxide curves can be derived by
calculating the stationary concentrations [A] and [Ea], inserting them into (27.57) and
then calculating the stationary concentration ACO2] by equalizing the rate of production
[
of [ACO2] given by equation (27.57) and the rate of reduction of this product by light.
In the Franck-Herzfeld theory, all carrier molecules, A, may be considered, for
kinetic purposes, as attached to a molecule of the sensitizer (chlorophyll), so that the
substrate molecules bound to A can undergo direct photochemical change; putting it
more cautiously, only those A molecules are taken into consideration in kinetic equations
that are attached to chlorophyll. Their total number can be designated as Ao. Simi-
larly, all chlorophyll molecules are supposed to carry acceptor molecules, A; putting it
more cautiously, only the absorption of light by those chlorophyll molecules that are as-
sociated with A is taken into consideration. We designate the total number of such
molecules as Chlo. This number probably can be reduced by certain inhibitors, such as
urethan or other narcotics, that displace the acceptor A from chlorophyll. (The same
applies, according to Franck, to "self-narcotization" by the unfinished product of
photosynthesis to which reference was made in chapter 24.)
In Franck's picture, the rate constant k* in equation (27.6) can be written as k*I:
(27.58) P = -nid[AC02]/dt) = A;*7[AC0.2]
Here, A;*7[AC02] is the rate of absorption of light by the acceptor A in combination

with CO2, k* being essentially an average absorption coefficient of the specimen under
investigation. The factor n is equal to 1 in the Franck-Herzfeld mechanism; but k*I
is, in the steady state, not more than one eighth of the total
rate of absorption.
If one assumes, as Franck and Herzfeld did, that this absorption coefficient is the
same whether chlorophyll is associated wath ACO2 or with any of the seven reaction
intermediates {cf. scheme 7.VA), then in the steady state the amounts of [ACO2] and
of seven intermediates must all be the same. This means that:
(27.59) Ao = ( = Chlo) = [A] + [A -002] + 8 [ACO2]
where Ao is the total available quantity of the acceptor A bound to chlorophyll.

Equalizing (27.57) and (27.58), we obtain:
(27.60) IACO2] = (k,K[\][EA][C02])/k*r
The stationary value of [Ea] can be calculated by equalizing the rates of reactions
27.55b) and (27.55c); this gives:
(27.61) [Ea] = A;:E°/(A:.[A-COo] + k:)

:
After inserting (27.61) into (27.60), one can use (27.56) and (27.59) to eliminate [A] and
—
[A.CO2] and to obtain the desired equation for [ACO2] and according to (27.58) also
for P— in Because of the assumed two-step mechanism of
terms of [CO2], Ao and E^.
formation of [ACO2] (with the loose complex A-C02 as an intermediate), the resulting
—
equation is quadratic. Its solution which again represents a family of hyperbolae is —
a rather complex expression, and we do not need to quote it here.
For high values of both [CO2] and /, the expression for P approaches asymptotically
the value:
(27.62) PZTr.: = nKKElAo/ikaAo + K)

(with the lower index referring to [CO2] and the upper to /). In the two extreme cases,
when either kaAo ]^ K, or vice versa, expression (27.62) reduces itself either to nkj^l
(t. e., the maximum rate of reaction 27.55c), or to nA-aEÂn (the maximum rate of reaction
27.55b).
The relations are much simpler if the rate of reaction (27.55c) is assumed to be
rapid (compared with the rate of photosynthesis P), so that [â] is practically equal to
'
zero, and [Ea] to E^. In this case, one obtains an equation containing only first
power of P, the solution of which is:
(27.63) P = k,,KElA,[C02]k*l/{k*I + k*IK[C0.2] + 8 k^KEUCO,])
This is an equation of a family of hyperbolae (with / as parameter)
Half saturation (P = i/jPrnax.) is reached when:
(27.65) VJCO2] = k*mKkn + 8 k^KEl) = 1

(^ + ^Sk.El/kÎ) )
It is shifted, with increasing light intensity, toward the higher [CO2] values. The initial
slope of the curves (27.63) is:
(27.66) {dP/d[C02])o = nkaKElAo

Because, in formula (27.55b), the formation of ACO2 was assumed to be irreversible, the
slope (27.66) is independent of light intensity. (In other words, at very low carbon di-
oxide concentrations, all ACO2 formed is reduced by light, whatever the intensity of the
latter.) Thus, as stated before, the carbon dioxide curves represented by equations
such as (27.47) or (27.63) have more pronounced "Blackman characteristics" than the
curves obtained with the assumption of a dissociable ACO2 complex.
Of course, the hypothesis of a stable association of the acceptor A. with chlorophyll
is independent of the other postulates of the Franck-Herzfeld theory; the latter can be
combined also with the assumption of a dissociable ACO2 complex.
(e) Back Reactions in the Photosensitive Complex
So far, we have discussed the carbon dioxide curves only from the point
of view of the "preparatory" dark processes at the "reduction end" of
photosynthesis, since these are the stages of photosynthesis most closely
related to the "carbon dioxide factor."
The influence of the preparatory reactions at the "oxidation end"
—
INTERPRETATION OP CARBON DIOXIDE CURVES 931
(such as the possible preUminary enzymatic binding of water) as well as

the influence of "finishing" reactions (such as conversion of AHCO2 to a
carbohydrate, and hberation of oxygen) have so far been neglected; while
the role of reactions within the photosensitive complex proper was taken
into account only by assuming the rate of reduction of ACO2 to be equal
to the product fc*JAC02], where k% was considered a function of the illu-
mination intensity, I. No new source of carbon dioxide saturation was

added by this assumption; saturation remained determined entirely by
the four factors treated in detail in sections a-d (limited quantity of A,
slow diffusion, slow carboxylation and limited quantity of the carboxylase,
Obviously, however, carbon dioxide saturation can also be produced by

limitations of any of the other partial reactions in photosynthesis. For
example, msufficient amount of a catalyst needed for the preliminary trans-
formation of the reductant (H2O, H2, H2S. .) might have the same "ceil-
.
ing" effect on the carbon dioxide curves as the limited amount of the en-
z>Tne (carboxylase) that catalyzes the prelmiinary transformation of car-
bon dioxide itseK.
be gained by trying to write out equations for carbon di-
Little could
oxide curves that would include the effect of a Umited supply of the reduc-
tant. On the other hand, a few words may usefully be said about the in-
fluence on the carbon dioxide curves of a limited supply of light.
The kinetic equations that follow from the consideration of the prob-
able forward and back reactions within the photosensitive complex proper
will be derived in chapter 28 (p. 1020 ff.). They show, as expected, that
the rate of absorption of light complex imposes a limit on the rate of
by this
photosynthesis that cannot be raised by increased supply of carbon dioxide
(or change in any other external factor). Consequently, the "light factor"
is in itself capable of producing a saturation effect in the carbon dioxide
curves. For example, if we consider equation (28.14), derived from reac-

tion mechanism (28.11), as an equation of carbon dioxide curves (i. e.,
if we treat 7 as a parameter), we find that, at high [CO2] values, the rate

approaches the maximum:
(27.67) Pmax. = nk*ICh\o
which is the rate of supply of light quanta multiplied by the number n of
carbon dioxide molecules that can be transfomied by a single quantum

perhaps 3^. Half saturation is reached, according to scheme 28. lA, at:
(27.68) ,/JAC02] = k'/kr
One could thus ask whether the interpretation of carbon dioxide curves
really requires the assumption of an acceptor A, supposed to be present in
limited quantities and carboxylated by a slow dark reaction; or whether

one could perhaps explain carbon dioxide saturation phenomena by ref-
all
erence to limited supply of light. The occurrence of phenomena such as

carbon dioxide "pick-up" after intense photosynthesis (Vol. I, page 206)
provides, however, direct evidence that a dark carboxylation reaction actu-
ally does occur, and that it has an effect on the rate of the over-all reaction
of photosynthesis. On the other hand, it is undoubtedly true that some, at
least, of the carbon dioxide curves, particularly those measured in weak

light, owe their hyperbolic shape entirely or preponderantly to the limited
rate of supply of light quanta.
Reaction mechanism (28. lA, eqs. 28.20), which we have used, provides
that the reaction of the primary photoproduct, HX-Chl-Z, with the oxi-
dant, ACO2 (rate constant, A,), competes with the "deactivating" reaction
that converts HX •
Chi Z back to X Chi HZ (rate constant k'). An alter-
• • •
native mechanism (28. IB, eqs. 28.21) also is discussed in chapter 28, in
which the "primary" back reaction of the photoproduct, HX-Chl-Z, is
ehminated by immediate reaction of HX •

Chi Z with
•bound) water,
(free or
converting it to HX-Chl-HZ. This simplified mechanism will be used in

chapter 28 to analyze another possible kinetic effect within the photosensi-
—
tive complex the accumulation, during strong photosynthesis, of the
chlorophyll complex in the changed, photoinsensitive form (HX • Chi • HZ in
the mechanism used). In deriving equation (28.14), the simplification
[HX Chi Z «; [X Chi HZ
• •
] ]
^
Chlo was made we do not make a similar
• •
;
assumption in respect to HX-Chl-HZ, but postulate that the "reduced"

form accumulates and brings about saturation of light curves. (With in-
creasing light intensity more and more chlorophyll complexes will be pres-
ent, in the steady state, in the photoinsensitive form, HX-Chl-HZ.) This
assumption leads to equation (28.27) for P as function of / and [ACO2], and
to eciuation (28.28), if the equilil)rium value (27.3) is substituted for [ACO2]
in (28.27). Considering (28.28) as equation of carbon dioxide curves (/ =
constant), we obtain the following expressions for these curves:
P — P k*I
'^'•*''
"p.„. " f/(l + K.[C0,1 + k,K,A,lCO,]
)
("•™' V.ICOJ - J; (s;j^,)

Equation (27.70) shows that the half-saturating carbon dioxide concentra-
tion rises with increasing light intensity, and that the equilibrium constant
Kg^ can be obtained by an extrapolation of 1/JCO2] to high light intensities
(linear extrapolation with 1/7 as abscissa) It will be recalled that in the .
case of the carbon dioxide supply limitation (c/. equation 27.22 or 27.33)
we had to obtain the same value by extrapolation to low light intensities
INTERPRETATION OF OARRON DIOXIDE CURVES 033
(/ = 0, and thusfc* = 0). We will return to the evaluation of K^ from car-
bon dioxide curves in section g.
(/) Acceptor "Blockade"
In comparing the equations obtained in the preceding sections, with

the empirical carbon dioxide curves, one has to keep in mind that, despite
the considerable complexity of some of these equations, they all embody
certain simplifjdng assumptions, and therefore cannot be valid except
within a Hmited range of conditions.
The only kinetic factors taken into account so far were slow diffusion
of carbon dioxide, slow carboxylation and limited quantities of the acceptor
A and of the carboxylase Ea. In section e we discussed the additional com-
plications that may be caused by the deactivation of the primary photo-
chemical product, HX-Chl-Z, in competition with its reaction with ACO2,
or by the accumulation of the photosensitive complex in the reduced form,
HXChl-HZ.
A single slow preparatory dark reaction with a rate proportional to
[CO2] plus the limited quantity of a single catalytic agent (e. g., Ea, A or
Chi) could suffice to account for the increase of P at low values of [CO2],
for the individual saturation of each carbon dioxide curve and for the oc-
currence of "absolute" saturation (i. e., saturation with respect to both
[CO2] and /). We know, however, that several catalytic steps of limited
maximum efficiency play a part in photosynthesis ; and, although some of

these steps are not directly associated with the assimilation of carbon
dioxide, a limitation of the rate of the over-all reaction, whatever its source,
must be reflected in the shape of the carbon dioxide curves, particularly in
the region where they approach "absolute saturation."
In Volume I, we outlined a general scheme of photosynthesis that in-
cludes, in addition to preparatory supply reactions, two other main types of
catalytic processes
— "finishing" reactions, associated with the conversion
of the first reduction products into carbohydrates and with the production
of molecular oxygen.
What happens to the first reduction product (which we will now desig-
nate as AHCO2) can affect the rate of photosynthesis in various ways.
If, for example, this product has to undergo a chemical transformation
before can be separated from the carrier A, this transformation may re-
it
ciuire a certain time, so that, in intense light, a considerable fraction of the

acceptor A can be "blocked" by AHCO2. Or else, the product AHCO2
may require a catalyst (^b) for its stabilization, and unless this catalyst is
available within a sufficiently short time, AHCO2 may react back (with
the oxidized photosensitive complex X-Chl-Z, or with the intermediates
of the oxidation of water, such as A'HO, or with some other celkilar oxi-
dants) to form ACO2 (or, A CO2).
according to a hypothesis of Franck, +
The latter possibihties will be discussed in section le of chapter 28.
We will briefly consider here the effects of the "acceptor blockade." The conserva-
tion equation (27.4) must in this case be replaced by:
(27.71) Ao = [A] + [ACO2] + [AHCO2]
Assuming that the restoration of the acceptor is a monomolecular process
(27.72) AHCO2
k'
—
'-
> A + HCO2
we can ACO2 and (assuming the
write the equations for the stationary concentration of
validity of 27.6) also for the rate of photosynthesis. Taking the simplest case that —
discussed in section a (carboxylation equilibrium undisturbed by slow diffusion or slow
carboxylation) —we obtain:
nA.K. [CO.W.
(27.73) P=
l+TValCOo]
(27.74)
(27.75)
The carbon dioxide curves (27.73) are thus hyperbolae the half saturation of which is
shifted, with increasing light intensity, toward the lower concentrations of carbon dioxide
(a shift opposite to that caused by slow diffusion or slow carboxylation; and apparently
not encountered in experimental curves). Because of the occurrence in the denomina-
tor of (27.73) of the product A;*[C02], the rate cannot exceed the "absolute maximum":
(27.76) PZ^. = n/cj'Ao
i. e., n times the maximum rate of restoration of the acceptor according to (27.72).
(g) Calculation of Carhoxijlation Constant from Carbon Dioxide Curves
one wants to use the carbon dioxide curves for the determination of
If
the carboxylation constant K^^, as was done in chapter 8 (Vol. I), one should
avoid the region of "absolute saturation," and determine systematically,

and with a high degree of precision, the carbon dioxide curves in the region
where the yield is still proportional to light intensity. In this way, one
could perhaps determine which of the several above discussed mecha-
nisms of carbon dioxide supply provides the best interpretation of the facts.
The experimental data available at present are neither exact nor systematic
enough for such an analysis.
Among the characteristics of the various theoretical equations derived

above for the carbon dioxide curves, which might be used for comparison
with the experiment, are the relations between half-saturating carbon di-
oxide concentration and light intensity (c/. equations 27.12, 17, 22, 33, 44,
45, 49, 53, 65,and 75), and between the initial slope and light intensity
(equations 27.11, 18, 23, 34, 46, 50, 54 and 66).
The value of 1/JCO2] is independent of k* {i. e., of light intensity) and
equal to \/K^ only to the extent to which the carbon dioxide curves are
proportional to the saturation curves of the ACO2 complex (eq. 27.12).
It has been suggested by some that this assumption is legitimate whenever
the carbon dioxide curves are found to be hyperbolic. Thus, Burk and
Lineweaver (1935), having satisfied themselves that the carbon dioxide
curves of Warburg, Harder, James, van der Paauw, and Emerson and
Green (Table 27.1) follow the hyperbolic saturation law, proceeded to
calculate from them the carboxylation constants K^ and obtained values
ranging from 1 X 10^^ to 10 X lO"'' room temperature). The
l./mole (at
corresponding free energies of carboxylation, AFa(= RT loge K^) are be-
tween — 6.9 and —8.3 kcal/mole.
was mentioned on page 908 that, according to Whittingham (1949),
It
the half-saturating CO2 concentration of Chlorella is shifted down to 0.5
or 1.0 X 10 -^ mole/1, if care is taken to avoid inhibition phenomena at
low CO2 concentration, by allowing 2-3 hour induction period (if the cells
were grown in high [CO2]) or, still better, by using cells gro^vn in low [CO2]
(e. g., in air).
The heat of carboxylation also was estimated by Burk and Lineweaver. From the
absolute rate values found by Emerson and Arnold, at 6° and 24° C. in flashing light,
they calculated AH a = 1.3 to 6.2 kcal/mole. However, Table 8. VIII shows that the
fixation of carbon dioxide by organic molecules is accompanied —
as is natural in reactions
in which small molecules are attached to larger ones— by a decrease in entropy, —TAS
being as large as +8 or even +16 kcal/mole at room temperature. Thus, if — AF of
carboxylation is 7-8 kcal/mole, - AH should be of the order of 15-20 kcal— considerably
larger than the estimate of Burk and Lineweaver.
Our derivations in sections b, c and d show that the carbon dioxide

curves may
be strongly affected by slow diffusion, or slow carboxylation,
without losing the hyperbolical shape. Equations (27.17, 22 and 33) show
that these two factors cause i/2[C02] to increase linearly with increasing
light intensitij. The constant K^ can in this case be obtained by linear ex-
trapolation of 1/JCO2] to / = 0. Figure 27.9 shows that the data of

Harder, Hoover and co-workers, and Smith (compare Table 27.1) extra-
10"®
polated in this way, give 1/2CO2 values in the neighborhood of 5 X
ilf, and thus K^ values of about 2 X lOS corresponding to AF = —7.9
kcal. /mole.
936 CONCENTRATION FACTOHS CHAP. 27
However, the linear extrapolation procedure is not always reliable.

While slow diffusion and slow carboxylation tend to shift the half-saturat-
ing concentration upward with increasing light intensity, other influences,
such as the "acceptor blockade" (equation 27.75), or the deficiency of
catalysts, may shift it downwards. Equation (27.43) shows, for example,
that a limited amount of the carboxylase Ea may cause 1/JCO2] either to
increase or to decrease with increasing light intensity, depending on whether
Kf, is smaller or larger than K^. In this case, extrapolation of 1/2 [CO2] to
zero light intensity should still give the correct value of K^, but systematic
data will be needed to make the required nonlinear extrapolation possible.
<
llJ
o
z _
O V.
O a,
O E
O
<
<
CD
Doto of Hoover et al
<
X I r I I I
2 4 6 8 10 12 14 16 18 20 22 24
LIGHT INTENSITY, klux
Fig. 27.9. Extrapolation of 1/JCO2] to / = 0.
under certain conditions the value of i/^ [CO2] may have no rela-
Finally,
tion to the carboxylation constant at all. This is obviously true if the
ACO2 complex is practically undissociable. Equation (27.49), for ex-
ample, contains only the kinetic constants k^, and 14 Similarly, in section
e, where we postulated that the carbon dioxide saturation is caused by
"detautomerization" of the photocomplex HX-Chl-Z, equation (27.68)

showed 1/2 [CO2] to be a function of the kinetic constant k^ and kr only.
To sum up, the constant 1/Ks, may be ^ 1/JCO2] or <C [CO2], 1/2
and the carbon dioxide cui-ves may nevertheless be hyiêrbolae. The

ol)servations of the "pick uptake of C*02 in the dark speak
up" and of the
ill fa^'()r of a reasonably stable ACO2 complex, and thus against the second
~^
possibility. Consequently, the above calculated value of iva ('^ 5 X 10
niole/1.) and of AF( — 7.9 kcal/mole) are to be considered as upper,
rather than lower, limits; in other words, the ACO2 complex is either as
:
stal)le as or even more stable than indicated by these values. We recall
that in chapter 8(Vol. I) a compilation of experimental data showed the

known free energies of carboxjdation of compounds of the type of (RH +
CO2 -» RCOOH) to be about +
10 kcal/mole. It was also mentioned that
Ruben had suggested attributing the extraordinary stability of the ACO2

compound in photosjTithesis to a coupling of carboxylation with an exer-
gonic transphosphorylation —the
transformation of a "high energy" into
a "low energy" phosphate ester. Another explanation of the same type
would be to assume the coupling of carboxylation Avith a parallel or con-
secutive exergonic oxidation-reduction, a coupling that actually occurs in
the uptake of carbon dioxide catalyzed by certain enzymes derived from
yeast and bacteria (as described by Lipmann and Tuttle 194.5; see also
Ochoa 194G).
There is one obvious experiment by which the carboxylation constant
derh-ed from the carbon dioxide curves of photosynthesis could be checked
— precise measurements of the carbon dioxide uptake in the dark as a func-
tion of the concentration [CO2] in the medium. This could be done by
means of radioactive carbon, or by ordinary analytical methods. So far,
no such measurements have been made. The observations of Ruben and
co-workers (c/. Vol. I, chapter 8) seem to indicate, however, that the com-
plex ACO2 is dissociated in vacuum, albeit very slowly, and thus possesses
a finite, although perhaps very small, dissociation pressure (c/., however,
chapter 36 for the interpretation of C*-measurements).
(h) Are Experimental Carhon Dioxide Curves Hyperbolae?
be noted that all theoretical carbon dioxide curves discussed in

It will
this chapter were hyperbolae. Smith (1937) analyzed the empirical car-
—
bon dioxide curves and concluded in contrast to Burk and Lineweaver
(1935)— that many of them reach saturation more rapidly than a hyperbola
approaches its asymptote. He has therefore attempted to obtain a better
analytical representation of the carbon dioxide curves by changing the ex-
ponents in equation (27.9). The empirical equation:
(27.77) P/VpL.. - P' = K[CO,\
was found by him to fit satisfactorily his own results, as well as those of
Warburg, Emerson and Green, and Hoover, Johnston and Brackett. In
the saturation region, where (Pmax. - P) <
P, equation (27.77) should not
differ much from the simpler quadratic relationship
(27.78) P/(Pm.x. - P) = K'[COiV
Exponential functions often are used in the interpretation of curves that rapidly
reach saturation. Harder (1921) tried to represent his results by the formula:
:
(27.79) (p„,,. - P)/P^,,, = e-const.[CO.]
(analogous to Mitscherlich's and Baule's revised formulations of Liebig's "minimum law"

of fertilization).A similar formulation was suggested by Brackett (1935):
(27.80)
^— " ^ = const.
,-![C0.] -(P/6)+<^1
max.
The correction term P /b in the exponent was intended to account for the fact that the
carbon dioxide curves approach asymptotically, at low [CO2] values, a straight line with
a finite slope rather than the axis of ordinates. The correction term c was attributed to
respiration. The exponential as a whole was supposed to represent the "true amount
of CO2 available at the site of photosynthesis" —
the first two terms describing the supply
by diffusion from outside, and the third that by respiration. The occurrence of a con-
centration factor in the exponent, unusual in reaction kinetics, was associated with
Beer's law of light absorption. However, since carbon dioxide is not the light-absorbing
species in photosynthesis, it is not clear why
concentration should appear in the ex-
its
ponent, and the equation of Brackett (as well as that of Harder) is not likely to represent
more than an empirical approximation formula fitting some of the experimental results.
If it is true, as asserted by Smith (1937) that the carbon dioxide curves

definitely cannot be represented by hyperbolae, the question arises as to
what could cause their deviation from the hyperbolic form, and lead to a
more sudden saturation. It is easy to show that the relation between P
and [COo], which was expressed by quadratic equations in the several cases
when two reaction steps were assumed to lead from the external carbon di-
oxide to the complex ACO2, will be represented by a cubic equation if a
third intermediate process is added {e. g'., B + AC02-^ BCO2 + A; BCO2 +
light products -^ photosynthesis). Since the equation representing
[BCO2] as a function of [CO2] will contain higher powers of [BCO2], but
only the first power of [CO2], the carbon dioxide curves will deviate from
hyperbolae in the direction of a slower (rather than of a more rapid) ap-
proach to saturation.
An obvious way to obtain carbon dioxide curves that approach satura-
tion more rapidly than hyperbolae is to assume carboxylation equilibria
involving two (or more) carbon dioxide molecules, e. g.,
k
(27.81) 2 CO2 +A .
' ^ A(C02)2
K
or more generally
(27.82) nCOi +A ,
' ^ A(CO.>)„
In this case, the thermodynamic carboxylation equilibrium condition (as-

suming that the several carbon dioxide molecules in the complex are inde-
pendent of each other) is:
(27.83) [A(C02)„] = XaAo[C02]V(l + A^fCOa]")
:
CARBON DIOXIDE CONCENTRATION AND FLUORESCENCE 939
consequently
(27-8^)
[ACCOO-l.a.. - [A(CO.)] - ^"^^^^J
One may use this derivation as starting point for speculations in which
the "condensation" of carbon dioxide into a (7„ compound (perhaps, with
n = 3 or 6) is assumed to be achieved, not after photochemical reduc-
tion (or between successive photochemical reduction steps), but already
in the preliminary, nonphotochemical carboxylation stage. One could
quote in this connection the observations of Van Rysselberghe and co-
workers (1946), who found indications that carbon dioxide is reduced, at
the cathode of a polarograph, after preliminary formation of a poljaneric
adsorption complex containing six CO2 molecules.
Studies of the Hill reaction (chapter 35) and of the C(14) uptake in
light (chapter 36) indicate three possible additional complications of the
kinetics of carbon dioxide uptake in photosynthesis:
There may be several carhoxylations involved, e.g., one of the
(i)
C2 -^ Cs and one of the C3 -^ C4 type. Since, in the steady state, both

must proceed at the same rate, this complication may not change the
kinetic derivations too radically. When, for some reason, one carboxyla-
tion becomes a rate-limiting step, the other must be slowed doAVTi too, by
the blocking action of its accumulated products.
{2) Carboxylation may he coupled with hydrogenation ("reductive
carboxylation," typified by direct conversion of pyruvic to malic acid, and
already mentioned on p. 937). In other words, reaction (27.2), instead
of being kinetically independent, may be combined with the first step in
(27.5A). This, too, should not necessarily change the kinetic derivations
in a radical way, since "coupling" probably means a fast sequence of two
reaction steps, perhaps catalyzed by two active groups in the same protein
molecule.
(3) The acceptor. A, 7nay he a product of photosynthesis, disappearing in
the dark. This would make Ao a function of the rate of photosynthesis,
P, and would necessitate reconsideration of the kinetic equations based on
a constant value of Ao.
B. Carbon Dioxide Concentration and Fluorescence*

In chapter 24, when discussing the fluorescence of chlorophyll in the
Uving cell, we mentioned the close relationship often found between the in-
tensity of fluorescence and the rate of photosynthesis; we stated that,
because of this relationship, the measurement of fluorescence has become an
important tool in the study of the kinetics of photosj^nthesis. Conse-
* Bibliography, page "JGS.
quently, we proposed to review the effects of various external factors on the

intensity of chlorophyll fluorescence in vivo parallel with the presentation
of the influence of these factors on the rate of photosynthesis.
In following this plan, we have now to describe the changes in the in-
tensity of fluorescence of living plantcells, associated with variations in the
supply of carbon dioxide.

The general finding appears to be that reduction or complete stoppage
of the carbon dioxide supply usually affects the yield of fluorescence, ^, in
a certain range of illuminations. It has no effect on the yield of fluorescence
in very weak and probably also none in very strong light, but the
light;
latter generalization is in need of experimental confirmation. In purple
bacteria, the commonly observed increase of ^ upon removal of carbon di-
oxide sometimes gives place, with increasing light intensity, to the opposite
effect (c/. fig. 28.30) Since no systematic measurements of ^ for variable
.
[CO2] have been carried out, the plotting of "carbon dioxide curves" of
fluorescence, F = /[CO2], is not possible. Instead, "hght curves" have
been drawn, showing the intensity of fluorescence as a function of light
intensity, with [CO2] as a parameter, usually for two different concentra-
tions of carbon dioxide only (or simply "with carbon dioxide" and "with-
out carbon dioxide"). Several such curves will be reproduced in chapter
28 (c/. figs. 28.25, 28, 29 and 30).
We anticipate here some of the facts to be presented there : The yield
of fluorescence, ^p, generally remains constant (^î) up to or beyond the
intensity region in which photosynthesis becomes saturated with light;
but sooner or later, it increases more or less gradually, finally to reach a new
steady level, ^2- We designate the intensity at which the transition begins,
as /', and that at which it ends, as I" The effect of removal of carbon di-
.
oxide apiêars to be a downward shift of this transitional range. Thus,

McAlister and Myers (1940) found that in 4% CO2, the yield of fluorescence
of wheat leaves was constant up to 600 kerg/(cm.^ X sec.) (kerg = 10^ erg)
while, in 0.03% CO2, the yield increased (by about 15-20%) between 200
and 600 kerg/(cm.2 X sec.) {cj. fig. 28.25).
Similarly, Franck, French and Puck (1941) found that the steady state
fluorescence of a leaf of Hydrangea, at 7 = 7 kerg/cm.- sec. was about 20%
higher in carbon dioxide-free air than in air containing 5% CO2. At 71
kerg/cm.- sec, on the other hand, the yield of fluorescence in 5%C02liad
increased so strongly that now a change to 0.03% CO2 had no noticeable
effect.
Working with a culture of diatoms (Nitzschia sp.), Wassink and Kersten
(1945) found the yield of fluorescence in intense light, 50 kerg/cm.- sec,
to be higher in the absence than in the presence of carbon dioxide. Figure
28.28 indicates that, in this case, the difference is brought about by a de-
,
CARBON DIOXIDE CONCENTRATION AND FLUORESCENCE 941
dine of <p carbon dioxide-supplied algae, rather than by an increase

in the
of (f carbon dioxide-starved cells (as in figs. 28.25 and 28.30 A).
in the
Wassink, Katz and Dorrestein (1942) noticed a similar effect of carbon
dioxide deficienc.y on the yield of fluorescence in purple bacteria; however,
the change was much
pronounced than that caused by the rationing or
less
denial of the reductants (cf. below, section D2). In the absence of a reduc-
tant (a condition that cannot be paralleled in green plants), the removal of
carbon dioxide had no effect at all on the fluorescence of Chromatium (cf.
fig. 28.29) In the

. presence of reductants (such as thiosulfate or hydrogen)
the fluorescence of carbon dioxide-starved bacteria was, however, con-
siderably stronger than that of carbon dioxide-supplied cells (c/. fig. 28.30A).
This difference occurred in the range from 2 to 30 kerg./cm.^ sec. At light
intensities above 30 kerg, the fluorescence curves with and without carbon
dioxide again approached, and finally even crossed each other, so that (p
became higher absence than in the presence of carbon dioxide. In

in the
other words, fluorescence was now higher when photosynthesis was pos-
sible than when it was suppressed (This is a good illustration of the fact
!
that the relation between photosynthesis and fluorescence is not a simple

competition; cf. i)age 820.)
In chap. 28 we will discuss several possible explanations of the change
in intensity of chlorophyll fluorescence in strong light. One, is to attrib-
ute this change to the accumulation of the photocomplex, X- Chi -HZ
(a complex of primary oxidant chlorophyll +
primary reductant)+
in a changed form due to the incapability of the catalytic dark reactions to
keep pace with the primary photochemical process. The changed forms
of the photocomplex can have a different fluorescence yield, first, because
they are photostable (i. e., cannot undergo the primary photochemical
photoprocess (e. g., X- Chi -HZ ^
XH-Chl-Z), and, secondly, because they
have an intrinsically different capacity for dissipating the excitation energy.
An alternative or additional cause is, according to Franck, the forma-
tion of a "narcotizing" substance, by reaction of excess oxidation inter-
mediates with metabolites this narcotic displaces the reactants from the
;
photocomplex and thus causes changes in the yield of fluorescence.

Absence of carbon dioxide slows down the restoration of the primary
oxidant, HX or ACO2, after it has been reduced by light. It must thus
favor the accumulation of the photocomplex in a form such as HX-Chl-Z
or HX-Chl-HZ, and this accumulation will find its expression in a change
(usually an increase) in the jaeld of fluorescence.
Franck and Herzfeld (1941) were inclined to consider the effect of car-
bon dioxide on fluorescence of plants as proof that carbon dioxide (in the
forai of a compound with an acceptor, ACO2) is a direct part of the photo-
sensitive complex (i. e., that X in X- Chi -HZ is identical with ACO2).
:
It was argued, however, in Volume I (page 167) that ACO2 and X- Chi -HZ
can be separated by intermediate oxidation-reduction systems, and,
nevertheless, an exhaustion of ACO2 may cause an accumulation of the pri-
mary oxidant, X, in the form HX, and consequent change in the intensity
of fluorescence. (A strike of longshoremen in America can cause tin ore to
accumulate at the pit heads in the East Indies.)
Fluorescence observations on purple bacteria produced new evidence
bearing upon the relation of carbon dioxide to the fluorescent pigment
complex.
Wassink, Katz and Dorrestein (1942) were strongly impressed by the
above-mentioned observation that changes in the concentration of re-
ductants affect the fluorescence of purple bacteria more strongly than
changes in the concentration of the oxidant (CO2), and that the former ef-
fect persists in the absence of carbon dioxide, while the latter disappears
when reductants were absent. They concluded that carbon dioxide does
not come into (direct or indirect) energy exchange with excited chlorophyll
at and that the excitation energy is taken up (directly or indirectly)
all,
by the reductants (H2S, H2S2O3, H2 in purple bacteria, H2O in green

. . .
plants). They thought that the small observed effects of CO2 on fluores-
cence are without real significance — an unjustifiable simplification.
Franck and his co-Avorkers (1947, 1949), on the other hand, interpreted
the enhancing effect of the absence of reductants on the chlorophyll
fluorescence of purple bacteria as an indirect action caused by accumula-
tion of unreduced oxidation intermediates of photosynthesis ("photoper-
oxides"), and narcotization of the chlorophyll apparatus by the products
of action of these peroxides on cell metabolites. Using this picture, they
gave the following interpretation of the effect of [CO2] on yield of fluor-
escence, its variability with light intensity, and its disappearance in the
absence of reductants (in purple bacteria)
Lowering the CO2 concentration has an effect on the yield of fluorescence
if it makes the rate of photosynthesis [C02]-limited. When this is the
case, the chlorophyll complex becomes free (depleted of the oxidant,
ACO2), and light energy absorbed by it cannot be used for the primary
photochemical process. This itself should change the yield of fluorescence
(p. 941).Howêver, Franck considers this "denudation" effect as of minor
importance, as far as fluorescence is concerned, compared to the "self-
narcotization" which follows it. He postulates that whenever the photo-
sensitizer complex isdeprived of carbon dioxide, photoxidation sets in (c/.
Vol. I, chapter 19), and produces a "narcotizing" intermediate (probably
a carboxylic acid) which settles on chlorophyll.
This mechanism cannot come into play in purple bacteria, since these
are studied under anaerobic conditions; this explains why carbon dioxide
deprivation has a comparatively slight effect on fluorescence in these
.
CONCENTRATION OF REDUCTANTS 943
organisms. The deprivation of rediictants, on the other hand, has a strong

effect,because abundant "narcotic" is produced in light by the action of
accumulated, unreduced photoperoxides.
One may ask: why, then, should the fluorescence-stimulating effect
of the deficiency of reductants persist also in the absence of carbon di-
oxide? No photoperoxides should be formed under these conditions.
Franck explains this paradox by questioning the efficiency with which
CO2, produced by fermentation, is removed in these experiments. He
points out that some hydrogen is taken up by purple bacteria in light even
if no extra carbon dioxide is provided in the medium. (Incidentally, fer-
mentation could produce "narcotizing" acids also directly and not via —
the reduction of the fermentation-produced carbon dioxide in light.)
Removal of external carbon dioxide may have no effect on fluorescence in
the absence of reductants, because enough CO2 is produced by fermenta-
tion to prevent the photochemical process from becoming " [C02]-limited";
instead, it remains "[reductant]-limited" {i. e., its rate and the concentra-
tion of the narcotic —
and thus also the intensity of fluorescence remain —
limited by the rate of the reaction between the photoperoxides and the
reductants)
be noted that Franck's concept differs from the first picture
It will
(presented on p. 941) in 3 ways: (1) the ultimate oxidant itself, ACO2,
rather than an intermediate, X, is supposed to be coupled with chlorophyll;
(3) no ultimate (or intermediate) reductant, ZH, is supposed to be asso-
ciated with chlorophyll, in such a way that its depletion, too, can cause an
increase in the yield of fluorescence ; and (3) all strong changes in the yield
of fluorescence are ascribed to narcotization by "half-oxidized" metabolites,
rather than to the depletion of reactants. These three assumptions are in-
dependent of each other; the third one, in particular, which is perhaps the
most important feature of Franck's interpretation, can be combined, if
desired, also with the picture of the primary photochemical process as light-
induced tautomerization of the complex X- Chi HZ ( > HX.Chl.Z).
C. Concentration of Reductants*
1. Efifect on Rate of Carbon Dioxide Reduction in Bacteria
The ultimate reductant in ordinary photosynthesis of green plants is

water. The activity of water in the cells can be changed by direct hydra-
tion and dehydration; or by immersion into solutions of different osmotic
pressure. Both treatments have a considerable effect on photosynthesis.
However, this effect cannot be treated as a kinetic phenomenon obeying
* Bil)liography, page 963.
the mass action law, since related to changes in permeability and other
it is
colloidal properties of the protoplasm and the cell membranes, which af-
fect, to a varying degree, all activities of the living cell. Dehydration ef-
fects were therefore discussed in chapter 13 (Volume I, page 333), where
we dealt with various physical and chemical inhibitions and stimulations
of photosynthesis.
It was mentioned there (page 334) that one of the ways in which de-
hydration may inhibit photosynthesis is bj'^ its influence on the stomata.
Since the primary purpose of the stomata is to regulate transpiration, they
100
2
o
< 75--
<
o 50-
<
b 25 -
LlI
q:
curve" has the same typical "saturation shape" as the curves representing
the rate of photosynthesis as function of carbon dioxide concentration;
half saturation is reached at ca. mm.
(2.5%) H2, full saturation above
20
75 mm. (10%). Wassink et al. (1942) found, with Chromatium in nitrogen
containing 5% CO2, no signs of hydrogen saturation up to about 2% (cf. fig.
27. 11 A); single experiments at higher pressures indicated that saturation
was reached probably at about 10%, certainly below 15%. (The rates at
15% and 30% H2 were identical.) These hydrogen concentrations are
approximately 100 times higher than those required for saturation of photo-
synthesis with carbon dioxide (about 0.03% CO2 is needed for half satura-
tion). In analogy with the interpretation of carbon dioxide saturation.
E Scale of concentratjon
E 200 10 times reduced
O
o
Ll.
O
UJ
<
I-
0.
3 100 200 300 400
AMOUNT OF Hj/VESSEL, mm^
Fig. 27.11. Rate of carbon dioxide reduction by Chrornatium (after Wassink 1942):
(A) effect of [H2]; {B) effect of [H2S]. 5% CO2 in N2; pH 6.3; 29° C; strong light.
given in the first part of this chapter, the simplest explanation of hydrogen
saturation is to assume reversible formation of a hydrogen acceptor com-
pound, with a dissociation constant of the order of 0.02 atmosphere (as
compared with 3 X 10 ~^ atmosphere for the carbon dioxide acceptor com-
pound). Thermodynamically, reversible hydrogenation (i. e., hydrogena-
tion with energy close to zero) presents no difficulties, since the free energies
of hydrogenation of organic compounds can be either positive or negative,
depending on the degree of resonance stabilization of the individual com-
pound in the hydrogenated and the dehydrogenated form (c/. Vol. I, page
217). Kinetically, however, the problem is less trivial, since in vitro, no
example is known of organic compounds behaving like metallic palladium,
i. e., taking up hydrogen under high pressure and releasing it when the
pressure is reduced.
Quantitatively, the results of French with Streptococcus varians and of
Wassink with Chromatiujn are similar enough to justify the suggestion
that the hydrogen acceptor is the same in both species. This common ac-
ceptor may be either the enzyme hydiogenasc, or the compound, desig-
nated in chapter 6 by Ah (i. e., the compound assumed there to be hydro-

genated to AhH2 by the mediation of the hydrogenase).
However, in interpreting the hydrogen saturation of photoreduction,
one has to keep in mmd that hydrogenation equilibrium is only one of the
two possible explanations. As in the case of the carbon dioxide curves,
the hydrogen curves —
may also be affected or completely determined by —
kinetic influences, such as slow rate of hydrogen supply and slow hydrogena-
tion (in the linearly ascending part), and limitations of light supply, of
oxidant supply, or of the availability of a catalyst, in the horizontal part of
the curves, which follows saturation.
iVJVJ
ro .
e
E
O
o
u.
o
UJ
a.
3
As with variable [CO2], the effect of variations in the concentration of

the reductants disappears in weak Hght when the supply of light quanta
becomes the rate-determining factor (c/. fig. 28.5B).
Photoreduction with mixed reductants offers an interesting kinetic
problem. Wassink, Katz and Dorrestein (1942) conducted some experi-
ments in which Chromatium cells were supplied with both hydrogen and
thiosulfate (at pH 6.8). They used each reductant "in excess" (meaning
that the quantity of each alone would have sufficed to produce saturation)
and calculated (indirectly, from manometric measurements) the relative
amounts in which the two reductants were consumed. The average was
about two molecules of carbon dioxide reduced at the cost of thiosulfate
for one molecule of carbon dioxide reduced at the cost of hydrogen. If
the two acceptor systems (for H2 and H2S2O3) are separate, this result may
mean either that the cells contain twice as much of the thiosulfate acceptor
system as hydrogen acceptor system, or that the first acceptor re-
of the
acts (in the hydrogenated state) twice as rapidly with the activated photo-
complex as the second one. On the other hand, if only one hydrogen ac-
ceptor is present, and the two donors compete in supplying it with hydro-
gen, then the result, as reported, is not very significant, since, in this case,
the relative utilization of hydrogen and thiosulfate would depend on rela-
tive concentrations (even if both reductants are present "in excess" the —
only information provided).
In chapter 22, we mentioned the spectroscopic experiments of the same
investigators thatmade them think that the composition of the photocom-
plex X-Bchl-HZ (Bchl = bacteriochlorophyll) may itself be specific for
each reductant, i. e., that bacteria contain a multiplicity of complexes,

X- Bchl' -HZ', X- Bchl" -HZ" • • •, adapted to the utihzation of the several
reductants R'H, R"H, • • • • (This hypothesis was suggested to explain
the multiplicity and varying relative intensities of the absorption bands of
bacteriochlorophyll m
vivo.) Wassink and co-workers now argued that, if
thiswere true, the photoreduction of carbon dioxide by a mixture of reduc-
tants would be additive, rather than competitive. Since rate measurements
indicated competition (the rate of total gas consumption in the presence of
both reductants always was lower than in pure hydrogen and larger than
in pure thiosulfate), the Dutch investigators concluded that their earlier
explanation of the spectroscopic phenomena wâs incompatible with the
results of kinetic studies. However, the argument would only be fully con-
clusive were definitely known that, under the conditions of the experi-
if it
ment, the rate of photoreduction was limited by the amount of the reduc-
tant available for reaction with the photocomplex. It was mentioned above
that saturation of the over-all rate with respect to the reductant can often
be due to a limitation elsewhere in the photosynthetic apparatus, e. g., to
S
the deficiency of a "finishing" catalyst such as Eg. Whenever this is the

case, the rate of photoreduction cannot he increased by increasing the avail-
able quantity of the reductant or by adding a second reductant and thus put-
ting to work the (otherwise idle) photocomplexes specifically adapted to it.
In kinetic work with purple bacteria, one has to keep in mind their
capacity to utilize organic materials —including intercellular ones —as re-
ductants. Competition rather than additivity seems to be the rule in this

case too. The "sigmoid" shape of the light ciu'ves of hydrogen consump-
tion, noted both by French, with Streptococcus varians, and by the Dutch
observers, with Chromatium (cf. figs. 28.8), can perhaps be interpreted as a
consequence of such competition: In weak light, intracellular reductants
(pei'haps sugars or their derivatives) supply all the hydrogen necessaiy for
the slowly i^roceeding ])hotoreduction of carbon dioxide, and, therefore,
only very little external hydrogen is used. In stionger light, the diffusion
supply of hydrogen donors proves insuffi(;ient, and the more
internal
rapidly diffusing molecular hydrogen takes over as the main reductant.
To minimize the role of internal reductants, and thus to obtain light
curves without a sigmoid initial section, Wassink (1942) has attempted to
starve the bacteria before the experiment; however, he found no significant
change in results.
The experimental material on the "reductant curves" of j^hotoreduc-
tion is as yet rather limited and no attempts have been made to represent
these curves analytically. If one accepts the general scheme of photo-
synthesis given in scheme 7.1 (Vol. I, page 153), the kinetic role of the re-
ductants appears symmetrical to that of carbon dioxide. An analytical
treatment of the effect of reductants on the over-all rate would thus have to
deal with the same type of partial processes as were treated in the analysis
of the carbon dioxide factor, namely, supply by diffusion, preparatory
catalytic dark reactions (such as binding of hydrogen by an acceptor with
the help of the hydrogenase), lilieration of the acceptor from the primary
oxidation product, and "finishing" dark reactions (such as stabilization of
the primary oxidation product). According to Franck's concept, re-
peatedly mentioned before, the finishing dark reactions on the oxidation
side (which consist in the elimination of the primary oxidation products —
—
"photoperoxides" either by their conversion to molecular oxygen or by
their reduction with reductants such as hydrogen or hydrogen sulfide)
have the peculiar property that their failure to keep pace with the primary
photoprocess leads not merely to the loss of a large proportion of primary
products by back reactions but also to a reaction between them and oxidiz-
able metabolites. This side reaction produces, according to Franck, a
"narcotic," capable of enveloping chlorophyll and stopping the primary
photochemical process.
—
EFFECT OF REDUCTANTS ON FLUORESCENCE 949
The question whether the participation of water as reductant in photo-

sj'nthesis involves some preUminary transformations similar to those of
carbon dioxide remains open. The abundance of water in all cells may be
advanced convenient assum]:)tion that, even if a transforma-
in favor of the
tion of this kind hydration of a "water acceptor" is needed before
c. g., —
water can act as a hydrogen donor, the rate of this reaction is high enough
to prevent it from playing a limiting role in photosynthesis. However, if
the binding of water, which we have sj'mbolized by H2O
—> {H2O) in
scheme 7.1, and which we may now describe by the equation:
(27.87) H2O + A' , A'H.O
in analogy to equation (27.2), isan enzymatic process, the limited amount

of the enzyme (particularly in an appropriately inhibited state) may well
])ecome a rate-limiting factor, despite the overabundance of the reactant
H2O in the cell.
2. Effect on Yield of Fluorescence
Hydration and dehydration of plant have a strong influence on the

cells
intensity of chlorophyll fluorescence in vivo; but in this case, as in that of

gas exchange, it is difficult if not impossible to distinguish between the
(undoubtedly possible) direct kinetic effects, and the indirect disturbances
caused by changes in the colloidal structure of the pigment-protein-lipide
complex. We must therefore refer here to the description of the relevant
phenomena in chapter 24.
The study again becomes much more fruitful when purple bacteria are
used. The supply of reductants, such as H2, H2S or H2S2O3, has been found
to strongly affect the yield of fluorescence of bacteriochlorophyll in these
organisms. The light curves of fluorescence given in figs. 28.31, 28.32 and
28.33 (taken from the work of Wassink, Katz and Dorrestein 1942, on
Chromatium) illustrate this phenomenon. The plots represent the fluores-
cence intensity, F, (not yield, <p) as a function of the incident light intensity
/, with the concentration of the reductant. [HR], as parameter. Figure
28.31 shows the comparative effect of three different reductants; figure
28.32, a set of measurements with different concentrations of thiosulfate;
and figure28.33 a similar set with different hydrogen pressures. In
both cases, increased concentration of the reductant causes an extension
of the initial, linear part of the fluorescence curve (which corresponds to the
"low light" yield of fluorescence, ip\). Figure 28.34 shows the critical
intensity," I^ (defined in fig. 28.27) as a function of the thiosulfate concen-
tration; the curve has a great similarity nith the corresponding gas ex-
change curve (fig. 27.12). It shows that deprivation of the reductant can
.
950 CONCENTRATION FACTORS CHAP, 27
lower the beginning of the transitional range from 11 to 2 kerg/cm.^ sec.

In discussing the yield of photosynthesis in relation to the factor [CO2],
we repeatedly encountered effects attributable to the exhaustion of the
molecular species CO2 in the immediate neighborhood of the plants; this
disturbance could be reduced (a) by buffering with bicarbonate, (6) by
using unicellular algae, to increase the surface of gas exchange and (c) by
stirring. With hydrogen, the solubility of the gas in water is much
smaller than that of carbon dioxide, and no buffering is possible; there-
fore, exhaustion effects can be expected to occur even more easily, and
could perhaps not be fully avoided even with unicellular organisms, such
as purple bacteria. Intense stirring is the only help available; the com-
paratively rapid diffusion of hydrogen in water ma}^ help to maintain uni-
form distribution. Wassink and co-workers (1942) have observed that,
in the presence of 15% Ha in the gas phase, the fluorescence of Chromatium
in strong light soon rapidly increased by 25-20% (about one minute)
after the cessation of shaking (fig. 28.33 would make even a larger increase
easily understandable)
According to the Dutch authors, the influence of [CO2] on the fluores-
cence of bacteria disappears if the supply of reductants is stopped (fig.
28.29). Inversely, however, the effect of reductants on fluorescence re-

mains considerable even if carbon dioxide is withheld {cf. fig. 28.35).
Wassink and co-workers interpreted this difference as confirmation of their
general concept that the reductants participate directly as "energy accep-
tors" in the photochemical process, while carbon dioxide does not react
with the primary photoproduct at all. Franck and Herzfeld, on the other
hand, assumed direct participation of carbon dioxide (in the form of a com-
plex, ACO2) in the primary photoprocess and quoted the effect of carbon
dioxide removal on fluorescence as evidence of this participation (cf.
above, page 941), while ascribing the (much stronger) effect of the ab-
sence of reductants to the indirect mechanism of "self-narcotization" in
consequence of accumulation of unreduced "photoperoxides." Later
(cf. page 942), Franck suggested that the effect of CO2 deficiency also is
due mainly to narcotization (caused by the products of photoxidation).
Without using the concept of narcotization, we can explain the effects
of reductants on the basis of the picture of a "photocomplex," X- Chi -HZ,
which undergoes primary photochemical conversion to HX-Chl-Z, fol-
lowed by dark reactions that transfer H from X to CO2, and supply H
to Z from H2O (or from one of the "substitute" reductants, such as H2 or
H2S2O3) While CO2 starvation may lead to the accumulation of the photo-
.
complex in the "reduced" form, such as HX-Chl-HZ, the absence of re-

ductants may lead to the accumulation of the "oxidized" form, X-Chl-Z;
when both CO2 and the reductants are deficient, the tautomeric form, - HX •
-
INORGANIC IONS 951
Chi Z, may be accumulated in strong light. Each of the four forms should
•
have its own characteristic yield of fluorescence, limited by the rate of

internal energy dissipation and, in the case of the normal form X- Chi -HZ,
also by the competition of fluorescence with the primary photoprocess
(X Chi -UYj -\- hv-*
• Chi Z) HXThe conditions under which the three
• .
forms can be expected to predominate in strong light, are summarized in

the following table.
Conditions of accumulation
Form Formula Oxidant (CO2) Reductant
Tautomeric HXChlZ
Reduced HX-ChlHZ - +
Oxidized X-Chl-Z + -
Which form will be accumulated in the presence of very strong light
when both the oxidant and the reductant are in good supply (case ++)
cannot be predicted a depend on which of the supply
priori, since this will
processes first fails primary photoprocess.

to keep pace with the
If the influence of carbon dioxide deprivation on fluorescence is due to
the accumulation of the form HX-Chl-HZ, while the effect of "RH starva-
tion" is caused by accumulation of the form X-Chl-Z, the absence of an
effect of carbon dioxide in can be interpreted as evidence
RH-starved cells
forms HX-Chl-Z and X--
of a similarity in the fluorescent capacity of the
Chi Z (or, perhaps as indication that in the absence of both carbon dioxide
•
and reductant, the complex accumulates in the same form X-Chl-Z, as in

the absence of reductants alone).
As mentioned on page 943, Franck's hypothesis, that all strong in-
creases of fluorescence are evidence of narcotization of the chlorophyll com-
plex, is not incompatible with the interpretation of the primary photo-
chemical process as hydrogen transfer in the complex, Chi HZ it only X . . ;
implies that the differences in the capacity for fluorescence of the various
forms of this complex (X- Chi -HZ, HX-Chl-Z, X-Chl-Z, and HX-Chl--
HZ) are less significant than that between all of them and the "narcotized"
form.
D. Concentration of Inhibitors*
1. Inorganic Ions
The effect of the hydrogen ion concentration on photosynthesis was dis-

cussed in chapter 8 (Vol. I), and also in the first part of the present chapter,
from the point of view of the correlated shifts of the carbonate-bicarbonate

952 CONCENTRATIOiN FACTORS CHAP. 27
carbon dioxide equilibrium. What we wanted to know —and did not quite
succeed in finding out —was whether the rate of photosynthesis is deter-
mined uniquely by the concentration of free carbon dioxide molecules,
[CO2], in the immediate surroundings of the cell, or whether the carbonic
acid ions also contribute to photosynthesis directly (and not merely because
they serve as a reserve for rajiid replacement of neutral carbon dioxide
molecules). One of the complicating factors was that a change in [HCOs"]
at constant [CO2] could not, and cannot be achieved without a change in
[0H~], i. c, a variation of the pH; and while some algae, such as ChlorcUa,
25U 700
200 600
o 500
o
o
<-)
A High light intensify
o LOW light intensity
o
UJ 400
a. <
3 Q.
300
200
Fig. 27.13. Effect of pH on carbon Fig. 27.14. Influence of pH on rate of

dioxide reduction with thiosulfate by carbon dioxide reduction by Chromatium
Chromatium (after Wassink, Katz and with hydrogen (5% CO., 15% H.2) (after
Dorrestein 1942): 5% CO2; 1% thio- Wassink, Katz and Dorrestein 1942).
sulfate; 29° C; strong Hght.
are capable of efficient photosynthesis over a wide range of acidities (say

from pH 4.5 to 10), this does not prove that the observed changes in rate
can be attributed entirely to variations of [CO2] and not to variations of
pH. Some unicellular algae (e. g., Hormidium) are definitely injured by
alkaline media.
The observations in this field, described in Volume I (pages 339-340)
can now be supplemented by the results of Wassink, Katz and Dorrestein
(1942) with purple bacteria. When reductants such as thiosulfate or hy-
drogen sulfide (or inhibitors such as cyanide) are used, the pH effect is com-
INORGANIC IONS 953
plicated b}^ changes in the ionic dissociation of these weak acids (in addition
to the conipUcations caused by the dissociation of carbonic acid).
Figure 27.13 shows the effect of pB. on carbon dioxide reduction by
Chromatium in 1% thiosulfate. The curve, obtained in phosphate buffers,
shows a shght maximum of P at pH 6.3, followed by a sharp decrease. The
effect disappears at low light intensities (as do all effects not connected
with the primary photoprocess). With hydrogen as reductant, the effect
was quite different (fig. 27.14); the rate, P (in strong light), increased
steadily with increasing alkalinity.
; : 079 X lO** erg/cm^ sec
70
pH
Fig. 27.1.5. Effect of yjH on fluorescence of Chromatium (after Wassink, Katz and
Dorrestein 1942): {A) 5% CO2, 1% thiosulfate, 29° C, strong light; (5) 5% CO2,
\b% H2, 25° C, strong and weak light.
One factor that may contribute to the decline of P with increased pH in
thiosulfate is the decrease in concentration of undissociated H2S2O3 mole-

cules; this may
be the main species that penetrates the cells and is used
there as the reductant. According to figure 27.12, at 1% thiosulfate, the
concentration of the reductant is not a "limiting" factor at pH 6.3. Whether
it could become such a factor when alkalinity is higher, remains to be inves-
tigated.
The increase in P with pH, as observed with hydrogen as reductant,
could perhaps be looked upon as new evidence of the direct participation
of HCOs" ions in i)hotosynthesis —
one were inclined to give weight to
if
such evidence. Alternatively and more plausibly, both the decline of P

with pH in the case of thiosulfate, and its increase in the case of hydrogen,
pH dependence of the enzy-
could be interpreted as evidence of different
matic processes by which the two reductants are supphed to the photo-
complex.
The influence of pH on the fluorescence of bacteriochlorophyll also was
studiedby Wassink and co-workers. With thiosulfate, the fluorescence
was about 30% more intense at pH 7.6 than at pH 6.0 (fig. 27.15A), a re-
sult that may
be attributed to a lower supply of effective reductant (un-
dissociated H2S2O3). As expected, the trend is reversed with hydrogen
(fig. 27.15B), where the enzymatic supply of the reductant appears to be
better in the more alkaline solution.

The effects on the rate of photosynthesis of various other inorganic ions
also were described in chapter 13. The only systematic data, showing the
rate in relation to the concentration of the inhibiting ion, were obtained by
Greenfield (1941, 1942); some of his curves were reproduced in figure 34
(Vol. I, page 341). Among new results pertinent to this field we may quote
the observations of Warburg and Liittgens (1946) that the reduction of
quinones by broken chloroplasts in vitro requires the presence of chloride
ions (c/. Chapter 35).
2. Poisons and Narcotics

In chapter 12 (Volume I) we discussed the influence of various inhibitors
on the rate of photosynthesis in a qualitative way, although some quantita-
tive data also were given, such as a curve showing the rate of oxygen libera-
tion by Chlorella as a function of the concentration of phenylurethan (fig.
30, page 323). Systematic kinetic study should include also rate measure-
ments with varying amounts of poisons, and some such measurements
have recently been reported, particularly by Wassink, Katz and Dorrestein
(1942). These investigators found that the photoreduction of carbon di-
oxide by purple bacteria {Chromatium D) is as sensitive to cyanide as is
the photosynthesis of Chlorella. With thiosulfate as reductant, some in-
hibition was observed even in weak light, such as 2 kerg/(sec. cm.^), while
in reduction with molecular hydrogen (as in normal photosynthesis), the
cyanide effect disappeared completely in weak light, / < 4 kerg/(sec. cm.^).
Wassink and Kersten (1945) found a considerable effect of hydrogen cya-
nide also on the photosynthesis of diatoms in weak light (~2 kerg/sec.
cm.-), a result which differs from many observations on green algae and
higher plants.
Figure 27.16 shows the relation between P and [KCN], in Chromatium
(with hydrogen or thiosulfate as reductant) and in Nitzschia. Photo-
synthesis of Chromatium is half-inhibited by about 2.5 10 ~' per cent X
KCN (with either hydrogen or thiosulfate). The pH (6.3 to 7.6) has an ef-
fect on inhibition in thiosulfate, but none in hydrogen, which makes the at-
CONCENTRATION OF INHIBITORS 955
tribution of its effect to a difference in the activity ofHCN molecules and

CN- ions (c/. Vol. I, page 301) doubtful. Figure 27.1GB shows that photo-
s}Tithesis of Nitzschia is half-inhi])ited by about 1.5 X 10"^% or about 2 X
10-5 mole/1. KCN. For similar figures for Chlorella, Hormidium and other
algae, see V (Vol. I, page 305).
Table 12.
The cyanide on the stea,dy fluorescence of Chromatium is shown

effect of
in figure 28.45. It is somewhat complex, but, in general, amounts to an
300
lO .
o
o
<
Q.
Z3
956 CONCENTRATION FACTORS CITAP. 27
in ordinary photosynthesis, hj^droxylamine also produces, albeit only

higher concentrations, two other inhibiting effects on the reduction —
of carbon dioxide which no oxygen is
by "hydrogen-adapted" algae (in
liberated),and on the "de-adaptation" of such algae. Both effects could

be explained by the assumption that hydroxylamine has an affinity either
800
700
600
E
E 500 \- —
• Scale of concentration
N \ 10 times enlorged
X
400
O
o
U-
o
300
Q.
3
200
100
U 01 2 3 0.4 0.5 6
NH2OH •
HCI CONCENTRATION
Fig. 27.17. Influence of hydroxylamine chloride on carbon

dioxide reduction by Chromaiium (after Wassink, Katz and
Dorrestein 1942): 5% CCDo; 15% H.; pH 7.1G; 29° C;
strong light. NHÔH-HCl concentration in per cent.
directly to the primary oxidation product Z (in photocomplex X- Chi -HZ),

or to anenzyme with which Z must react to prevent this primary product
from being lost by back reactions.
New experiments of Wassink and co-workers (1942) showed that the
influence of hydroxylamine hydrochloride on the photoreduction of carbon
dioxide by purple bacteria {Chromatium) is somewhat stronger than the
above mentioned effect on the similar reaction in hydrogen-adapted
Scenedesmus. (The following figures show this relation: Normal photo-
synthesis of Scenedesmus, 50% inhibition at 5 X 10~^ mole/1.; photo-
rONCENTRATION OF INHIBITORS 057
reduction by adapted Scenedesmus, no drop below 50% at any concentra-

tion up to 3 X 10-- mole/l.; photoreduction by Chrornatium, 50% inhil)i-
tion at 3 X 10-^ mole/l., {cf. fig. 27.17). In experiments with bacteria
(in contrast to those of Weller and Franck, 1941, on Chlorella; cf. Vol. I,
]mge 312) no inhibition by hydroxylamine was found (cf. in weak light
fig. 28.110 and fig. 28 in Vol. I). The difference is understandable if the
effect of hydroxylamine on photosynthesis in weak light is due to its action
on the oxygen-liberating enz^^me (designated as Eo or Ec in Vol. I); a
possible "autocatalytic" mechanism of this effect was discussed on p. 312.
IB)
600 - 200-
500 150
E
E 6 \ Scale of concentration
OJ £ \ 10 times enlarged
X 400 100
+ O \
eg O
O \
O \
300-
li. 50
o \.
<
I- \
< a
I- 200 3 \.
^^-
-50
-100
0.1 0.2 0.3 0,4 0.5 1 02 0.3 04 05
NaNj CONCENTRATION, % NaNj CONCENTRATION,"/,
Fig. 27.18. Influence of sodium azide on rate of photoreduction of carbon

dioxide by Chrornatium (after Wassink, Katz and Dorrestein 1942): (A) 15%
hydrogen; (i5) 1% thiosulfate. 5% CO.; pH 6.3; 29° C; strong light.
In the absence of reductants, hydroxylamine hydrochloride was found

to have no effect on the fluorescence of Chrornatium, up to a concentration
of 0.5%. In the presence of reductants, hydroxylamine causes, in general,
an increase in the intensity of fluorescence; however, this effect becomes
marked only above 0.05%, while the reduction of carbon dioxide is half
inhibited by as little as 0.03% of the poison (cf. fig. 28.46). Thus, the
inhibition of bacterial photosynthesis by hydroxjdamine also seems to be a
complex phenomenon, the first stage of which has no effect on fluorescence,
\yhile the second stage causes an increase in the fluorescence yield.
%
Sodium azide. Only one casual observation of the inhibition of photo-

synthesis by azide was mentioned in Volume I (page 318). Since then, the
effect of this tyi:)ical poison for heavy metal catalysts on the gas exchange
and fluorescence of Chromatiurn has been studied quantitatively by Was-
sink, Katz and Dorrestein (1942). Figures 27.18A and 27.18 B show the
rates of photoreduction, with molecular hydrogen and thiosulfate, respec-
tively, as functions of azide concentration. The half-inhibiting concentra-
tion is about 0.02% sodium azide with hydrogen and about 0.01% with
thiosulfate as reductant. Strangely enough, particularly strong inhibition
was found in weak light (fig. 28.1 ID).
700
600-
500
400
o
300
<
CL 200
3
as 10 15 20 2.5 3.0
ETHYLURETHAN CONCENTRATION,
Fig. 27.19. Effect of ethylurethan on photoreduction of
carbon dioxide in Chromatiurn (after Wassink, Katz and
Dorrestein 1942): 5% CO2; 15% H2; pH 7.6; 29° C; strong
light.
The effect of azide on fluorescence also was different from that of the
other enzyme poisons, such as cyanide and hydroxylamine. In the first
place, fluorescence was strongly affected even in the absence of reductants

(fig. 28.47A). In the second place, the typical effect was a considerable
lowering of the yield of fluorescence, particularly at the higher light intensi-
ties (fig. 28.47B).
All three observations (inhibition of the gas exchange at low light in-
tensities, effect on fluorescence in absence of reductants, lowering of fluores-
CONCENTRATION OF NARCOTICS 959
cence intensity) indicate that this poison does not, or not merely, interfere
with the enzj^matic supply to the photosynthetic apparatus of the oxidant
(carbon dioxide), or of the reductant (such as hydrogen), but affects the
primary photocomplex, X- Chi -HZ, directly, possibly by displacing the
reductant HZ in this complex. Such a close association with the photo-
complex could be expected to lead to the decomposition of the azide in
light,an expectation which might, perhaps, be tested by experiments.
Urethans. In chapter 12 (Vol. I, page 321) the "narcotizing" effect of
phenylurethan on photosynthesis of Chlorella, which appears to be equally
strong at all light intensities, was illustrated by figure 30, taken from War-
burg's early work on
Chlorella (1920). This figure showed half inhibition
at about 2 10~* mole/1.; Table 12.VIII, taken from the same paper,
X
indicated hah inhibition at 5 X 10~^ mole/1, for phenylurethan and 0.22
mole/1, for ethylurethan.
An inhibition curve for Chlorella, given by Wassink and co-workers
(1938) (fig. page 323) indicated half inhibition by about 2.5 per cent,
31,
or about 0.02 mole/1, ethylurethan, and the measurements of Wassink,
Katz and Dorrestein (1942) on Chromatium (fig. 27.19) gave about 1.3%,
or 0.01 mole/1., as half-inhibiting concentration, at 29° C. Wassink and
Kersten (1945) found about 2% as half -inhibiting concentration for the dia-
tom Nitzschia dissipata.
One peculiar characteristic urethan on Chromatium
of the effect of —
similar to that of azide —
is the enhanced inhibition at low light intensities,
leading to a more pronounced sigmoid shape of the light curves of photo-

reduction in inhibited cells (fig. 28. HE).
Theoretically, a weaker inhibition at the higher light intensities {i. e.,
light curve systems of chapter 26) could be explained if one

"type 3"; cf.
would assume that the narcotic partially covers the chlorophyll-bearing

"photocomplex," but leaves free the enzyme that determines the limiting
yield of photosynthesis in strong light. In this case, the decrease in in-
hibition would be brought about by continued increase, with increasing
light intensity, of the rate of photosynthesis of narcotized cells — in the in-
tensity range in which photosynthesis in noninhibited cells is light-saturated.
Figure 28. HE shows, however, that light saturation occurs at the same in-
tensity on both curves, so that this explanation appears inadequate.

In Nitzschia, according to Wassink and Kersten (1945), the inhibition
by urethan is somewhat less strong in weaker than in stronger light.
The influence of ethylurethan on fluorescence of Chromatium is like
that of azide. As with azide, addition of urethan causes the fluorescence

to decrease in intensity in the absence of reductants (fig. 28.50A).
When reductants, such as hydrogen or thiosulfate, are present, and the
fluorescence of nonnarcotized cells is thus considerably weakened, addi-
960 CONCENTKATIOX FACTORS CHAP. 27
tion of urethan causes an increase in F (of. fig. 28.50B), so that the intensity
of fluorescence finally becomes about the same with and without reductant.
It is noteworthy that in the completely narcotized state {e. g., with 3%
ethylurethan, when the gas exchange is completely inhibited; cf. fig. 27.19)
the characteristic curvature of the fluorescence curves of bacteria disap-
pears (fig. 28.50B).
These by the assumption that lU'ethan reacts
results can be explained
directly with the primary photochemical complex XBchl-HZ, and that
the product of this interaction is characterized by a yield of fluorescence
about halfway between those of the forms X-Bchl-HZ and X-Bchl-Z.
Oxygen. In chapter 13 (Vol. I, page 328) we described the inhibiting
effect of excess oxygen on photosynthesis. The light curves of photosyn-
thesis and fluorescence in the presence and absence of oxygen will be dis-
cussed in chapter 28. The only systematic measurements with varying
oxygen pressures permitting the plotting of an "oxygen curve," P = /[O2],
were made by Warburg (1920); the results were reproduced in figure 32.
This figure indicated the steepest rate decline between and 20% oxygen;
it was mentioned that this was contradicted by Wassink and co-workers
(1938), who found no difference between the rates at and 20% oxygen,
but a considerable drop from 20 to 100% oxygen.
Concentration Factors
A. Experimental Carbon Dioxide Curves
1804 de Saussure, T., Recherches chimiques sur la v^gHaiion. Paris, 1804.

1865 Boussaingault, J. B., Covipt. rend., 60, 872.
1873 Bohm, J., Sitzber. Akad. Wiss. Wien, Math.-naturw. Klasse. AM. I, 68, 171.
1881 Stefan, M. J., ibid., Abt. II, 83, 943.
1885 Kreusler, U., Landw. Jahrb., 14, 913.
1887 Kreusler, U., ibid., 16, 711.
1895 Blackman, F. F., Trans. Roy. Soc. London, B186, 503.

1896 Ewart, A. J., /. Linnean Soc, London, 31, 364.
1900 Brown, H. T., and Escombe, F., Trans. Roy. Soc. London, B193, 223.
1902 Brown, H. T., and Escombe, F., Proc. Roy. Soc. London, B70, 397.
Chapin, P., Flora, 91, 348.
1903 Treboux, 0., ibid., 92, 49.
Pantanelli, E., Jahrb. wiss. Botan., 39, 167.

1904 Demoussy, E., Cmipt. rend., 139, 883.
1907 Xathansohn, A., Ber. sacks. Ges. Wiss., Math.-naturw. Klassc, 59, 211.
1910 Heiiaer, 0., Flora, 100, 451.
Nathansohn, A., Der Stoffwechsel der Pjlanze. Quelle & Meyer, Leipzig.
1911 Aiigelstein, U., Beitr. Biol. Pflanz., 10, 87.
Blackman, F. F., and Smith, A. M., Proc. Roy. Soc. London, B83, 389.
BIBLIOGRAPHY TO CHAPTER 27 9()1
Renner, 0., Ber. dent. Botan. Ges., 29, 125.

1914 ami Reinau, E., Chem. Ztg., 38, 545.
fflein
1918 Brown, H. T., J. Chem. Soc, 113, 559.
1920 Freeman, G. F., Botan. Gaz., 70, 190.
1921 Harder, R., Jahrb. wiss. Botan., 60, 531.
Lundeg&rdh, H., Sirnsk botan. Tid., 15, 46.
Sierp, H., and 'Sonck, K., Jahrb. wiss. Botan., 60, 4.59.
Wilmott. A. J., Proc. Roy. Soc. London, B92, 304.

1923 Iljin, V. S., Flora, 16, 360.
1924 Chapman, R. E., Cook, W. R. I., and Thompson, X. L., Xew PhytoJogist,
23, 50.
Eundeg&rdh, H., Der Kreislaiif der Kohlensdvre in der Natiir. Fischer,
Jena, 1924, p. 127.
Schroeder, H., Flora, 117, 270.
1925 Noack, K., Z. Botanik, 17, 481.
1926 Noack, K., Natururissenschafteii, 14, 383.
Noack, K., Z. ongew. Chem., 39, 302.
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Shutov, D. A., Planta, 2, 132.
Dahm, P., Jahrb. wiss. Botanik, 65, 314.
Bode, H. R., Jahrb. idss. Botanik, 65, 352.
1927 Reinau, E., Praktische Kohlensauredungimg in Gartnerei und Landwirt-
schaft. Springer, BerUn, 1927.
Romell, L. G., Flora, 121, 125.
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Sierp, H., and Seybold, A., Planta, 3, 115.

Geiger, M., Jahrb. vriss. Botan., 67, 635.
1928 Huber, B., Ber. deut. botan. Ges., 46, 610, 621.
James, W. 0., Proc. Roy. Soc. London, B103, 1.
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Maskell, E. J., Proc. Roy. Soc. London, B102, 488.
1929 Bergamaschi, M., Atti. reale accad. nazl. Lincei, [6], 9, 238.
Bergamaschi, M., Atti ist. botan reale univ. Pavia, (4) 1, 117.
1930 Arens, K., Playita, 10, 814.
van der Honert, T. H., Rec. trav. botan. neerland., 27, 149.
Bazyrina, K., and Chesnokov, V., ibid., 11, 463.
White, H. L., Ann. Applied Biol., 17, 755.
1931 Harder, R., Keppler, E., and Reuss, H., Gartenbauwiss., 5, 389.
1932 Bazyrina, K., and Chesnokov, V., Trav. inst. biol. Peterhof, 9, 58, 103;
Proc. Leningrad Soc. Nat. Sci., 61, 279, 323.
Schoder, A., Jahrb. vnss. Botan., 76, 441.
Boysen-Jensen, P., Die Staffproduktion der PJlanzen. Fischer, Jena, 1932.
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van der Paauw, F., Rev. trav. botan. neerland., 29, 497.
1933 Hoover, W. H., Johnston, E. S., and Brackett, F. S., Smithsonian Inst.

Brinkman, R., Margaria, R., and Roughton, F. J. W., Phil. Trans. Roy.
Soc. London, 232, 65.
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1934 Emerson, R., and Green, L., J. Gen. Physiol, 17, 817.
Livingston, B. E., and Beall, R., Plant Physiol, 9, 237.
1935 Barker, H. A., Arch. Mikrobiol, 6, 141.
Singh, B. N., and Lai, R. N., Plant Physiol, 10, 245.
Singh, B. N., and Kumar, K., Proc. Indian Acad. Sci., Bl, 909.
St&lfelt, M. G., Planta, 23, 715.
Miller, E. S., and Burr, G. 0., Plant Physiol, 10, 93.
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No. 15.
Burk, D., and Lineweaver, H., Cold Spr. Harb. Symp. Qu. Biol, 3, 165.
Brackett, F. S., ibid., 3, 117.
Weishaupt, C. G., Dissertation, Ohio State Univ. 1935.
1936 Newton, R. G., Thesis, Univ. London, 1936.
Arens, K., Jahrb. wiss. Botan., 83, 513, 561.
1937 Suessenguth, K., Botan. Archiv, 38, 480.
Smith, E. L., /. Gen. Physiol, 20, 807.
1938 Overkott, 0., Z. ges. Naturw., 3, 480.
Hartel, 0., Jahrb. wiss. Botan., 87, 173.

Richter, A. A., Cornpt. rend. acad. sci. USSR, 18, 59.
Emerson, R., and Green, L., Plant Physiol, 13, 157.
Gessner, F., Jahrb. wiss. Botan., 86, 491.
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1939 Katunsky, V. M., Bull acad. sci. USSR, Ser. biol, 1939, 85.
Overkott, 0., Botan. Arch., 39, 389.
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1940 Livingston, R., and Franck, J., Am. J. Botany, 27, 449.
1941 Heath, 0. V. S., and Penman, H. L., Ann. Botany, 5, 455.
Ballard, L. A. T., New Phytologist, 40, 276.
Franck, and Herzfeld, K. F., J. Phys. Chem., 45, 978.

J.,
Franck, J., and French, C. S., J. Gen. Physiol, 25, 309.

Katz, E., Wassink, E. C, and Dorrestein, R., papei' at Spectroscopy
Symposium, Chicago, 1941.
C., Katz, E., and Dorrestein, R., Enzymologia, 10,
285.
1942 Wassink, E.
and G. R., Plant Physiol, 19, 370.
1944 Thomas, M. D., Hendricks, R. H., Hill,
Verduin, and Loomis, W. E., Plant Phtjsiol, 19, 278.

J.,
1945 Lipmann, F., and Tuttle, L. C, J. Biol. Chem., 158, 505.

1946 Wassink, E. C., Enzymologia, 12, 33.
Steemann-Nielsen, E., Nature, 158, 594.

Freeland, 0. R., presented at A.A.A.S. meeting, Boston, Dec. 1946.
Van Rysselberghe, P., Alkire, G. J., and ]McGee, J. AI., /. Am. Chem. Soc,
68, 2050.
Tseng, C. K., and Sweeney, B. M., Am. J. Botany, 33, 706.
Ochoa, S., in Currents in Biochemical Research. Interscience, N. Y., p. 165.
1947 Ruttner, F., Osterr. Botan. Z., 94, 265.

1948 Fuller, H. J., Am. Midland Xaturalist, 39, 247.
Osterlind, S., Nature, 161, 319.
Ruttner, F., Osterr. Botan. Z., 95, 208.
1949 Verduin, J., "Diffusion through ]Multiperf orate Septa," in Photosynthesis
Iowa State College Press, Anies, 1949, p. 293.
in Plants.
Thomas, M. D., and Hill, G. R., "Photosynthesis under Field Condi-
tions," ibid., p. 19.
Gabrielsen, E. K., Nature, 163, 359.
Warburg, 0., Burk, D., Schocken, V., Korzenovsky, M., and Hendricks,
S. B., Arch. Biochem., 23, 330.
Osterlind, S., Symbolae Botan., Upscd., 10, Xo. 3.
Whittingham, C. P., Thesis, Cambridge Univ.

1950 Osterlind, S., Physiol, plantarum, 3, 353, 430.
B. Carbon Dioxide Concentration and Fluorescence
1940 McAlister, E. D., and Myers, J., Sm. Inst. Pub. Misc. Coll., 99, No. 6.
1941 Franck, J., and Puck, T. T., J. Phys. Chem., 45, 1268.
French, C. S.,
Franck, J., and Herzfeld, K. F., ibid., 45, 978.

1942 Wassink, E. C, Katz, E., and Dorrestein, R., Enzymologia, 10, 285.
1945 Wassink, E. C, and Kersten, J. A. H., ibid., 11, 282.
1947 Shiau, Y., and Franck, J., Arch. Biochem., 14, 253.
1949 Franck, J., "The Relation of the Fluorescence of Chlorophyll to Photo-
synthesis," in Photosynthesis in Plants. Iowa State Coll. Press, p. 293.
C. Concentration of Reductants
1937 French, C. S., J. Gen. Physiol, 20, 711.

1938 Eymers, J. G., and Wassink, E. C, Enzymologia, 2, 258.
1942 Wassink, E. C, ibid., 10, 257.
D. Concentration of Inhibitors
1938 Wassink, E. C, Vermeulen, D., Reman, G. H., and Katz, E., Enzymologia,
5, 100.
1941 Weller, S., and Franck, J., /. Phys. Chem., 45, 1359.
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1945 A\;issink, E. C, and Kersten, J. A. H., ibid., 11, 282.

1946 Warburg, 0., and Liittgens, W., Biokhimija, 11, 303.
Chapter 28
THE LIGHT FACTOR. I. INTENSITY *
A. Light Curves of Photosynthesis

The between photosynthesis and the quantity of Hght available
relation
to the plants was investigated for the first time in 1866, when the Russian
botanist Volkov (Wolkoff) counted the oxygen bubbles evolved by sub-
merged aquatic plants at different distances from a sun-illuminated, frosted
glass Avindow. He found that the rate of gas evolution was proportional
to the intensity of illumination. In 1883, Reinke in Germany extended
similar measurements to stronger illuminations and observed that, when the
light intensity approached that of full simlight, the "light curves" (i. e.,
curves in which the rate of photosynthesis was plotted against the intensity
of incident light) bent, and finally became horizontal. He had thus dis-
covered the phenomenon of ligJit saturation.
Ewart showed that, if the illumination is increased
(1896, 1897, 1898)
still beyond the saturating value, the rate begins to decrease
further, far
again, and photosynthesis may even be completely inhibited.
The initial increase of the rate with light intensity, the "light saturation"
that follows and the ultimate "light inhibition" of photosynthesis were
again observed by Pantanelli in 1903; at that time, it was natural to in-
terpret these results in terms of the theory of "cardinal points" {cf. fig.
26.1). Blackman and Matthaei Blackman and Smith (1911)

(1905) and
pointed out, however, that photosynthesis requires no minimum light
intensity (at least, if one considers the true rate, corrected for respira-
tion, rather than the rate of net gas exchange). Furthermore, it shows a
broad "saturation plateau" instead of a sharp optimum. Therefore, they
argued, light curves can better be explained by means of Blackman's theory
of "limiting factors" {cj. fig. 26.2) than by reference to the three "cardinal
points."
Singh and Kumar (1935) and Lubimeuku (193G) observed that the light curves of
some land plants are sigmoid in shape, and Lubimenko saw in this the proof of the exist-
ence of a "light threshold" of photosynthesis; but this conclusion runs contrary to the
results of all the other observers. On the other hand, sigmoid-shaped light curves ap-
pear to be the rule with purple liartcria {rf. French 1937; Wassink, Katz and Dorrestein
1942).

904
GENERAL REVIEW 005
Not with the qualitative similarity between the empirical

satisfied
light curves and the broken lines predicted by the theory of limiting factors,
Blackman insisted on a quantitative agreement, and thus precipitated the
controversy to which we have referred in chapter 26. He insisted that no
decline in rate occurs at high light intensities, unless injuries are brought
about by overheating, and denied the gradual character of the transition from
the linearly ascending part to the horizontal part of the light curves.
Blackman was
prolial)ly right in suggesting that the inhibition of
photosynthesis by excessive be attributed to destructi^•e processes
light
alien to the intrinsic kinetic mechanism of photosynthesis. (However, we

believe these processes to be photoxirlations, rather than thermal reactions
caused by overheating; this theory of light inhibition was discussed in
chapter 19, when we described the phot oxidation phenomena in living
plants.) on the other hand, impossible to accept the second contention

It is,
of Blackman— that the linearly ascending part of the light curves goes over
abruptly into the horizontal part. All precise observations confirm that
light saturation is reached asijmptotically, sometime over an extended range
of fight intensities (cf. the early criticism of Blackman's interpretation by
Brown and Heise 1917, 1918). It was shoT^^l in chapter 20 (cf. also fig.
28.20) that the inhomogeneity of light absorption, which is inevitable even

in single chloroplasts, not to speak of multicellular systems, should in itself
suffice to make practical observation of Blackman's angular light curves
impossible —even
these curves correctly represented the relation between
if
light intensity and rate of photosynthesis in a uniformly illuminated vol-

ume element. Application of the general laws of reaction kinetics shows,
however, that even in the ideal case of completely uniform light absorption
Blackman's broken lines could represent only a first approximation, which
may be more or less satisfactory, depending on specific conditions.
1. General Review
Table 28.1 lists the most important determinations of the light curves of
photosynthesis carried out since the time of Blackman.
A remark mu.st be made on the units of light intensity used in these measurements.
For white light, lux (meter candles), or foot candles, have been and still are widely used.
A foot candle is equal to 10.8 meter candles; one meter candle corresponds {cf. chapter
25, page 838) to about 4.5 erg, or 1.4 X lO'^ quanta, or 2.3 X
10^'^ einstein of photosyn-
thetically active light (400-700 mju), falling each second on a square centimeter of the
illuminated surface. light, the intensity is usually given in ergs (or calories;
For colored
1 cal = 4.2 X square centimeter per second, or in watts per square centi-
10^ erg) per
meter (1 watt = 10^ erg/sec). We will use the abbreviations klux for thousand lux,
and kerg for thousand erg. In comparing the results obtained in light of different color,
the most appropriate measure of intensity is the number of incident quanta (A';,^), or the
number of einsteins (1 einstein = 6.1 X 10" quanta) falling per second on one .square
' ^
966 THE LIGHT FACTOR. INTENSITY CHAP. 28
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LIGHT CURVES OF PHOTOSYNTHESIS 967
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968 THE LIGHT FACTOR. I. INTENSITY CHAP. 28
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Fig. 28.3. Light curves of water moss Fonlinalis

aniipijretica 23-24° C. for different values of
at
[KHCO3], inwhite light (after Harder 1921). 1%
KHCO3 corresponds to [CO.] = 1.1 X 10 -« mole/1.
tn
UJ
o
150
£ 100
E
CM
O
<3
Fig. 28. G. Light curves of

Chlorella at different temperatures
(after Wassink, Vermeulen, Reman
and Katz 1938).
* 1.75 X IC*
INTENSITY, erg/cm. sec.
25,000
Pig. 28.7. Light curves of

Chlorella at different tem-
peratures (after Noddack and
Kopp 1940). [CO2] = 7.87
X 10 ^ mole per Uter. 1 HK
= 940 erg/(cm.^ sec). Up-
per figure, white light; lower
figure, red light.
10 15
INTENSITY, HK
pH 6.3, l%thiosulfate
250
200 -
150
I
o
100
50
GENERAL REVIEW 975
O no inhibitor
• 05 ml. 50%
200 urethan/ml.
O no inhibitor
E
• 2.5 X 10"" mole/hter E 100
600 NHjOH-HCl
a:
iLl
400
>
<
^ 200
150 X 10'
20 40 60 80 100
INTENSITY
RELATIVE LIGHT INTENSITY
Fig. 28.9B. Light curves of inhibited Fig. 28.9C. Light curves of in-
Chlorella cells showing that NH20H-HC1 is hibited Chlorella cells showing that
effective at all light intensities (after Weller ethylurethan is effective at all light
and Franck 19-11). intensities (after Wassink, Vermeulen,
Reman and Katz 1938). Intensity is
in erg/cm.^ sec.
2.00 F
n
S lOOi-
0.50 -
o
O 0.25 -
X
Q.
0.13
350 700 1600 4000 22000 350 700 1600 4000 22000
LIGHT INTENSITY, lux
Fig. 28. 9D. Light curves of inhibited Chlorella cells sliowing that CuSO^ is
effective, NiSO^ ineffective at low light intensities (after Greenfield 1942).
976 TflE LIGHT FACTOE. I. INTENSITY CHAP. 28
i 1.45 X 10*
INTENSITY, erg/cm sec
Fig. 28.9E. Light curves of Chlorella cells showing O2

effect (after Wassink, Vermeulen, Reman and Katz 1938).
- - o without cyanide
250 250
A 0003% KCN
-
200 200
E
E
z"
o
o
>
LlJ
3 xlO" 3 xlO*
INCIDENT INTENSITY, erg/cm.^ sec.
Fig. 28.10. Light curves of inhibited diatoms (Nitzschia) at 25° C. in Warburg

buffer No. 9, showing both ethylurethan and cyanide (?) to be effective at low light
intensities (after Wassink and Kersten 1945).
)
500
15% Hj, pH 6 3
,o'
INCIDENT INTENSITY, erg/cm sec
250
I % H2S2O3, pH 6 3
200 \- (S) ^,
without KCM
150 INCIDENT INTENSITY, erg/cm sec.
o
o
o 100
tr A
<
H .50 o'/0.00l5 % KCN
3
.9/ xlO"
600 300
1% H2S2O3, pH 6 3
250
1 2'7o
0187% NaNj
eti./lure'î"
^
xlO" X IC
/-.^^"
INCIDENT INTENSITY, erg/cm' sec. INCIDENT INTENSITY, erg/cm sec
Fig. 28.11. Light curves of inhibited purple bacteria (Chromatium) (after

Wassink, Katz and Dorrestein 1 942 ) ( H. or H2S2O3 as reductant, 5
. CO2, 29 ° C % .
HCN shows no effect in weak light with Ih. With HoSaOj, effect is shown also iu
weak light. NnÔII-IICl shows no effect in weak light (c/. fig. 28.9B). With
ethylurethan, the effect is particularly strong in weak light.
THE LIGHT FACTOR. INTENSITY CHAP. 28
978
15% Hs /
y
300 600
O pH 6 3 o pH 63 /
A pH 76 A pH 7.6 /
/
250 500 /
/
E
e /
400-
£
£
200
o
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150 / i~ 300- /
fe / u. /
LlI o //
< UJ
t < 200 //
100- / y-
Q.
/ 13
50^ /. 100 //
k
X 10' X 10^
I i_
INCIDENT INTENSITY, erg/cm^ sec
Fig 28 12 Effect of pH on rate of COo reduction by Chromatium (after Wassink,

Katz and Dorrestein 1942). 15% Ho (at right) or 1% H2S0O3 (at left) as reductant,
5%C02, 29°C.
300
20 40 60 80 100 120 140
INCIDENT INTENSITY
Fig. 28.13. Light curves in relation to age in Chlorella (after

Wassink and Katz 1939). Gas pha.se air, 29° C.
LIISTEAR RANGE 979
centimeter. For relationships between iV/,^ and intensity of illumination see chapter 25,
page 838.
Table 28.1 does not list the measurements of light curves in the presence
of various poisons, such as potassium cyanide, hydroxylamine or azide,
of narcotics, such as urethan, or of salts, such as copper sulfate. Several
curves of this type are, however, reproduced in figures 28.9-11; for addi-
tional information, we refer to chapters 12 and 13 in volume I, and to
chapter 37. In the latter, we will also describe the light curves of algae in
the state of (almost complete) anaerobic inhibition, which Franck, Prings-
heim and Lad (1945) were able to measure by the very sensitive phos-
phorescence method.
Figures 28.1-28.13 contain a selection of typical light curves. Attempt
was made to include curves for all types of plants higher land plants, —
aquatic higher plants, green and colored algae, diatoms and purple bacteria.
Figures 28. 1-28. 5A represent families of curves in which the carbon dioxide
concentration, [CO2], serves as parameter (strangely enough, no such set is
available for Chlorella). Figure 28. 5B shows light curves of purple bac-
teria for two concentrations of the reductant (thiosulfate) figures 28.6- ;
28.8 represent curve sets with temperature as parameter. Figures 28.9-

28. 1 1 illustrate the effect of inhibitors. The effect of pH (in purple bacteria)
is shown in figure 28.12, while figure 28.13 shows the influence of age.
Later in this chapter, some additional sets of curves will be given to illus-
trate the influence of inherited or acquired conditioning to strong or weak
light.
In chapter 26we discussed three types of curve sets, P = f[Fi] with a
parameter which
F2,can be anticipated in photosynthesis. Examples of
conditions under which each type can occur were given, for carbon dioxide
curves, Fi = [CO2], in chapter 27.
2. Linear Range
We will now consider some of the details of the light curves : the linear
range, the compensation point, the saturating light intensity and the maxi-
mum yield. Perhaps the most important quantitative characteristic of the
light curves is the initial slope, which determines the maximum quantum
yield; it will be discussed separately in chapter 29.
Figures 28.1,28.7, 28.9A,28.10and 28.14A,B show that many lightcurves
exhibit a practically exact proportionality between rate and light intensity
over a considerable range of intensities. This "linear range" is less clearly
delimited in figures 28.2-28.6. In the light curves of purple bacteria, it is
often obscured by an inflection (c/. figs. 28.8 and 28.11A-D). Data col-
lected in Table 28.11 indicate that (at room temperature and with an ample
II ' ^
080 THE LTGHT FACTOR. I. INTENSITY CHAP. 2S
o
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o
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Qj a;
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z
COMPENSATION POINT 981
suppl}' of carbon dioxide) the linear range usually extends up to 5 or 10

kerg/cm.2 sec, corresponding to 1-2 klux of white light. In some cases,
however, the first signs of curvature have been observed despite ample —
—
supply of carbon dioxide as earl}'- as at 1 kerg/cm.- sec, or 200 lux; while
in others, the linear increase continued up to 50 or even 100 kerg/cm.'^ sec,
i. €., 10-20 klux (c/. figs. 28.1 and 28.14B).
Theoretically, no exact definition of the linear range can be given, since
all light curves probably are hyperbolae (or curves of a higher order) and
can only approach straight lines asymptotically. A formal definition of
the upper limit of the linear range could thus be given only in terms of a
definite deviation from linearity.
200-
E
6
o
I-
<
C/5
<
_J
<
I-
<
5 10 15 20 2 4 6 8
LIGHT INTENSITY, einstein/cm.^ mm LIGHT INTENSITY
Hg. 28.14A. Approximate linearity of Fig. 28. 14B. Light curves in purple
light curves of Chlorella in white light up hactciia in Eymers
soilium light (after
to ca. 1300 lux (or G.5 kerg/cm.- sec.) and Wassink 1938) (showing linearity up
(after Emerson and Lewis 1941). to GO kerg/(cm.2 sec.)). Light hitensity
in (erg/cm.^ sec.) X 10^
Wassink (1946) gave incident intensities of monochromatic yellow light

at which the yield of photosynthesis of nine horticultural plants showed
16% deviation from proportionality {cf. Table 28.11).
Kok (1948,1949) and van der Veen (1949) found that the linear range
may consist of two segments, the lower one up to twice as steep as the up-
per one {cf. chapter 29, p. 1113).
3. Compensation Point
The compensation point is the light intensity Ic at which photosynthesis
is balanced by respiiation. so that the net gas exchange is zero.
;
One could also call "compensation point" the carbon dioxide concentration at which
the gas exchange becomes zero at a given light intensity {cf. chapter 27) or the tem- ;
peratui-e at which the gas exchange becomes zero at a given combination of the param-
eters Iand [CO2] (cf. chapter 31); but the word is seldom used in either of these two
ways. Sometime, the designation "upper compensation point" is applied to the second
crossing of the curves of photosynthesis and respiration, which may occur either at very
high light intensities or at "superoptmial" temperatures (cf., for example, fig. 31.1).
When the carbon dioxide supply is not too low, compensation occurs
within the linear range of the light curve, where the slope of the latter is
determined by the maximum quantum yield of photosynthesis and the rate
of light absorption {i. e., the optical density of the specimen). Probably
{cf. chapter 29) the maximum quantum yield is approximately the same for
all species (at least, when all cells is not always the
are fully active —which
case, e. g., in "aged" cultures). Differences in the compensation points
found under these conditions must therefore depend mainly or exclusively
on two factors: rate of respiration and optical density of the specimen.
Respiration is proportional to the concentration of cells in a suspension
Table 28.III
Compensation Point of Leaves and Thalli
Authority Plant 7 , lux Temp.
HIGHER LAND PLANTS
Boysen-Jensen, Mliller (1929) Fraxinus excelsior (shade leaves) 200 20° C.

(sun leaves) 700 20° C.
Fagus silvatica {shside leases) 150 20° C.
(sun leaves) 500 20° C.
MOSSES
Boysen-Jensen, Miiller (1929) Marchavtia pohjmorpha 100 20° C.

Stalfelt (1939^) 6 species (winter), average 390" About 11° C.
6 species (summer), average
LICHENS (symbiotic GROWTHS OF ALGAE AND FUNGi)
Boysen-Jensen, Miiller (1929) Peltiqera canina 4200 ''

20° C.
Stalfelt 19391) 12 species (winter), average 1020 13° C.
(
12 species (summer), average 1160
AQUATIC HIGHER PLANTS AND MOSSES
Plaetzer (1917) Elodea (summer)

COMPENSATION POINT 983
Table 28. Ill (continued)
AQUATIC HIGHER PLANTS AND MOSSES (continued)

The relation between the kmetic properties of sun-adapted (heho- I

phiHc) and shade-adapted (umbrophiHc) plants will be discussed on page |
987; that between warmth-adapted (thermophilic) and cold-adapted
(cryophihc) plants, in chapter 31. As shown by Harder 's data in Table
28. Ill, the effects of adaptation to weak light and low temperature differ
in sign — the first one reduces respiration and thus shifts h toward weaker
illuminations, while the second one enhances repiration, and thus shifts h
toward more intense light.
Algae that live deep under the sea, particularly red algae, are adapted
both to weak light and to low temperature. The effect of umbrophilic
adaptation predominates, however, and the compensation points of these
algae generally are lower than those of the surface algae. Without low
compensation points, these organisms could not develop 100-120 meters
under the sea (the lowest level from which organisms have been recovered
by dragnet) because the intensity of illumination at 120 meters depth is of
the order of only 200 lux (see data of Seybold 1936, in chapter 22).
As discussed in more detail in chapter 15 (page 424), the deep-sea algae
are adapted not only to low light intensity, but also to predominantly blue-
green light. If the compensation points of the green surface algae and
the colored deep-sea algae were compared in blue-green light, the lower
compensation points of the latter probably would appear even more strik-
ingly than in Table 28. III.
In general, the compensation points of different species shown in the
table are comparable only if the experiments were carried out under closely
similar conditions (same carbon dioxide concentration, temperature and
previous history of the plants), since otherwise the intensity of respiration
of a given species may vary wddely. Plants allowed to photosynthesize
efficiently for some time often accumulate assimilates and then respire
many times stronger than similar plants "starved" for an extended period
of time (c/. Harder's data in Table 28.III). Such special conditions may
perhaps explain the very low /, values found by Plaetzer for some aquatic
plants.
The between respiration and photosynthesis at low light intensi-
ratio
ties is generally changed in favor of respiration by an increase in
tempera-
ture (cf. chapter 31); thus, higher temperature must cause an upward
shift of the compensation point (of. fig. 28.15 and Table 31. III). Narcotics
have a similar influence, since they, too, reduce photosynthesis (at all light
intensities) much more than respiration (c/. chapter 12). En-
effectively
zyme poisons (e. g., cyanide) may have a lesser or even opposite effect,
because their influence on photosynthesis in weak light usually is rather
small (c/. figs. 28.9A, 28. 11 A), while most of them strongly inhibit
respiration. In certain algae (e. g., some Scenedesmus strains), the effect
SATURATING LIGHT INTENSITY 985
of cyanide on respiration is stronger than on photosynthesis, even in strong

Hght; organisms of this type, addition of cyanide causes a strong down-
in
ward shift of the compensation point (c/. chapter 12). Indications of a
peculiar difference between cyanide effects of photosynthesis above and
below the compensation point were mentioned in chapter 12 (Vol. I, p. 308).
Reduced supply of carbon dioxide decreases photosynthesis without af-
fecting respiration. If, in consequence of carbon dioxide deficiency, the
light curves begin to bend in very weak light, the compensation point
may be shifted to high light intensities (c/. fig. 28.15), or never reached
(3) (l)(2)
Fig. 28.15. Shift of compensation point with changing carbon dioxide

concentration. (1) — >-
(2) decreasing [CO2]; (3) -*- (1) increasing tem-
perature.
at all. This case was mentioned in chapter 26, when we spoke of the ex-
periments of Chesnokov and Bazyrina (1932) and Miller and Burr (1935),
in which gas balance was observed at light intensities of the order of 20
klux. Miller and Burr (1935) noticed that, in this "carbon dioxide-limited"
range, the compensating light intensity was independent of temperature.
This means that the temperature coefficient of the carbon dioxide supply
process (diffusion or carboxylation?) was practically equal to that of respira-
tion.
4. Saturating Light Intensity
When Reinke discovered tlu^ light saturation of photosynthesis, he

found it to occur at an intensity close to that of sunlight at noon (*So = ap-
proximately 60 klux in moderate zones; cf. chapter 25). However, the

saturating intensity varies widely from species to species and specimen
to specimen. One reason for this is difference in optical density. Satura-
tion begins when the most exposed chlorophyll molecules receive a certain
hght and becomes complete when the most deeply shaded molecules
flux,
obtain this saturating intensity. The intensity of incident light at which

this complete saturation occurs obviously must depend on whether we use
a thick or a thin leaf, a dense or a dilute cell suspension. This "density
effect" already was described in chapter 25 and will be again discussed
later in this chapter (page 1007).
Even with the density effect eliminated —either experimentally, by
using optically very thin objects, or by calculation {cf. fig. 28.22) —the
saturating light intensity still remains dependent, for a given species, on
internal factors such as age and adaptation (to strong or weak light), and
external variables, such as carbon dioxide supply and temperature. The
effects of carbon dioxide concentration are illustrated by figures 28.1 to
28.5, those of temperature by figures 28.6 to 28.8. Using the notion of
"ceilings" introduced in chapter 26 (page 869) we can say that everything
that lowers the ceiling imposed on the over-all reaction of photosynthesis
must shift the saturation toward lower light intensities. This may be a
decrease in [CO2], a decrease in available reductants (in purple bacteria),
or a decline in the amount
of one of the catalysts. The temperature effect
is complex, because changes in temperature affect all ceilings simultane-
—
ously those imposed by diffusion, as well as those caused by enzymatic
reactions of limited maximum yield.
Among the internal factors affecting the saturating light intensity, the
most important is adaptation to strong or weak light. Shade-adapted
plants often are darker green, i. e., contain more chlorophyll (per unit area
or unit volume) than the corresponding light-adapted species or individuals.
This difference in optical density would in itself be sufficient to cause
changes in the shape of the light curves: Darker, shade-adapted plants
are more efficient light absorbers, and their light curves should there-
forehave a steeper initial slope. If the higher optical density is due to in-
creased concentration of the pigment (with the concentration of all other
constituents of the catalytic apparatus remaining the same), the saturation
rate, related to unit volume of cells (or to unit area of leaves, assuming the
leaf thickness is constant), should be the same for heliophilicandumbrophilic
varieties; while the saturation rate related to U7iit amount of chlorophyll
should be lower in the darker specimens.In practice, conditions are more
complicated, because shade leaves often are thicker, and shade cells do
grow larger than their heliophilic counterparts. These relationships will
be discussed in more detail in chapter 32. The experimental result we

SATURATING LIGHT INTENSITY 987
want to quote now is that the saturation rate of umbrophiHc plants usu-
ally is much lower than that of the heliophilic plants, even if related to
unit volume or unit area (not to speak of the rate per unit chlorophjdl con-
tent). This indicates that adaptation to weak light involves, in addition
to an increase in pigment concentration, a decrease in the amount of one or
several catalysts that exercise a rate-limiting influence in photosynthesis.

Coupled with steeper initial rise, this lower "ceiling" on the rate of photo-
synthesis in shade plants often leads to a very early light saturation. While
the light curves of sun-adapted plants may continue to rise at or even be-
yond 100 klux (cf. data of Singh and Kumar, Smith, Boysen- Jensen, and
Gabrielsen in Table 28.1), the light curves of shade plants may show
saturation at light intensities as low as 1 klux (cf. figs. 28. IG and 28.18).
2-
Peltigera
'
I
Marchantia
o
If)
O
CSJ
0,^
^(J
o
a. -I
E
_L
9 II 13 15 17 19 21 23
klux
Fig. 28.16. Light curves of net gas exchange of an umbrophiHc moss (Mar-
chantia) and a hehophiUc hchen (Peltigera) (after Boysen-Jensen and IMiiller 1929).
The former is hght-saturated at 1 klux; the latter at or above 20 klux.
The difference between the shapes of the light curves of heliophilic and
umbrophilic land plants was observed by Weis (1903), who compared
first
the shade plant Poly podium with the sun plant Oenothera. This phenom-
enon was also investigated by Lubimenko (1905,1907,19081.2,1928,1929),
Boysen-Jensen (1918, 1929), Boysen-Jensen and Miiller (1929^) and Lunde-
gardh (1921, 1922), among others. Typical results are illustrated by
figures 28.16, 28.17 and 28.18. The first of these figures refers to an umbro-
philic moss (which is compared with a heliophilic lichen) the second com- ;
pares shade-adapted specimens of two aquatic plants with sun-adapted

individuals of the same species and the third contains a comparison of the
light curves of a shade-adapted leaf and a sun-adapted leaf on the same
plant (see also Table 28. IV). We see that the effect of phylogenetic adap-
tation (fig. 28.16) is similar to that of the individual adaptation of whole
plants (fig. 28.17) or single leaves (fig. 28.18). The figures in Table 28.IV
further show that the respiration of sliade-adapted plants is weaker than
that of the sun-adapted specimens.
Fig. 28.17. Light curves of photosynthesis of shade plates (S) and sun plants
(L) of the same species (after Gessner 1937). Former saturated at 40 klux; latter
onlv far above 80 klux.
9 II 13 15 20 30 40
klux
Fig. 28.18. Light curves of sun leaf (a) and shade leaf (b) of
Fagus silvatica (after Boj^sen-Jensen and Miiller 1929). Former
saturated at or above 30 klux; latter at 3 klux.
AB.SOLTITK MAXIMUM RATE 9S9
Bohning (1949) noted that the rate of photosynthesis of shade-adapted

leaves on trees of Pyrus malus decHned in continuous ilkimination of 32
klux from an value of about 20 mg. to < 5 mg. C02/(hr. X 100 cm. 2)
initial
after 20 days. Sun-adapted trees, on the other hand, showed no dechne

during a similar period of continuous illumination, even in 50 klux. Kramer
and Decker (1944) compared the light curves of white pine with those of
three hardwood trees, and noted that the first one behaves as a heliophile
and the deciduous trees as umbrophiles. This supports a previously sug-
gested explanation of the fact that young deciduous trees "squeeze out"
young pine trees on the floor of a forest.
Table 28.IV
and Respiration of Shade Leaves and Sun Leaves
Photo.synthesi.';
OP THE Same Plant (after Boysen-Jensen and Muller 1929)
Species
<
Co
O
HI
(G
B
>: g
00 M
o
O
n K
< P^
O
to
p:3
&
S
H
J
O
m
ABSOLUTE MAXIMUM RATE 991
^^
a 9
H H
(3 4
Eg
o
o
>
a
o
before "carbon dioxide inhibition" or "light inhibition" becomes apparent.

We have therefore assumed that, independently of any inhibition, certain
intrinsic internal factors (such as limited availability of certain catalysts)
impose an "absolute" ceiling {i. e., a ceiling independent of both [CO2] and
/) on the maximum rate of photosynthesis.
Determination of this maximum rate is of interest from the practical
point of view (estimation of absolute and relative efhciency of different
plants as producers of organic matter) as well as from the point of view
of the kinetic mechanism of photosynthesis. However, the two aspects
call for different methods of comparison. The practical problem can best
be answered by using unit surface as the basis of rate determination (since
what one wants to know is how much organic matter can be harvested
from a unit area covered with plants of different species) From the point
.
of view of a theorist, comparison should be based on unit cell volume, or

unit chloro'phyll content, rather than on unit area. Willstatter and Stoll
(1918) designated the maximum quantity of carbon dioxide that can be
reduced in unit time by unit quantity of chlorophyll in a cell or tissue as
"assimilation number" Table 28. V), and the shortest time in which
{va in
one molecule of chlorophyll can reduce one molecule of carbon dioxide
{Ta in Table 28. V) as "assimilation time." (These constants will be ana-
lyzed in chapter 32.)
We designated, in chapter 27, the maximum rate of photosj'nthesis, at
a given light intensity, reached with saturating concentrations of carbon
dioxide, by Pmax.l we can use the symbol p™^'' for the maximum rate
reached, at a given carbon dioxide concentration, when light intensity be-
comes saturating; and the symbol Pmax! for the "absolute" maximum rate,
obtained when both carbon dioxide supply and light intensity are saturat-
ing.
Table 28. V shows that for the leaves of land plants the values of PmTx.
generally are of the order of 20 mg. COa/hr. 100 cm.^ of leaf surface, and
sometimes reach 80-90 mg. Even aurea very low
leaves, despite their
content of chlorophyll, constitute no exception. Only some algae and
aquatic plants investigated by Kniep (1914), Emerson and Green (1934)
and Gessner (1938) fell far short of this production. A yield of 20-80 mg.
—
CO2/IOO cm. 2 hr. assuming it is reached in light of 40 Idux, 80% of which
is —
absorbed by the leaf corresponds to the conversion into chemical en-
ergy of 4 to 16% of absorbed light energ}^ (in the photosynthetically active
region, 400-700 m/x), and thus to a quantum yield between 0.018 iyio) and
0.07 (H4)- (This estimate is based on factors given in chapter 25.) The
relation of these yields, obtainable in strong light, to the maximum quan-
tum yields observed in weak light will be discussed in chapter 29. In the
case of aurea leaves, the quantum yield in the light-saturated state appears
;
ABSOLUTE MAXIMUM RATE 993
to be higher, .since roughly the same yield of carbon dioxide reduction is
obtained here with a lower light absorption. However, the average ab-
sorption of white light by aurca leaves can vary, depending on their actual
chlorophyll content, from as low as 30% or less, to as high as 75% of that
of normal green leaves of the same species. An estimate of the quantum
5'ield in the light-saturated state requires therefore that absorption de-
terminations and yield measurements be performed on the same specimens.
The fact that aurea leaves may absorb only a slightly smaller propor-
tion of incident light than normal green Seybold and Weiss-
leaves, caused
numbers" (table 28. V)
weiler (1942) to consider their higher "assimilation
—
as irrelevant (and not as assumed by Willstatter and Stoll as a sign of —
exceptionally high capacity for photosynthesis). However, the capacity
for photosynthesis f». the lighl-saturaled state, P"^^'., is not a function of the
efficiency of light absorption, but a measure of the amount of a limiting
enzyme present in the cells. The values of Pmax.' for aurea leaves show that
in these leaves an abnormally low chlorophyll content is accompanied
not
by a proportional reduction in the content of the rate-limiting enzyme.
Yields obtained by Noddack and Kopp (1940) with Chlorella pyren-
oidosa, if most land
related to dry weight, are higher than those given for
plants in Table 28. V. However, because of the high concentration of
chlorophjdl in Chlorella (3-4%, instead of 0.5 to 1% in leaves), the assimila-
tion numbers are not higher, but somewhat lower, and the assimilation
times somewhat longer than those given by Willstatter and Stoll for the
leaves of the higher plants.
Like the maximum quantum yield (at low light intensity) the, maximum
rate of photosynthesis (in strong light) is a constant of the plant, i. e., it is
independent of the optical density of the selected material. The only ex-
ternal factor that affects it (apart from the presence of poisons or inhibi-
tors) is temperature (as illustrated by figs. 28.G-28.8). It is difficult, if not
impossible, to define the absolute maximum rate of photosynthesis also
as a function of temperature. In short experiments, the highest rates can
be obtained, with plants adapted to moderate conditions, at about 35° C.
but, in prolonged experiments, "heat inhibition" is apt to occur even at
temperatures as low as 22-25° C. (c/. chapter 31). We have used, in
Table 28. V, mostly values obtained at 18-20° C, which are certainly smal-
ler than the highest efficiencies of which most of the investigated plants
were capable at higher temperatures, at least for short periods of time.

The maximum rate of photosynthesis of a species or individual plant
depends on adaptation to strong or weak light. As described on p. 986,
shade-adapted species or individuals generally have a lower "ceiling rate,"
indicating a decreased content of a rate-limiting catalyst. In addition,
they often show an early onset of light inhibition (c/. fig. 28.19).
max.
Since inhibition by excess light chapter 19), the P ma.^.
is a time effect {cf.
values of shade-adapted plants change with the duration of illumination.

We recall in this connection the time curves that Harder (1933) found for
Fonfinalis antipyretica(fig. 26.8). The general impression made by these
complex curves was that photosynthesis declined with time {i. e., the plants
suffered light injury) whenever the illuminating light was more intense
than the light to which the specimens were accustomed during the growth
period.
It was noted on page 987 that in the shade-adapted plants the apparent
lower content of the enzyme responsible for the absolute saturation of
photosynthesis is coupled with a higher content of chlorophyll. We will
encounter, in chapter 32, other cases in which the content of the rate-
limiting enzyme appears to be independent of that of chlorophyll (Chlorella
cells grown in strong or weak light, cf. Tables 25.1 and 28. V; green and
aurea varieties of land plants which were mentioned above, cf. Table 28.V
and fig. 32.2) as well as cases in which these two concentrations change in
the same direction {Chlorella cells made chlorotic by iron deficiency, cf.
figs. 32.3 and 32.4).
The shape of the light curves of shade-adapted plants has been much discussed in the
ecological literature, particularly in relation to the photosynthetic production of aquatic
plants at different levels under the surface. Even green algae, or submerged higher
plants, found only a few meters under the surface, which should not have acquired ex-
treme umbrophilic characteristics, were observed to produce a maximum of oxygen when
placed at a certain depth, and to show light inhibition when exposed to direct sunlight.
This, however, might have been, at least in part, a thermal effect. Much more pro-
nounced optima on yield vs. depth curves were reported for the photosynthetic efficiency
of colored (brown or red) algae at different levels under the sea.
Ruttner (1926) and Schomer (1934) observed that several aquatic higher plants
{Elodea, Myriophyllum, Cerathopyllum) had a maximum efficiency 1-5 meters under
the surface. Curtis and Juday (1937) found similar optima for the green algae Ana-
boena and Gloethea (in 9-10 meter depth). Van der Paauw (1932) found that Hormi-
dium grown in a light of 2000 lux suffered light inhibition at 5000 lux. On the other
hand, Gessner (1938) found no "optimum" in the light curves of shadow-grown or sun-
grown Elodea plants in lamp light up to 30,000 lux. He tried ultraviolet light (360-400
m.y.) to imitate sunlight, but this, too, produced no inhibition. He suggested that the
reported depth optimum of Elodea may be caused by chromatic adaptation (to bluish-
green light) rather than by intensity adaptation. However, this explanation is im-
plausible since it implies that photosynthesis can be inhibited by the addition of red and
blue-violet light to green light, which has never been observed. Perhaps, carbon dioxide
supply conditions are more favorable at a certain depth than on the surface, and this
causes the rate to increase with increasing depth, as long as illumination remains suf-
ficient for light saturation.
Particular attention has been paid to the maximum efficiency and light
inhibition of colored algae in relation to their vertical distribution in the sea.
Engelmann suggested (see chapter 15, page 420) that the color of brown.
—
ABSOLUTE IVIAXIMUM RATE 995
and especially of red, algae is the result of chromatic adaptation to the

predominantly bluish-green light that prevails deep under the sea; Berth-
old (in 1882) and Oltmanns (in 1905), on the other hand, thought that
colored algae are adapted not so much to the spectral composition of light
in their natural habitats as to its loiv intensity. The ensuing controversy
which led to almost complete vindication of Engelmann's theory of chro-
—
matic adaptation will be discussed in chapter 30. However, the funda-
mental importance of chromatic adaptation for the composition of the
pigment system of deep-sea algae does not mean that these algae are not also
adapted to low light intensity and do not use the same mechanism shifts —
in relative concentrations of red, blue and green pigments for chromatic —
as well as for intensity adaptation (c/. Harder 1923).
6000
5000
Cladophora rupeslris, green
Fucus vesiculatus, brown
Ys ~^^hodymenia palmato, red
\ .^
60 75 117 140 197

S/90 LIGHT INTENSITY S/2
Fig. 28.19. Typical light curves of red, brown and green algae
(after Montfort 1929). Light intensity in relative units and frac-
tions of full sunlight. Equal fresh weights of algae used.
The response of colored algae from different depths to intense illumination has been
among others, by Maucha (1924, 1927), IMarshall and Orr (1927, 1928), Ehrke
studied,
1933, i.^
(1931), Curtis and Juday (1937) and particularly by Montfort (1929, 1930,
1934, 1936). Figure 28.19 shows a typical "optimum" curve, obtained by the last-
named investigator. Montfort noticed that algae from one and the same level often
show different resistance to strong light: Some red algae, containing much phycocyanin
(such as Rhodymenia palmata), continued to synthesize effectively on the surface, while
others, found in the same depth, but containing mainly phycoerythrin (such as Dehsseria
alata) suffered a "sunstroke" and died. The surface-living, almost pure-green form of
the blue alga Gigarlina behaved like a typical shade plant, whereas the violet, deep-water
form of the .same species, I'ich in j)h3'cocyauin, maintained its jjhotosj'nthesis at full ef-
ficiency even in direct sunlight.
6. Maximum Rate and Average Rate of Photosynthesis

under Natural Conditions
The curve corresponding to [C'02] = 0.03% i.s theoretically not more

important than any other light curve of photosynthesis, but its saturation
value has a considerable interest because it represents the maximum rate
of production of organic matter hy land plants in the open air. (In dense
growth, or under otherwise abnormal conditions, the concentration of car-
bon dioxide may vary between 0.01 and 0.1%, and this must affect the
maximum rate of photosynthesis in some natural habitats.)
It was stated in chapter 27 that with 0.03% carbon dioxide, and in
intense light, the supply of carbon dioxide has a considerable rate-limiting
influence, and the saturation value may therefore be below the "absolute"
maximum, P|||"' at the same temperature. Table 28. VI contains some
relevant experimental data (for a more extensive table, see Stocker 1935).
Most figures in this table represent the net consumption of carbon dioxide.
For strongly photosynthesizing plants, the corresponding values of true
photosynthesis are 10-15% higher; but for weakly photosynthesizing
plants (e. g., the arctic plants investigated by Miiller) the difference may
be much larger, as illustrated by the figures in parentheses. Table 28. VI
contains some striking contradictions, which remain to be elucidated.
There is a general contrast between the P""' values found by Boysen-
Jensen and co-workers (usually 1-10 mg. C02/hr. 100 cm.-, with the largest
single values not exceeding 20-25 mg.), and the much larger values reported
— often for the same species and under similar climatic conditions—by
Kostychev and other Russian plant ph3^siologists (usually 10 40 mg.
COo/hr. 100 cm.'-, with the largest single values reaching 80 or 100 mg.).
Only in the case of arctic plants is there an approximate agreement between Boysen-
Jcnsen's co-worker Miiller, and Kostychev and his co-workers. In the case of sun-
adapted plants from moderate zones, the average of Danish measurements (section Ba of
the table) is 13 mg., that of Swedish measurements (section B6), 16 mg., and that of
English, Japanese and German measurements (with the exception of the early determina-
tions of Sachs carried out by the half-leaf starch method), 10 mg. The average of the
Russian analyses, listed in section Be, is as liigh as 24 mg. The results obtained by
Kostychev, Bazyrina and Vasiliev (1927) by the determination of the synthesized assim-
ilates did not differ significantly from those obtained by the same group by determina-
tion of absorbed carbon dioxide.
It was mentioned on page 908 that Kostychev and co-workers attributed

the lower values of Boysen-Jensen to insufficiently rapid gas circulation.
PHOTOSYNTHESIS RATK UNDER NATURAL PONDTTIONS 007
This was denied by Boysen-Jensen and Miiller (1029); but one notices
in Table 28. VI that the newer measurements Danish school have
of the
given somewhat higher values than those of 1018-1920, and thus reduced
the discrepancy between the averages in sections Ba and Be to a factor of
about 2.
In section C, containing plants from arid zones, we find a similar dis-
crepancy l>et ween the result of Harder and co-workers in Algeria, and Wood
in Australia (1-10 mg./hr. 100 cm.-), and those of Kostychev and Kardo-
Sysojeva in Central Asia (20-70 mg.).
In section D, practically all the listed values fall into the range 1-10 mg. (no Russian
measurements are listed here, c/. however, the data of Kostychev and Kui'saiiov for
the subtroiiical vegetation of the Black Sea littoral in .section Be).
In the group of alpine jilants (section E), Monch (1037) and the Russians
agree in finding the highest yields ever recorded under natural conditions.
Earlier, Henrici (1018) had reported, for the alpine plant BelUs perennis,
a yield of 232 mg. C02/hr. 100 cm.^ This value appears so incredibly
high that we did not include it in Table 28.VI; but even the results of
Blagoveshchenskij (1035) and Monch (1037) (00-100 mg./hr. 100 cm.^)
indicate remarkablj^ high quantum jaelds (of the order of one CO2 mole-
cule reduced per twenty quanta, in light of 80 klux, and \vith not more than
0.03% CO2 present).
It shouldnot be assumed that the carbon dioxide concentration was exactly 0.03%
in all measurementslisted in Table 28. VI. In Blagoveshchenskij 's experiments in the
Pamir, for example, the [CO2] assays varied between 0.01 and 0.02%, and the highest
yields were obtained at the latter concentration (which is. still considerably below the
normal value of 0.03%). Stocker found, in the undergrowth of the tropical forest,
[CO2] values up to 0.04%. (Compare also data given in Chapter 27, page 902.)
To sum up, it is certain that plants growing in moderate climates can

reduce, in their natural habitats and under favorable conditions, 20 or 30
mg. CO2 per hr. per 100 cm.- of leaf surface; but it is much less certain
—
whether any plants desert and alpine plants not excluded are capable of —
yields up to 100 mg./hr. 100 cm.^, as is suggested by the measurements of
Kostychev, Monch and Blagoveshchenskij.
We will now discuss the relation between the maximum yield of photo-
synthesis of which leaves are capable under favorable natural conditions,
and the average production of organic matter by whole plants or large
plants assemblies.
Land plants in the open air, exposed to the sun, can be expected to main-
tain the above-estimated rate of photosynthesis (about 20 mg./hr. 100
cm.^) for a considerable part of the day (barring such phenomena as the
"midday rest" ;
cf. chapter 26). The intensity of illumination is sufficient,
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PHOTOSYNTHESIS RATE UNDER NATURAL CONDITIONS 999
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riTOTOSYNTTnESIR RATE FNDETl NATURAL CONDITIONS 1001
or almost sufficient, for light saturation during most of the day, unless the
sky is heavily overcast. On the other hand, the carbon dioxide supply,
under natural conditions, may often be less constant than it is in labora-
tory experiments with a circulating gas containing 0.03% carbon dioxide
and this may
cause considerable variations in the rate of photosynthesis.
In quiet open air, a carbon dioxide-deficient air layer will form around the
plants, and cause a decline in rate, while the exhalations of the ground,
which contain carbon dioxide produced by the decay of organic matter and
by the respiration of the roots, can be caught in the foliage, and can provide
an increased supply of carbon dioxide (c/. page 902). A certain amount of
carbon dioxide, of the same origin, also may reach the leaves with the trans-
piration flow from the roots page 910).
(c/.
Thus, variations in the supply of carbon dioxide, under natural condi-

tions, may be considerable, and these variations, more than alterations in
the intensity of illumination, may cause the rate of photosynthesis of
land plants to vary from location to location, and to fluctuate with time
in a given location. The same appears to be true of the multicellular
aquatic plants. As mentioned on page 878, Gessner (1937) found that the
dechne in rate of photosynthesis of higher aquatic plants with time, re-
ported, among others, by Arnold, was largely caused by insufficient circu-
lation of water. Although the carbonates contained in natural waters
(particularly hard waters) represent a considerable reserve of carbon di-
oxide, the diffusion of dissolved electrolytes is so slow that a carbon dioxide-
deficient alkaline layer can easily be formed around a submerged plant.
Gessner (1938) found {cf. Table 28.V) a rather low value (7.0 mg./hr. 100 cm.^)
for the maximum rate of carbon dioxide reduction by Potomageton crispus in hard tap
water; the value for Potomageton perfoliatus was even lower (4.8 mg. C02/hr. 100 cm.^).
These yields are less than one third of those of land leaves in ordinary air, and five to
ten times smaller than the maximum yields produced by land leaves provided with an
abundant supply of carbon dioxide. This indicates that Gessner's water plants might
have been in a "carbon dioxide-limited" state, despite the relatively high carbonate con-
tent of the medium. (Gessner attributed the low yield of aquatic plants per unit area
to the fact that these leaves consist of only a few layers of cells; however there is no in-
dication that the light absorption in the leaves of aquatics could be three or five times
smaller than in ordinary leaves.)
Unicellular algae, w4th their extremely favorable ratio of surface to

volume, are unlikely to feel any deficiency of carbon dioxide supply, at
least as long as the average carbonate concentration in the medium is high
and stirring sufficiently intense. Since these algae form a vast majority of
the organisms living in the sea, we can conclude that the total photosynthe-
sis of the marine flora — in contrast to that of the continental flora — is not
much affected by limitations of carbon dioxide supply.
PHOTOSYNTHESIS RATE UJJDER NATURAL CONDITIONS 1003
In addition to vagaries of carbon dioxide supply, other external factors,

such as variations in temperature and humidit}'-, and internal changes re-
sponsible for permanent "aging" and temporary "resting" of plants, also
affect the average rate of photosynthesis under natural conditions. And
lastly, variations in the brightness of the daylight, even though less im-
portant than one is at first inchned to believe, certainly affect the yield of
photosynthesis, particularly in the case of species adapted to strong hght.
We estimated in chapter 1 the average conversion yield of incident solar
energy as 1.5 to 6% (assuming 20 klux as the average intensity of illumina-
tion). We will now compare this estimate with the results of several inves-
tigations in which the determination of the yield of photosynthesis under
natural conditions was combined with the measurement (or estimation) of
the solar radiation that fell upon the plants during the same period. These
investigations can be divided into two groups experiments of short dura-
:
tion (several hours), and studies lasting several weeks or months. Table
28. VII contains the results of three short-time experiments. The figures
of Brown and Escombe were obtained in the same investigation that gave
the low absolute yields listed in Table 28. VI; the conversion yields were
correspondingly low (of the order of 1% of incident energy, or an estimated
2.5% of the visible radiation absorbed by the plants). The figures of Pure-
vich and Bose are considerably higher, and can be placed alongside the
higher absolute reduction rates found by Willstatter and StoU in 5% carbon
dioxide (Table 28. V), and by many recent investigators in ordinary air
(Table 28. VI).
Table 28.VII
Energy Conversion under Natural Conditions
Author
in ordinary air, and 30-40 mg. in air enriched with carbon dioxide, and not
the much higher yields found Monch.) We
by Blagoveshchenskij, or
and Bose
therefore suggest that in Table 28. VI, too, the results of Purevich
should be given preference over those of Brown and Escombe. However,
the whole problem is in need of renewed and more exact experimental
analysis, which alone could link the rate of photosj^nthesis under natural
conditions (strong illumination and limited carbon dioxide supply) to
the much better known rates in weak light and in the presence of an ample
amount carbon dioxide.
of
We nowturn to experiments of longer duration, in which the total yield
of photosynthesis of an assemblage of plants was compared, over an ex-
tended period of vegetation, with the integral of insolation over the same
period.
Noddack and Komor (1937) studied two plots of grass, one of 9 m.^
and another of 74 m.^ In two consecutive periods of 20 days each, they
measured the total solar radiation falling on these two plots, I dt; after S
this, the grass was mowed, dried and combusted, and the heat of combus-
tion, AHf, was measured. Here are the results: total irradiation in 20
days, 2.6 X 10^ cal./cm.^ (average irradiation 0.0015 cal./cm.^ sec, or ap-
proximately 6000 lux) ; -pro-portion of incident energij stored in the hay, first
plot, Mic/f I dt = 0.67% (first period) and 0.80% (second period), sec-
ond plot, 0.41% and 0.64%, respectively.

In these measurements, the growth of the root system was not taken into
consideration. This correction is difficult to estimate; but it should bring
the average value of AHc/Sl dt up to almost 1%, and e (= lÊJ fA dt)
close to 2.5%.
In comparing these results with those of the short-time experiments
listed in Table 28.VH, one has to consider that some obvious factors tend
to decrease the long-time average value of energy conversion by a large
assemblage of plants growing under natural conditions, compared with
that of a few isolated plants or leaves, averaged over a few hours of full
sunlight but that other less obvious factors may act in the opposite direc-
;
tion. Such favorable factors are the lower average light intensity (which
decreases S^
without reducing strongly the rate of photosynthesis),
<^f
and, possibly, partial retention of the respiratory gases in the dense foliage,
permitting reutilization of exhaled carbon dioxide. On the unfavorable
side we can anticipate that in a large assemblage of organisms a certain
proportion will not be in a healthy state, and others will be "resting";
sometimes the temperature will be too low for maximum photosynthetic
efficiency; it will bo so high as to cause inhibition (cf. chapter
sometime
31). leaves will be in the shade of others, at least part of the day.
Some
These unfavorable influences seem to predominate, judged by the fact
PHOTOSYNTHESIS BATE UNDER NATURAL CONDITIONS 1005
that the average yields of Noddack and Komor are two to three times
smaller than the most reliable short-time averages in Table 28. VII.
The measurements of Noddack and Komor represent the only available
parallel large-scalemeasurements of irradiation and production of organic
matter by plants. There is, however, no dearth of estimates, often on a
much larger scale, based on agricultural and meteorological statistics. They
have been mentioned in chapter 1, where we used them for the estimation
of the total yield or organic synthesis on earth. We will now consider
these estimates somewhat closer. Putter (1914) took the insolation data
from observations of the l^rightness of daylight carried out by Weber in
Kiel, Germany, over a period of several years, and used the relation 1 lux
= 6.3 erg/cm. sec. to calculate the corresponding energy flux.
=^ He cal-
culated, for the total irradiation over a year, 35.3 kcal./cm.^, corresponding
to an average illumination intensity of about 7000 lux.
For the calculation of the heat of combustion of the synthesized organic
matter Putter used estimates of "exceptionally high" crops from agricul-
tural yearbooks; he then added the (estimated) heat of combustion of the
roots and stubbles, and subtracted the heat of combustion of the seed.
Thus he obtained values for AHc] for the calculation of S
Idt, he inte-
grated Weber's data over the periods of vegetation of the several crops, and
subtracted the energy of infrared radiations (above 1 m) (It would be more
•
reasonable to subtract all radiations above 0.7 n, since light between 700
and 1000 niM probably is not used for photosynthesis at all, cf. chapter 30.)
Table 28.VHI shows some of Putter's results. In the last column, the
conversion yields are corrected for losses by respiration, estimated at 15%
of the weight increase due to photosynthesis during the vegetation period.
Table 28.VIII
Energy Conversion by Field Plants (after Pi tter 1914)
—
experimental yields of Noddack and Komor, and about equal to the highest
short-time averages of Purevich. Putter attributed these comparatively
high values to conditions that favor large-scale field experiments (particu-
larly to thecarbon dioxide supply from the ground) However, it seems more
.
likely that his conversion yields were overestimated. An error by a factor

of about 1.5 could have been caused by the use of too low a factor for the
conversion of lux into energy units (in chapter 25 we stated that, in sun-
light,one lux corresponds to about 10 erg/cm.^ sec, while Putter used a

factor of 6.3). Another error in the same direction may have been intro-
duced by the comparison of exceptional crops with average insolation data.
Spoehr (1926), who made calculations similar to those of Putter, but
took into consideration only the grain in the field crops and the utilizable
timber in the forests, obtained much lower values of energy conversion
c. g., 0.13% of total incident radiation for a wheat field, and 0.35% for a
forest of fast-growing eucalyptus trees. Similar figures were obtained, for
forest trees, by Boysen-Jensen (1932). These calculations were intended
to estimate the practical efficiency of plants as converters of solar energy
so that stalks, husks and roots of the wheat plants, and leaves and roots
of the trees were neglected altogether; but the consideration of these
terms could scarcely more than double the calculated conversion yields —
which would thus become comparable to the results of Noddack and Ko-
mor, but could never approach the much higher figures of Putter. To sum
up, it seems that 1% of total incident solar energy, and about 2.5% of ab-
sorbed visible radiation, represent a fair estimate of the average utilization
of light energy by field crops and forests, during the summer vegetation
period, under moderate climatic conditions. The average quantum yield
of photosynthesis under these conditions is of the order of 0.01 (1 molecule
COo reduced per 100 visible quanta absorbed).
Analysis of the data on plankton production in the sea (of. Table l.II)
led Riley (1941) to the conclusion that the average utiUzation of light energy
falling on the surface of the sea is between 0.6 and 0.8%,* i. e., similar to
average utilization of light by fields and forests. However, in the ocean,
vegetation develops more or less uniformly throughout the year; and ex-
cept for the part of the Arctic seas covered by ice, there are no large barren
regions in the ocean comparable to the deserts or glaciers on the surface of
the earth. These differences weigh heavily in favor of the oceans as the
main producers of organic matter on earth (of. Table l.III).
In chapter 1 (page 9) we made one more step and calculated the
total production of organic matter on earth by assuming that the average yield
of conversion of the energy of visible radiations absorbed by the plants is
2% (corresponding to 0.8% of the total incident light energy). Now,
* Lanskaja and Sivkov (1950) gave much higher figures, 3-14%.
INTERPRETATION OP LIGHT CURVES 1007
after having analyzed the foundations of this estimate, we feel certain that
its order of magnitude, at least, is secure —even though the figure given may
be in error bj^ as much as a factor of two.
7. Interpretation of Light Curves
(a) Influence of Inhomogeneity of Light Absorption
The nonimiform illumination and supply of reactants in photosynthesis

were discussed in general terms in chapter 26 (section 2). In chapter 27,
while dealing with the carbon dioxide curves, we noted that these curves
can be strongly affected by concentration gradients, which arise, particu-
larly during intense photosynthesis, between the external medium and the
immediate neighborhood Similarly, the light curves
of the chloroplasts.
may present a strongly distorted picture of the intrinsic relationship be-
tween the rate of light absorption and the yield of photosynthesis, because
a considerable gradient of light intensity often must exist between the light-
exposed and the shaded chlorophyll molecules. Even within a single
chloroplast the rate of light absorption may decrease by a factor of five or
ten from the light-exposed to the shaded side ; or, in the case of diffuse il-
lumination, from the surface to the center of the plastid. In suspensions

containing millions of cells, as well as in leaves or thalli, the heterogeneous
nature of light absorption is further enhanced by the mutual shading of
the numerous chloroplasts (of. fig. 26.5). Consequently, the light curves
of different specimens of one organism, even if they all have the same con-
tent of all the relevant pigments and catalysts and are investigated under
the same external conditions, maj^ nevertheless differ in shape, depending
on optical density {i. e., the number of cells per square centimeter in an
algal suspension, or the thickness of a leaf or thallus). The assumption of
equal content of catalysts may be incorrect; for example, the cells
itself
of the spongy parenchyma may be adapted to weaker light (and thus con-
tain less of certain catalysts) than the leaves of the palisade tissue.
Let us consider, as the simplest example, two suspensions of identical
cells —one optically thin (e. g., transmitting 80% of incident light), the other
optically dense (e. g., absorbing 80% of incident light). There is no reason
(aside from the phenomena of "self-inhibition" by metabolic products
mentioned in chapter 25) why these suspensions should differ in the maxi-
mum quantum yield at low light intensities, or in the maximum yield per
chlorophyll molecule in strong light. However, the transition from the
linearly ascending part to the horizontal part of the light curves wll be
sharper in the optically thin system (where saturation occurs more or less
simultaneously in all cells), and more gradual in the optically dense system,
where saturation sets in at the surface of the vessel and spreads inward with
increasing intensity of iHumination.
Figure 28.20 shows, in a quaUtative way, the expected differences in
the plots of the absolute rate of photos>Tithesis, P, and the relative satura-
tion, p/p™'^^-^ against the incident energy flux, I, and the absorbed energy,
la.
If the rate of photosynthesis, P, is plotted against the incident light
intensity, /, as independent variable (curves 1), the initial slopes of the
R Dense
'Thin
Fig. 28.20. Effect, of optical density of a cell suspension on shape of light curves.
Heavy and thin double arrows represent the "linear range" of dense and thin suspen-
sion, respectivel}'; y is the angle that determines the maximum quantum yield.
curves vary in proportion to optical density, but the extension of the linear
range must be practically independent of optical density (since the curve of
the dense suspension must bend as soon as saturation begins in the surface
layer). But if P
plotted against the absorbed energy, la (curves 2),
is
the initial slopes must be the same, but the linear range of the thin suspen-
sion must be shorter than that of the dense one. On the other hand, if we
plot the relative saturation, p/p"^^^-^ against either/ or/g, (curves 3 and 4),
the curves of the thin suspension will remain linear much closer to full
saturation.
As an illustration, figure 28.21 shows the light saturation curves,
INHOMOGENEITY OP LIGHT ABSORPTION 1009
p/pmax. ^ j^js^^
q£ ^^^.q chlorella suspensions of different density {cf. Table
25.1) given b}^ Their relationship is in agreement with
Eichhoff (1939).
the prototype of figure 28.20 (curve 5). Katz, Wassink and Dorrestein
(1942) attempted to reduce analytically the Ught curves obtained with
three suspensions of bacteria (Chromatium, D) of different concentration
to a single curve showing the average yield per cell as function of average
iUumiiiation. In a 2 cm. deep absorption vessel, the "dense" suspension,
-
0.8
Q.
2 4 6
LIGHT INTENSITY
Fig. 28.21. Light curves of a thin and a dense Chlorella

suspension, in red hght, X = 6500 A (after Eichhoff 1939).
Intensity in "energetic meter candles" (HK) (page 1098).
with a concentration of 30 "Trommsdorff units"/ml. (concentration 3)

absorbed about 80% of incident light of a sodium lamp, the "medium"
suspension (concentration 1; 10 Trommsdorff units), about 60%, and the
"thin" suspension (concentration \; 2>\ units), about 30%. Figure 28.22A
shows the empirical light curves of these three suspensions, P = f{I)- They
have the relative positions anticipated in figure 28.20 {!) (except for the
sigmoid initial shape, which is characteristic of the Hght curves of purple
bacteria).
Near 7 = 0, the order of the three curves is reversed. The probable reason for this
is that, at a given incident light intensity, the average illumination is lowest in the densest
suspension; hydrogen consumption (which we think, is re-
therefore, the deficiency of
sponsible for the sigmoid shape) maintained, in the dense suspension, up to higher in-
is
tensities than in the dilute one, and tliis influence apparently overcompensates that of
stronger absorption.
THE LIGHT FACTOR. I. INTENSITY CHAP. 28
1010
yield
Figure 28.22B shows the same three curves, replotted to represent
per single cell. The densest suspension now shows the lowest yield.
However, all three curves appear to approach (as expected) the same limit-
ing yield athigh light intensity; thus, except for the sigmoid shape, they
1100
cone 3
cone I
1000 cone '/3
cone control, run

1
together with cone 3

cone control, run
I
900 together with cone '/3
800
:e 700
o 600
o
u. 500
<
a.
400
3
300
200
INCIDENT INTENSITY, erq/cm sec.
Effect of cell concentration on light curves of photosj-athesis

(Katz,
Fig. 28.22.
Wassink and Dorrestein 1942).
are of the type 3 in figure 28.20. (The latter refers to P/P"''-^; but, since
pmax. must be proportional
jg proportional to the number of cells, P/P"^^^-
to the yield per cell.) Figure 28.22C, finally, shows the yields per cell as
function of average intensity of illumination throughout the vessel (in A
and B, the abscissae were the incident light intensities, as measured at the
INHOMOGENEITY OF LIGHT ABSORPTION 1011
front wall of the vessel). The conversion is made by reducing the abscissae
in the ratio a/c, where c is the concentration and a the per cent absorption,
by 0.8/3 = 0.27 for the dense suspension, 0.6/1 = 0.6 for the medium
{i. e.,
suspension, and 0.3/0.3 = 1 for the dilute one). This treatment causes
the curves for c = 0.3 and c == 1 to coincide almost exactly; but the last
point on the c = 3 curve still shows considerable deviation.
There are two obvious reasons why one cannot expect the reduction method used to
be completely successful. In the first place, the averaging cannot be quite correct, be-
cause the cells are not actually exposed to the "average" light intensity, but some are
illuminated with stronger, and some with weaker light. This would not matter if the
yield were proportional to intensity; but, if the yield declines with increasing intensity
(as it does in the saturation region), the yield that corresponds to a given average intens-
ity will be lower whea the spread of actual intensities is wider, i. e., in the more concen-
trated suspension.
A second complication arises from the stirring of the reaction vessel, which causes
the cells to come successively into light of different intensity. The effect of this varia-
tion is complex; it belongs to the group of phenomena (induction; photosynthesis in
alternating light) which will be treated in chapters 33 and 34. Only if the illumination
cycles are much shorter than the periods required for the completion of all dark processes
of photosynthesis can one expect the cells to work, in alternating light, with the same
efficiency as in steady light with the same average intensity. The known periods of
dark reactions, associated with photosynthesis, include at least one with a period as short
as T = 0.01 sec. at room temperature; stirring is not usually rapid enough to send each
cell through the whole cycle of intensities within 0.01 sec. (c/. chapter 29, page 1106).
Consequently, the cells in the stirred vessel are illuminated with an alternating light the
average frequency of which is smaller than 1/r. While the frequency of intensity
variations is identical for all three suspensions, their amplitude is the larger the denser
the suspension. Because of induction phenomena, the highest yield at a given average
illumination is obtained in continuous light (cf. fig. 34.5); consequently, the efficiency
losses caused by intermittency will be highest in the densest suspension. We have
thus found two reasons, each of which may explain the deviation from the average of the
last point in the c = 3 curve in figure 28.22C.
It may
be useful to note that the changes in the illumination of individ-
ual cells, caused by stirring, may be discontinuous. The absorption by a
single chloroplast {i. e., in the case of Chlorella, a single cell) is so strong
that the only significant intensities of illumination to which a cell is ex-
posed may be those with no cells or with a very small number of cells (1 or
2) between itand the light source. The scattering of light in the suspen-
sions tends to smooth over these discontinuities.
With respect to the inhomogeneity of light absorption, two cell suspen-
sions with the same number of cells per square centimeter, but with a dif-
ferent concentration of chlorophyll within each cell, offer a case similar to

that of two suspensions of identical cells, but different dilution. Whether
the light curves of such two specimens a picture similar to that
will present
sho-wn in figure 28.20, depends on their content of the catalyst that limit
the rate in strong light. In the case for which figure 28.20 was drawn, the
catalyst content could be assumed to be proportional to the content of
chlorophyll (in other words, the "assimilation numbers" could be taken as
identical), since the two suspensions differed only in quantity and not in
quality of the cells. When, however, changes in optical density are brought
about by âriations in the pigment concentration ivifhin the cells, the "as-
similation number" does not necessarily remain constant. We will deal
with these relationships in chapter 32; of the three cases discussed there
(umbrophilic and heliophilic plants, 28.16-28.18; green and aurea
cf. figs.
varieties, cf. and normal and chlorotic plants, cf. fig. 32.4),
fig. 32.2;
only the last one is characterized by approximate constancy of the as-
similation numbers, and thus leads to light curves such as those in figure
28.20. In other words, in this case only does the intracellular content of
the rate-limiting catalyst change proportionally to the content of chloro-
phyll.
(b) General Shape of Light Curves
We now leave the effects of inhomogeneity of light absorption and in-

quire into the intrinsic shape of the light curves. All kinetic interpreta-
segment of the light curves corre-
tions agree that the initial, almost linear
sponds to a state in which the primary photochemical process is so slow
that the catalysts which participate in the nonphotochemical steps can
suppl}'^ the substrates needed for, and transform the intermediates formed
by, the primary process, without depletion of the former or accumulation

of the latter. Only the light curves of purple bacteria generally show a
sigmoid-shaped initial part; the probable reason for this was discussed on
page 948.
In the linear section, the quantum yield of photosynthesis has its high-
est value along a given light curve. (In sigmoid hght curves, the highest
quantum jdeld is in the point where the tangent to the light curve passes
through the origin of the coordinates.)
This yield may correspond to actual utilization of all absorbed light quanta for
photosynthesis, or to a certain, not further reducible proportion of quanta wasted by
complete or partial inactivity of a certain number of cells, or of a certain fraction of
chlorophyll. A similar irreducible loss of energy can be caused by back reactions, if the
proportion of photochemical products that they destroy is independent of the rate of
formation of these intermediates (cf. page 1037, and chapter 29, page 1137).
The light curves bend toward the hoi-izontal when the rate of the pri-
mary photochemical reaction ceases to be slow compared with the maxi-
mum possible rate of one or several of the nonphotochemical processes as-
sociated with photosynthesis. As demonstrated in chapter 26, the limit-
GENERAL SHAPE OF LIGHT CURVES 1013
ing influence of a bottleneck reaction in a "catenary series" generally be-

comes felt long before the rate of the over-all process has reached the maxi-
mum speed of which this hmiting reaction is capable. Consequently, the

light curves must approach saturation asymptotically rather than sud-
denly "hit the ceihng" (even if we forget for the time being about the ef-
fects of inhomogeneity which further enhance the grad-
of light absorption,
ual character of saturation). For the same reason, the maximum rate
reached in the light-saturated state will often be considerably lower than
the "ceihng" imposed by the limiting process.
As to the nature of the processes that can cause hght saturation, the
general alternative is between "preparatory" and "finishing" reactions.
These two types of dark processes have been fu^st discussed by Warburg,
and Willstatter and Stoll, respectively. Because all transformations that
occur in photosynthesis must be cychc as far as chlorophyll and other cata-
lysts are concerned, the questionwhether a reaction takes place "before"
or "after" the primary photoprocess is not always as easy to answer as one
would at first imagine. We will assume that a dark reaction precedes the
photochemical step, retardation prevents the occurrence of this step
if its
(and thus also all the succeeding ones), and that a dark reaction /o^/oius the
primary photochemical process, if the latter takes place in any case, and the
effect of the limited rate of the dark reaction is merely to cause an accumula-
tion of the primary photoproducts. Since experience shows that no large
accumulation of oxidation intermediates occurs in photosynthesis (this is
evidenced by the abrupt stoppage of oxygen production after the cessation
of illumination), we must assume that the primary oxidation products
("photoperoxides") are unstable; unless rapidly removed or chemically
stabilized by a "finishing" process, they apparently disappear by back reac-
tions.
The uptake of carbon dioxide may sometimes continue for about 20 sec.
in the dark {cf. Vol. I, page 200, and Vol. II, chapter 36). This may
mean that some intermediate reduction products survive for that length
of time, or that the carbon dioxide acceptor, A, requires it to become re-
carboxylated. (It may also be that the COa-acceptor is itself a reduction

intermediate of carbon dioxide cf. chapter 36.)
The two mechanisms of light saturation can thus be de-
alternative
scribed as starvation, which causes an "idling" of the primary photochemical
mechanism, and constipation, which blocks the elimination of the primary
products and compels most of them to return to their initial form.
As described before, the distinction between the effects of prepara-
tory and finisliing dark reactions becomes still more diffi(.*ult, if we follow
Franck in theassumption that one of the finishing reactions "backfires," so
that its slowness, like that of the preparatory reactions, affects the composi-
tion of the photosensitive complex. According to Franck, this is the re-

action that converts the intermediate oxidation products, formed by hght,
into free oxygen (or sulfur, or other final oxidation products formed in
bacterial photosynthesis). When this reaction fails to keep pace with the
primary photochemical process, the intermediate oxidation products
("photoperoxides") accumulate in amounts sufficient to oxidize certain
metabolites, thus forming a product of narcotizing properties (perhaps an
organic acid). The latter is adsorbed on the photosensitive complex, and
this retards or stops altogether the primary process.
Each partial nonphotochemical process of limited maximum rate im-
poses its o^^^l "ceiling" on the over-all rate of photosynthesis; and, since
the influence of such a ceiling is felt long before it has actually been reached,
the saturation value of photosynthesis in strong light may be affected not
by one limiting process, but by several such processes—particularly since
the maximum capacities of different parts of the photosynthetic apparatus
appear to be of the same order of magnitude (as one would expect of a well-
adjusted catalytic system).
In the general discussion of the kinetic curves of photosynthesis in
chapter 26, three types of curve sets, P = f{Fi) with Fg as parameter, were
described and designated as the first (or "Blackman") type, the second (or
"Bose") type and the third type, respectively (see figures 26.2, 26.3 and
26.4) We recall that curves of the first type must arise when the parameter
.
Fz determines the maximum rate of a partial process that does not depend
on the independent variable, Fi. This process then imposes a horizontal
ceiling on the curve P = f{Fi), but does not affect its initial slope. In curve
sets of the third type the parameter affects the initial slope of the light
curve, but not its saturation level; this type results when F2 codetermines
the rate of a process that is also a function of the independent variable, Fi.
In curve systems of the second type, the parameter F2 affects both the initial
slope and the saturation level. Carbon dioxide curves offered examples of
allthree types, depending on the nature of the parameter {cf. page 868).
Since most parameters do not affect the rate of the primary photochemical
process, and therefore do not change the initial slope of the light curves,
the P =/(/) curve systems usually are of the first type, i.
e., the various
curves coincide at low light intensities, but diverge at saturation. Such

are most of the curve systems observed with carbon dioxide concentration
as parameter (figs. 28.1, 28.2, 28.4, 28.5 and 28.5A), the only exception
being Harder's Fontinalis curves (fig. 28.3). The two light curves of
Chromatium with thiosuJfate concentration as parameter (fig. 28.5B) have
the same general appearance, and the curve systems with temperature as
parametei-, illustrated by figures 28.6, 28.7 and 28.8, are of the same type.
The efiect of inhibitors, however, is uneven and some results are contra-
GENERAL SHAPE OF LIGHT CURVES 1015
dictory. It has been suggested, as a generalization of empirical results,

that catalyst poisons affect only the saturation level, thus producing light
curve systems of the first type, while narcotics depress also the initial slope,
thus giving curve systems of the second type. However, not all experi-
mental results conform to this rule.
In figures 28.9 A,B, the curves of Chlorella in the presence of cyanide are,
as expected, of the Blackman type; but the hydroxylamine curves (as ob-
served by Weller and Franck) are of the Bose type. With the diatom
Nitzschia (fig. 28.10) the effect of cyanide was different: A distinct depres-
sion was observedat all light intensities between 2 and 30 kerg/cm.^ sec.
(However, the per cent inhibition increased with increasing light intensity,
for example, 0.003% KCN caused an inhibition by 35% at 2.4 kerg, by
45% at 13 kerg and by 57% at 27 kerg.) Other observations of cyanide
inhibition of photosynthesis in weak light were quoted in Volume I (page
309). The light curves of cyanide-inhibited purple bacteria, as observed
by Wassink and co-workers, show a similar "semi-Bose" behavior; re-
versing the results obtained by Weller and Franck ^^^th Chlorella, the bac-
teria exhibited a Blackman type behavior toward hydroxylamine!
The light curves of urethan-inhibited Chlorella (as given by Warbiu'g,
and by Wassink and co-workers, respectively) appear to be of the second
type, according to rule; but the third type is not entirely excluded. Bose
type curves were found also with urethan-inhibited Nitzschia (fig. 28.10),
although in this case the per cent inhibition was not constant, but rose with
increasing light intensity. Wassink's curves, showing the effect of urethan
—
on purple bacteria (fig. 28. HE), exhibit the reverse change the depression
is more pronounced in wêak light than in strong light.
The effect of oxygen on the photosynthesis of Chlorella (fig. 28. 9E;

cj. also fig. 33, page 329, Vol. I) is of the Bose type, and the same is true of
the effect of copper sulfate, while that of nickel sulfate apparently is of the
Blackman type (fig. 28.9D). The curves of purple bacteria with pH as
parameter (fig. 28.12) are of the Bose type with thiosulfate, and of the
Blackman type with hydrogen as reductant.
Theoretically, one can easily understand why specific catalyst poisons
such as cyanide, should affect only the saturation level and not the initial
slope of the light curves: The former is determined by the rate of a dark
catalytic reaction, the latter by the rate of supply of light quanta. With
the poisoning becoming more and more complete, the inhibition can be
expected to spread to lower and lower light intensities. If one assumes (as
suggested in Vol. I, page 307) that cyanide inhibits most strongly the
carboxj'lating enzyme, Ea, the observed differences in the sensitivity of dif-
ferent species may be attributed to variations in the amount of this en-
zyme. Some plants may contain a considerable reserve of Ea, and there-
fore show poisoning effects only in strong light; in others, the concentra-
tion Ea may
be just sufficient to maintain photosynthesis in the absence of
cyanide and very little inactivation is needed to cause marked retardation
even in moderate or weak light. However, even in this case, the per cent
reduction of photosynthesis should remain smaller in weak light than in
light of saturating intensity (as was, in fact, observed \vith cyanide-poisoned
Nitzschia, cf. above). This hypothesis cannot explain an apparently uni-
form per cent reduction of photosynthesis at all light intensities by a typical
catalyst poison such as hydroxylamine (fig. 28. 9B). An ad hoc interpreta-
tion, suggested by Franck and co-workers, was described in Volume I (page
312).
What was said about the effects of catalytic poisons should apply also
to deficiencies of the reaction substrates (carbon dioxide in green plants,
carbon dioxide and reductants in bacteria). Their effects, too, should
gradually diminish, and finally disappear with decreasing light intensity.
The on the initial slope of light curves can be under-
effect of narcotics
stood one assumes that they are adsorbed on chlorophyll (or "chloro-
if
plastin") in such a way as to prevent the access of reactants or catalysts.

Consequently, in the partially poisoned state, only the chlorophyll mole-
cules free of these adsorbents are capable of properly utilizing the ab-
sorbed light quanta. If it is true that light saturation is due to the limited
amount of a catalyst, such as Ea or Eb, which is kinetically independent
of chlorophyll, the fact that the light curves obtained in the presence of
urethan appear to be of type 2 rather than 3 (z. e., that narcotics affect also
the saturation rate) requires special explanation. For example, it can be
postulated that the narcotic becomes adsorbed on the molecules of Ea or
Eb as well as on those of chlorophyll. Alternatively, it can be suggested
that, if the light curves of narcotized plants were followed to still higher
light intensities, approach the same saturation level as
they wôuld finally
the light curves of normal plants (i. e., they would actually prove to be of
type 3 rather than 2).
A third explanation, based on the idea that definite catalyst molecules
are "assigned" to definite chlorophyll molecules, and become useless when
the latter are "narcotized," will be discussed in chapter 32.
another phenomenon needs to be taken into consideration. It
Still
will be shown later in this chapter that a saturation level of photosynthe-

sis probably exists which is due to the distribution of the chlorophyll com-
plex, during photosynthesis, between the normal photosensitive form and
a changed (tautomeric, or reduced) form. The latter is formed as an inter-
mediate in photosynthesis, and requires a certain time for reconversion to
the original photosensitive form. While this saturation limit may not be
generally apparent in non-narcotized plants, because another limit (im-
: :
EFFECT OF PREPARATORY REACTIONS ON LIGHT CURVES 1017
posed by the deficiency of a finishing catalyst, Eb) is lower, the two may
be not too far apart. Therefore, in the narcotized state, when a large
fraction of the chlorophyll complexes is blanketed by the narcotic and
therefore inactive, the saturation level due to chlorophyll can become lower
than that due to the catalyst Eb- This will cause photosynthesis to be in-
hibited by narcotics even in strong light however, the per cent inhibition
;
will be smaller than in weak light. (This prediction is in agreement with

Wassink's findings on purple bacteria, but not mth his observations on
diatoms.)
(c) Analytical Formulation: Effect of Preparatory Dark Reactions
In chapter 27, a rather extensive effort was made to derive equations

for the function P = /[CO2] under different assumptions concerning the
preparatory dark reactions on the "reduction side" of the primary photo-
chemical process. The influence of light intensity was expressed in these
derivations{cf. equation 27.6) by assuming that the rate is proportional to
the concentration of the reduction substrate, [ACO2], and that the pro-
portionality factor, k*, is a function of the light intensity, /. The resulting
equations for P
were then applied to the analysis of the carbon dioxide
curves, by assuming constant values of the parameter / {i. e., of fc^)- The
same equations can, however, equally well be considered as analytical ex-
pressions of the light curves, P = f{I), with [CO2] as parameter. A speci-
fic assumption must be made in this case concerning the relation of k* to
I (e. g., by postulating that /:* is proportional to /, k* = k*I, cf. eq. 28.13).
The simplest equation for P in chapter 27 was equation (27.8). It was
based on the assumption of a dissociable carbon dioxide-acceptor complex
with no limitations on the rate of its formation. The corresponding light
cui'ves are linear (at least, if k* = k*I), and show no saturation effects.
(This is, due to the fact that, in the derivation of equation 27.8,
of course,
the equilibrium concentration of the compound [ACO2] was supposed to
be undisturbed even by intense photosynthesis.) The equation of these
straight lines is (using k* as independent variable)
(28.1) P = {nk*K,P^,{CO^])/{l + K.[CO,])
Their slope is proportional to [CO2] at low carbon dioxide concentrations

and approaches a maximum at high carbon dioxide concentrations
(28.2) (rfPM-*)max. = nAo
As soon as the assumption is made tliat the stationary concentration

[ACO2] is affected by the rate of consumption of this complex by photo-
synthesis {i. e., that the formation of ACO2 is not infinitely fast), the light
:
curves cease to be straight lines and become hyperbolae. We can refer

here to equation (27.16), which describes the combined effects of slow dif-
fusion and slow carboxylation, or to equations (27.21) and (27.31), which
express these two effects separately. In intense light, the hyperbolae
p = f(k*) approach one of the following two saturation levels, either:
(28.3) P"^""- = nkdlCOi]
if the rate of diffusion is the limiting factor; or:
(28.4) -?•"" = nk^ Ao[C02]
if the rate of carboxylation is limiting. It will be noted that both satura-

tion values are proportional to [CO2]; in other words, this approximation
provides for no "absolute saturation" with respect to both / and [CO2].
Half-saturation is reached, in the case of limitation by diffusion, at:
(28.5) r/k*r = {K,k,[C02] + 2 ki)/2 KM

and, in the case of limitation by carboxylation, at
(28.6) V/"* = ^^'

+ ^-'[COj]
In both cases, half-saturating light intensity increases linearly with carbon

dioxide concentration. The initial slopes of the light curves are the same
as those of the straight lines (28.1). This means that at low carbon di-
oxide concentrations, the light curve families are of the Bose type, the
Blackman type being approached at the higher values of the parameter
[CO2].
"Absolute" saturation follows, as usual, as soon as we postulate a rate-
limiting step, the maximum velocity of which is independent of both [CO2]
and but is determined entirely by the available amount of
light intensity,
a catalyst. Equations (27.40) or (27.51), derived for the case of limitation
by carboxylase deficiency, contain in their denominators terms proportional
to the product, k* X [CO2]; when light intensity and carbon dioxide con-
centration are both very high, this term becomes predominant and the
yield approaches the "absolute" saturation value:
(28.7) P'S,H: = nA-eaEoAo
which is the maximum rate of the catalytic formation of ACO2 by reaction
(27.37).
For the reaction mechanism discussed in section d of chapter 27 (non-
dissociable ACO2 complex attached to chlorophyll for the duration of the
eight photochemical steps, as in the Franck-Herzfeld theory), with the
simplification (28.8) used in deriving equation (27.03), we have:
(28.8) [EaIÊ:
EFFECT OF PREPARATORY REACTIONS ON LIGHT CURVES 1010
(28.9) P™-- = nA-aA'E!Ao[C02]/(l + A'ICOa])
(28.10) ,//-* = (Sk.KEnCO-,])/{l + K[C02])
(28.11) PZl: = nkJRlAo
(28.12) idP/dk*)o = nAo/8
with n probably equal to 1.

So far, we have considered the shape of light curves as determined ex-
clusively by preparatory reactions on the "carbon dioxide end" of photo-
synthesis (the possible rate-limiting factors being the constant of carbon
dioxide diffusion, the bimolecular rate constant of carboxylation and the
available concentration of the enzyme Ea). Analogous derivations can be
made for limiting influences on the "oxidation end," such as, the rate con-
stant of diffusion of reductants, the rate constant of their preliminary trans-
formations (e. g., of the binding of hydrogen to an acceptor) and the de-
ficiency of enzymes catalyzing these reactions (e. g., the hydrogenase).
In making these derivations, we could, for example, set the rate of photo-
synthesis proportional to the concentration of the primary oxidation sub-
strate such as the hypothetical "bound water," A'H20 or, more generally,
A'HR (instead of to the concentration of the primary reduction substrate,
ACO2, as we have done so far). However, we abstain from a detailed dis-
cussion of these possibilities, because, in the case of green plants, there is no

positive proof that a dark hydration reaction actually is needed to make
water available for the photochemical process. The abundance of water in
cells may make this hydration, even if it were needed, practically instan-
taneous. In the photosynthesis of purple bacteria preliminary transforma-

,
tions of reductants are known to occur, but no definite proof has as yet been
given that these transformations must be considered as preparatory reac-
tions {i.e., reactions providing the oxidation substrate for photochemical
process) rather than as finishing reactions removing the primary oxidation
products, formed by the photochemical oxidation of water. (The second
alternative favored by van Niel, Gaffron and Franck; cf. Vol. I, p. 168.)
is
It must, nevertheless, be borne in mind that the rather detailed considera-

tion of the preparatory processes "on the reduction side," and the compara-
tive neglect of the analogous processes "on the oxidation side" of the pri-
mary photochemical process, which is common
to most discussions of the
kinetics of photosynthesis, are not justified, being based only on our in-
ability to study the fate of water before its oxidation in photosynthesis, and
our present insufficient knowledge of the initial transformations of hydro-
gen and other reductants used by bacteria.
:
1020 THE LIGHT FACTOR. I. INTENSITY CHAP. 2S
(d) A nalytical Formulation : Effect of Processes in the Photosensitive Complex
Leaving aside the preparatory reactions "on the

effects of hypothetical
oxidation end," we return to equation (27.6), P = nA'*[AC02], for closer

consideration from the point of view of the Ukely mechanism of light par-
ticipation in photosynthesis. As mentioned before, the assumption, more
or less implicit in the derivations of chapter 27, was that k* is proportional
to I, the intensity of incident light
(28.13) K = ^*I
and consequently:
(28.14) P = nl:*l.\C(),] = 'nk*ri\C(h]
One remark, limiting the practical applicability of the analytical ex-

pressions derived in this section, must be made immediately. Kinetic
equations are based on the law of mass action they presume homogeneity
;
of the reacting system. The light intensity, /, is, however, not uniform
throughout a leaf or cell suspension; it varies even within a single cell or a
single chloroplast. This complication has been repeatedly mentioned be-
fore, and we it again on page 1044.
shall return to In the meantime, we
will proceed as if light absorption were uniform throughout the region
under consideration. This means that our equations will be strictly valid
only for optically thin layers. In the following equations, then, I must be
taken as meaning the light flux actually reaching a chlorophyll layer, and
not the light flux falling on the outer surface of the system. (These two
fluxes are proportional to each other, but the proportionality factor varies
with depth, as well as with the wave length of the incident light.) Practi-
cally, most if not all kinetic measurements have been made, not with opti-
cally thin pigment layers but with leaves, thalli or suspensions absorbing a
large proportion (sometimes up to 100%) of incident light. We will con-
sider on page 1044 to what extent kinetic relationships derived for optically
thin layers are changed through inteji,iation over the path of the light in the
system (and also over the differently absorbed components of non-mono-
chromatic light). The treatment of this problem is further complicated
by the structural effects discussed in chapter 22 (scattering and "sieve ef-
fect"). Still another complication arises in the treatment of cell suspen-
sions rapidly agitated during the measurement, thus bringing the indi-
vidual cells more or less periodically into light fields of different intensity.
were so intense as to cause each cell to slip through all the various
If stirring
time which is short compared to the "Emerson- Arnold
light fields in a
period" (about 10 -^ sec, at room temperature, cf. chapter 34), it would
PROCESSES IN THE PHOTOSENSITIVE COMPLEX 1021
have been permissible to take into account only the average ilhimination
and to consider the latter as identical for all cells. In other words, the rate
of absorption of light by each cell could be taken as equal to the rate of
total absorption in the suspension divided by the number of the cells in it.
No amount however, can mix the contents of the chloroplasts,

of stirring,
so that chlorophyll molecules situated deeper inside them always will re-
ceive less light than those situated on the illuminated surface. What is
even more important, the rate of stirring usually is quite insufficient to
make legitimate even the averaging of intensity for whole cells. Such
fast stirring is difficult to achieve; it is unlikely that Warburg and Burk
liad the right to claim that in their experiments 1006) stirring was
(c/. p.
so effective that only the average intensity of illumination was important.

Often, a danger exists that the periods spent b\' individual cells deep in the
suspension, lêtween two exposures to full light in the surface layer, could
1)6 long enough to cause induction losses during the subsequent expo-
sure (c/. chapters 29, 33 and 34).
These quahtative considerations show that the way to obtain light

curves of photosynthesis best suitable for kinetic interpretation is by using
optically thin suspensions or tissues. A limit to this procedure is, how-
ever, set by the fact that even single chloroplasts may absorb up to 50%
of incident light in the absorption peaks of chlorophyll (cf. fig. 22.35);
so that diluting algal suspensions until they al)8orb much less than that
amount green tissues such as green onion skins) may
(or using faintly
merely mean allowing a part of incident light to pass between the chloro-
plasts— without improving the uniformity of absorption within the plas-
tids. This uniformity can only be improved by employing cells poor in
pigment (chlorotic cells), or by employing light which is comparatively
weekly absorbed by chlorophyll (e. g., green light).
We are thus forewarned that the several equations of the light curves,
which will be derived below from alternative kinetic models of photosyn-
thesis, can be used for comparison with the experimental curves found in
the literature, only with strong reservations. We nevertheless consider it
worth while to continue with these derivations, as a step toward a more
quantitative study of the problem in the future. The latter will require
both improved theoretical treatment (including the effects of inhomogene-
ous structure and nonuniform light absorption), and, above all, precise
kinetic experiments on optically thin objects.
In analyzing the validity of equations (28.13) and (28.14), two alterna-
tive pictures must be considered. According to one, favored by Franck
and Herzfeld, the compound ACO2 is part of the "photosensitive complex"
proper and its reduction can therefore be considered the primary photo-
chemical process, e. g.;
:
(28.15) ACOs-Chl-A'HoO ,
" ^ AHCOz-Chl-A'HO
In considering this alternative, it is not necessary also to adopt Franck
and Herzfeld's complex mechanism, which involves eight consecutive
photochemical steps; essentially the same conclusions can be reached also
by considering a single photochemical step, such as reaction (28.15),
and leaving the completion of the process to nonphotochemical reactions,
such as dismutations and coupled oxido-reductions, as described in chapter
9, Volume I.
—
The second alternative for which certain arguments were adduced in
Volume I (page 166)— is that the compound ACO2 (and perhaps A'H20
as well, although the two assumptions are separable) is kinetically inde-
pendent of chlorophyll; its reduction is then a secondary process, a dark
reaction brought about by the products of the primary photochemical re-
action.
The analysis is simpler if the first alternative is chosen. If we assume
that the acceptor. A, is part of the chlorophyll complex, and that it takes
up or loses carbon dioxide without separating itself from this complex
(and that consequently, Ao = Chlo, and [ACO2] < Chlo), then all quanta
absorbed by the chlorophyll molecules carrying ACO2 must be effective
(as far as the primary photochemical process is concerned) while all —
ciuanta absorbed by chlorophyll carrying "bare" A are lost. The rate
of reduction of ACO2 is then k*I[AC02], as required by equation (28.14);

^*/[Chl] being the rate of absorption of quanta by chlorophyll in light of
intensity I (assuming that the absorbing capacity is not affected by asso-
ciation of chlorophyll with either ACO2 or A). This equation already was
used in chapter 27, section 7d (c/. equations 27.58-27.66).
If ACO2 is kinetically independent of the photosensitive complex, the
concentration [ACO2] cannot be limited to Chlo; equation (28.14) now
appears to indicate that the quantum yield of photosynthesis, 7 = P/h
(/a
= absorbed light energy), can increase indefinitely with increasing
[ACO2]. At least, this would be so if one would assume, as usual, that I^
is proportional to 7, I a,
= al, so that
(28.16) 7 = P/h = ank* [ACO2]
Of course, a certain limit to the increase of 7 is set by the fact that ACO2
must be <Ao, the total number of available acceptor molecules. How-
ever, there is no reason why this limit could not be higher than the maxi-
mum yield possible under Einstein's equivalency law. Therefore, a limi-
t Or 2 hu, if it is assumed that one quantum is used to transfer one hydrogen atom
from A'HoO to Chi, and another one to transfer the same atom from Chi to ACO2.
In (28.15), it is also assumed that the hydrogen donor is "bound water," A'HaO; and
that, like the hydrogen acceptor, ACO2, this donor is stably associated with Chi.
: : : — ,
tation on P/I^ must exist that is quite independent of the Umited quantity
of the carbon dioxide acceptor, A. One way in which this Hmitation can
arise is for fc* in equation (28.14) to become a function of [ACO2], such that
the product /c* X [ACO2] never exceeds a certain maximum vahie; another
is for /a to cease to be proportional to 7, i. e., for k* in equation (28.14) to be-
come a function of P.
The first phenomenon is a common occurrence in photochemical proces-
ses in vitro, where the photochemical secondary reaction competes with the
deactivation of the light-activated molecules. (The latter can occur by
by energy dissipation within the activated molecule, or by
fluorescence, or
energy transfer to other molecules.) The competition between a bi-
molecular photochemical reaction (rate constant k,) and one (or several)
monomolecular deactivating reaction (combined constant k') leads to a
yield equation (Stern- Volmer equation)
(28.17) y = kr[^]/{k' + Av[S])
according to which the quantum yield, 7, does not increase indefinitely

with the concentration [S] of the reactant, but approaches, at /bJS] ^ k'
a maximum value (for the primary process !)
(28.18) 7max. = 1
This maximum quantum yield is independent of the light intensity, /.
Comparison of equation (28.17) with (28.16) indicates that in this case

assuming that ACO2 is the reactant S
(28.19) ank* = kr/{k' + kAkCO-z])
i. e., k* is in fact a function of the concentration of the reactant, decreasing

with increasing [ACO2] in such a way that the product ak*n[kC02\ can
approach, but never exceed, 1.
In our schemes of photosynthesis (c/., for example, schemes 28. lA and

28. IB below), we do assume a competition of the secondary photochemical
reaction (such as 28.20b or 28.21b) with monomolecular deactivation,
(such as 28.20a'), but mean by the latter not the energy loss by fluorescence,
or by immediate energy dissipation in the light-absorbing complex, but
the slower back reactions that follow primary tautomerization. The
photochemically altered forms of the chlorophyll complex, HX.Chl.Z
(or HX.Chl.HZ), play the part of "long-lived activated states" discussed
in Volume I, p. 484. This changes the order of magnitude of the deac-
tivation constant, /c', from > 10^ or 10^ sec.~^ (the inverse of the life-time
of electronic excitation states in strongly light-absorbing molecules)
to perhaps as little as 10^ sec.~^; but formally, relation (28.17) remains
valid as long as the back reaction follows the monomolecular law.
The possibility that the "activated" complex can be deactivated before
: —
encountering a molecule with which it has to react (be it ACO2 or A'H20)

creates a new source of dependence of the rate on the concentration [ACO2]
(or [A'H20]).
We now consider the second anticipated phenomenon — the breakdown,
in strong light, of proportionality between irradiation and absorption.
This complication does not occur significantly in "ordinary" photochemis-
try, where the rate constant of the process by which the excited molecule
returns to the normal state is at least a million times higher than the rate
constant of light absorption, even in the strongest available Ught (order of
magnitude of the maximum frequency of absorptions: 10 sec.~\ of. page
838; order of magnitude of the rate constant of deactivation, by fluores-
cence or energy dissipation > 10^ sec. "0
: In strongest available light, the
•
photostationary concentration of activated molecules ceases to be negligible

compared to that of the normal molecules if the life-time of the activated
form exceeds 10"- sec. (light absorption: once every 0.1 sec; lifetime
of the activated state: 0.01 sec; therefore, stationary concentration of
activated molecules: 10%). If we assume such a longevity for the ac-
tivated state of the chlorophyll complex, a sizable proportion of this com-
plex will be present in the changed state during strong photosynthesis, and
this will lead to a lack of proportionalitybetween the "useful" light absorp-
tion, Ta, and the incident light intensity, I ("useful" meaning the absorp-
tion of light by chlorophyll complexes in the unchanged form assuming —
that, if the changed form does absorb visible light at all, this absorption
is photochemically "useless"). A new reason is thus added for the de-

pendence of the yield on light intensity.
We will
analyze these two phenomena (1) the competition of the "de-
tautomerizing" back reaction in the photosensitive complex with the photo-
chemical forward reaction; and {2) the depletion of the normal form
of thiscomplex during intense photosynthesis by using two simple —
mechanisms in which the photochemical forward reaction is assumed to
involve the tautomerized chlorophyll complex, HX.Chl.Z, and either the
carbon dioxide acceptor compound, ACO2 (mechanism 28.20), or the hydro-
gen donor, A'HR, where HR may stand for water, or for a "substitute" re-
ductant (mechanism 28.21)
k*I
(28.20a ami a') X-Chl-HZ ,
HX-Chl-Z
K
(28.201)) TlXChIZ + ACOo > X-Chl-Z + AHCO,
(28.20e) X-Chl-Z + A'HR ^ X -Chi -HZ + A'R
(28.20(1) AHCO2 + A'R > ACO2 + A'HR

This mechanism is represented in scheme 28. lA. The reason for assuming
the occurrence of the secondary back reaction (28.20d) will be discussed
later. If we assume, as suggested on page 1019, that /vo[A'H20] ^ Av[AC02],
the concentration of the oxidized form, [X.Chl.Z], can be neglected in
cqa XChlHZ H20(orHR)

1
+A (â) k'
+ A
(28.20a) (28.20a')
ACO2 HX-ChIZ A'HaO (or A'HR)
L (28.20 b))(/(.
AHCOz
r X-ChIZ
1
{28.20c)
))$
)(*«,
(£"b)
X-Chl-HZ
r ]
AHO(orAR)
(fc)
(28.20d
I \
A +C02 + H2O
(fo)
rA + {CHgO} A' + 02 (orA+R)
Scheme 28.IA. Photosynthesis according to equations (28.20a-d).
green plants (though perhaps not in purple bacteria), compared to that of

the tautomeric form, [HX Chi Z the latter is then the only one the ac-
. .
]
;
cumulation of which may affect the photosynthesis of green plants in

strong light.
It may be argued that, if the reaction of Z with water is so much more
rapid than that of HX with carbon dioxide, the sequence of the secondary
and c) should be reversed,
reactions (28.20b resulting in the following
mechanism (shown in scheme 28. IB):
k*I
(28.21a and a') X- Chi -HZ ± HXChlZ
k'
ko
(28.21b) HX-Chl-Z + A'HR -^ HX-ChlHZ + A'R
(28.21c) HX-Chl-HZ + AGO. -> X-Chl-HZ + AHCO2

k'
(28.21d) AHCO2 + A'R -> ACO2 + A'HR
In this case, the main form in which the chlorophyll complex accumu-
lates during intense photosynthesis is the reduced form, HX-Chl-HZ.
CO, X-CHIHZ HgO (or HR)
+A (â) k' + A'
(28.21a) (28.21a')
AGO, HXChIZ A'H20(orA'HR)
L J
r) ((28.2lb)
HXChlHZ
r A'HO(or A'R)
fr/)((28.2lc)
(Ec)
AHCO2
r 1
X- Chi- HZ
(£-9)
)Q(28.2ld)
A
r
-I- COa + HzO
(£0)
A ^ (CHgO}^ A'H-Og (orA'-hR)
Scheme 28.IB. Photosynthesis according to equations (28.21a-d).
We chose above equation (27.6) as starting point of our considerations

(and not a similar equation for the reaction of the light-activated complex
with the oxidant A'H20), and are now interested, first of all, in the possible
interdependence of A* and [ACO2], caused by the primary back reaction.
Therefore, mechanism (28.20) is more convenient for our purpose than
mechanism (28.21), (because in 28.20 the primary back reaction, 28.20a',
competes directly with the reduction of [ACO2]).
•
In discussing the consequences of the primary back reaction, on the basis

of mechanism (28.20), we will neglect the second anticipated phenomenon
—
the accumulation of tautomerized chlorophyll complexes in strong Ught.
(We thus introduce a new cause for carbon dioxide saturation, but no new
cause for light saturation.) Later, we will use mechanism (28.21) to con-
sider the second phenomenon (accumulation of HX-Chl-HZ in strong
Hght, and consequent light saturation), while in turn neglecting the pri-
mary back reaction.
By assuming [HX Chi Z «: [X Chi HZ ], conditions become formally
• •
] . .
analogous to those prevailing in "ordinary" photochemistry in vitro, as dis-

cussed above. From the reaction sequence (28.20a-c) we obtain, for the
photostationary concentration [HX-Chl-Z], the Stern-Volmer type equa-
tion:
(28.22) [HX-Chl-Z] = A;*/[X-Chl-HZ]/(A;' + kAkCO-A) ^

k*ICh.Wik' + kr[A.CO,])
and hence:
(28.23) P = nkrk*I [AC02]Ch\o/(k' + ^^[ACOa])
(in expected formal analogy to equation 28.1). Comparison with equation

(27.6) shows that A-*, while proportional to /, is now in fact also a function
of [ACO2]:
(28.24) k* = krk*I Ch\o/{k' + A-JACO2])
If [ACO2] increases indefinitely, P approaches the maximum rate:
(28.25) Pmax. = nA:*7Chlo = nZ aChlo
which corresponds to the maximum quantum yield, n. Similarly to the

yield (28.18),n is independent of Hght intensity.
Equation (28.23) shows that back reactions in the photosensitive com-
plex can explain the increase in yield with increasing [CO2] and the final
[CO2] saturation, even if the acceptor A is available in unlimited quantities
(e. g., if [ACO2] stands for dissolved carbon dioxide, the quantity of which
can be increased practically indefinitely by raising the partial pressure of
carbon dioxide over the system). In other words, equation (28.23) indi-
cates how a limited supply of light quanta can account for hyperbolic car-
bon dioxide curves, without the assumption of a limited amount of a [CO2]
acceptor, or slow diffusion, or slow carboxylation.
The effects of carbon dioxide supply can, of course, be superimposed
upon those of limited light supply, by introducing the corresponding ex-
pressions for [ACO2] into (28.23). Using for this purpose "static" equa-
tion (27.3), taking into consideration only the limited amount of the
i. e.,
[CO2] acceptor, will not lead to light saturation; the latter will be intro-
;
duced if one uses for [ACO2] one of the "kinetic" expressions, taking into
account the hmited rates of supply processes (carbon dioxide diffusion and
carboxylation) Absolute saturation, Pmll'. will result if it is assumed that
.
a maximum rate of supply exists which is independent of [CO2], e. g., as a

consequence of a limited amount of the carboxylating catalyst, Ea-
We will now consider the second phenomenon, which is without parallel
in "ordinary" photochemistry, and arises from the assumed longevity of
the "activated state" of the photosensitive complex: the accumulation of
these complexes in a changed form, and the consequent lack of propor-
tionality between the incident light intensity, /, and the photochemically
significant light absorption, I^. For this purpose, we use the alternative
mechanism (28.21), since the postulated practical instantaneousness of
reaction (28.21b) gives us some right to neglect in this case the primary back
reaction, (28.21a) {i. to assume /co[A'H20]
e., A;') ^
The steady state
concentration of the reduced form, [HX.Chl.HZ], is, under these condi-
tions :
(28.26) [HX-Chl-HZ] = k*I Chlo/(A-*/ + MACO,])

(implying that, in the absence of ACO2, all chlorophyll complexes would
go over, in light, into the reduced form, HX-C1il-HZ). The rate of photo-
synthesis is:
(28.27) P = /(/,v[HX-Chl-HZ][AC02] = nAvChloA-*/[ACO.>]/(A-*/ + AvlACO,])
Assuming further that the complex ACO2 is in equilibrium with free carbon
dioxide, and no diffusion gradient exists, so that [CO2] — [CO^la we obtain:
(28.28) P = nkrChU*IAoKAC0.2]/(k*I + A\[COo]^"*/ + A-.AoKalCOa])
an equation for the rate of photosynthesis when neither the preparatory

nor the finishing dark reactions, neither on the "reduction side" nor on the
"oxidation side," have a rate-limiting influence. Light saturation is in
this case due entirely to the limited amount of the acceptor, Ao; and car-
bon dioxide saturation, to the limited amount of chlorophyll, Chlo.
The light curves (28.28) are hyperbolae:
(28.29) P/(P'°^^- - P) = A-*/(l + A'alCO.,])/Av.-lo/va[CO,I
The half-saturating light intensity is:
(28.30) ,/./
= AvAoA\[CO,l/(A-* + A*A'JCO,])
Extrapolating to [CO2J = <», we obtain:

(28.31) y/c = AvAo/A-*
If the carboxyln tion equilibrium is not maintained in light, we have, more

generally, instead of (28.30)
(28.31a) y/ = kr[AC02]/k*
:
PROOKSSES IN TTTE PTTOTOSENSTTIVE COMPLEX 1020
Comparing equation (28.31a) with equation (28.2G), we note that, in the

particular picture we are considering now, "half saturation" with light
corresponds to the equal distribution of chlorophyll between the forms
X-Chl-HZ and HX-ChlHZ:
(28.32) [HX-Chl-HZ] = Chlo/2 = [X-Chl-HZl
we assume that reaction (28.21a) has a quantum yield of unity, the

If
constant k* must be equal to the frequency of light absorptions by a single

chlorophyll molecule. We recall that / is the intensity of light actually
reaching the volume under consideration, and that light absorption within
this \'olume is supposed to be so small that the rate of absorption is practi-
cally the same everywhere within it. Under these conditions:
(2S.r>3) k* = a In 10 X 10-^ = 2.3 X 10^ « (mole/cm. 2)
where a is the average molar absorption coefficient for the incident light.
This equation implies that / is measured in number of einsteins of light
falling per second on a square centimeter and Chlo is expressed in moles per
liter. According to page 838, for white light, 1 lux 1.4 X 10' ^ quanta/ ^
sec. cm. 2 = 2.3 X 10"'- einstein/sec. cm.^ We thus have:
(28.34) k* = 5.3 X IQ-^ a (mole/cm. 2)
applicable when I is expressed in lux (meter candles). Average absorption

coefficient of chlorophyll for visible light is of the order of 10^; this means
/^* X 10-^ or one photochemical act per second per chlorophyll mole-
i^ 5
cule in Hght of 20,000 lux.
Experiments indicate that i//o= is of the order of 5 X 10^ lux (cf. Table
28.1) assuming ^^
; 5 X IQ-^ and Ao~ ~
5 X 10"^ m./l. (roughly the con-
centration of chlorophyll in the plastids; cf. Vol. I, page 411), we obtain
for kr a value of 5. When both [CO2] and / are very large, the rate ac-
cording to equation (28.28) approaches the "absolute ceiling":
(28.35) PS: = nivChloAo (= nA:*Chlo ,/,/co)
which is the maximum possible rate of reaction (28.21c) reached when a/i
chlorophyll is in the state HX-Chl-HZ, and all acceptor is occupied by

carbon dioxide.
For low light intensities and ample supply of carbon dioxide, the light
curves defined by equation (28.28) approach asymptotically the limiting
straight line
(28.35a) P'/ =o = nA-*Chb/
The simplifying assumption that the carbon dioxide acceptor is, even
during intense photosynthesis, in equilil^rium with external cai'bon dioxide
could be dropped, and the more general expression (27.15) for [A-C02]
:
could be introduced into equation (28.27) instead of the equilibrium expres-

sion (27.3). However, the resulting equations, embodying the effects of
carbon dioxide supply limitations together with the limitation due to light
supply, would become too unwieldy for practical use.
One may ask whether equation (28.35) provides a sufficient explanation
of the "absolute saturation" of photosynthesis, i. e., of the maximum yields
discussed in section 5 of this chapter. We
can obtain from equation
(28.35) the follomng estimate of the maximum possible yield of photosyn-
thesis per chlorophyll molecule per second
(28.36) PSS:/Chlo = nkrAo ( = nk* yl^) ^ 0.025

or, for "assimilation time," T^:
(28.36a) Ta = Chlo/PS!S: ^ 40 sec.

The experimental values of "assimilation time" (c/. Table 28.V) actually
range from 10 to 100 sec. (with the exception of the remarkably smaller
values found for aurea varieties). It thus appears as if chlorophyll could
be the component of the photosynthetic apparatus the slow rate of restor-
ation of which imposes an "absolute ceiling" on the rate of photosynthesis.
However, this is not the only adequate answer to the problem of absolute
saturation, since flashing light experiments reveal the existence of a "finish-
ing" catalyst (Franck's "catalyst B"), which appears to be present in
an amount equivalent to about one two-thousandth of that of chlorophyll,
and to have a "working time" of the order of 0.02 sec. at room temperature;
the "ceiling" imposed by this catalyst is of the same order of magnitude
as the one derived in (28.36), since 2 X 10^ X 2 X 10 ~^ is the same as
1 X 40.
It is useful to show why the approximate agreement of the value (28.36)
with the experimental results is not a significant confirmation of the model
used in the derivation of this equation.
We will see below (page 1043) that in a rectangular hyperbola, the three
parameters which in general specify a hyperbola such as (a) the initial —
slope, (6) the abscissa corresponding to half-saturation and (c) the satura-
tion value of the ordinate —
are not independent of each other, but related
by equation (28.48C). What we did above was to insert the approximate
experimental value of two of these parameters (n ^^ 0.1 and i/J ^^ 10^ lux)
into equation (28.28), Avhich represents a rectangular hyperbola (of.
equation 28.29; the equation in parentheses in 28.36 is a special case of
28.48C) ; and an approximately correct value of the
to derive in this way,
third parameter, p™^'^-. This result merely shows that the light curves (or,
at least, the limiting light curve at high [CO2]) do approximate rec-
tangular hyperbolae. Any kinetic mechanism which leads to light curves
approximating rectangular hyperbolae will permit a correct estimation of

the third parameter from the experimental values of the two other para-
meters.
We chapter 32) evidence that the principal rate-
will find later (c/.
limiting reaction in photosynthesisis a dark reaction catalyzed by a cata-
lyst (Eb) which is present in the cells in a concentration only 0.05% of

that of chlorophyll. We will also see (page 1038) that this type of limita-
tion leads to light curves which are hyperbolae, but not rectangular hyper-
bolae. It remains to be seen whether light curves can be measured pre-
cisely enough to exclude one of the two mechanisms.
Is it possible for two or more bottlenecks to exist in photosynthesis,
each allowing the passage of about the same amount of reactants the —
maximum rate of passage through one bottleneck being determined by the
product of the concentration Chlo, the (approximately equal) concentra-
tion, Ao, the (bimolecular) constant, K, of reaction (28.21c) and the quan-
tum yield n (O.OS^ x 5 X 0.1 = 1.25 X 10"^); and the other, by the
product of the enzyme concentration Eb and its(monomolecular) rate
constant (2.5 X 50 = 1.25 X 10^^).
X 10"^ Such a coincidence seems
not implausible; could even be considered as admirable economy in
it
the allotment of catalysts to the cell. (Why have more of a certain

catalyst than can be utilized because there is not enough of another one?)
However, certain experimental results are not consistent with the as-
sumption that restoration of chlorophyll is the bottleneck which limits
(or "co-limits") the maximum rate of photosynthesis; these data indicate
that a chlorophyll molecule which has taken part in the primary photo-
chemical process needs much less than 4Qn (—4) sec. to return to the photo-
sensitive form. We mean here the observations of Willstatter and Stoll
(c/. Table 28. V; see also chapter 32, fig. 32.2), that aurea leaves have
P'^^''-values only slightly lower than those of ordinary green leaves,

although they contain only one third (or less) of the normal amount of
chlorophyll. This is obviously inconsistent with equation (28.35), and indi-
cates that P"^^"^- is determined not by the rate of restoration of the photo-
chemically tautomerized chlorophyll complex (rate constant kr in equation
28.35), but by the rate of transformation of a substrate by a catalyst (such
as Eb) that is kinetically independent of chlorophyll. The above-esti-
mated value of kr (— 5 (sec. mole)~i) is therefore merely a lower limit; in
fact, quantitativeobservations which aurea leaves indicate that the true
value of this constant (which determines how often a given chlorophyll
mole('uIe is available for the primary photochemical reaction) is at least
ten times higher. This means h >50 sec.-^ (for [ACO2] = 0.05 mole/
liter),assuming that the primary photochemical reaction is (28.21c).
Because of fundamental significance of these conclusions, a reinvestigation
of the kinetics of photosynthesis in aurea leaves seems desirable.
:
1032 THE LIGHT FACTOR. I. INTENSITY CHAP, 28
It that, according to equation (28.32), half saturation of

was noted
photosynthesis with light should take place, in the presence of excess car-
bon dioxide, when chlorophyll is distributed equally between the forms
X- Chi -HZ and HX-Chl-HZ. This state should also correspond to the
halfway point in the transition of fluorescence from the "low light" yield,
<Pi to the "high light" yield, ^2 (c.f. page 1049).
It has been argued that ac-
cumulation of one half the total quantity of chlorophyll in a changed state
during intense photosynthesis is unlikely, since no reversible change in the
spectrum of strongly illuminated green plants has ever been noticed. An
exact experimental re-examination of this statement remains desirable;
but even if it were confirmed, this might only mean that the spectrum of
the chlorophyll complex in the form which accumulates in strong light is
practically identical with that of the form present in darkness. This is
quite possible, since the difference in composition is supposed to lie not in
the chlorophyll molecule itself, but in the associated hydrogen donors and
acceptors.
One has to distinguish between this hypothesis and the assumption
(favored,among others, by Franck and Herzfeld) that the conversion of
X- Chi -HZ to HX-Chl-Z (or of ACOs-Chl-A'H.O to AHCO^-Chl- A'HO
to use Franck's picture) requires two quanta, with an intermediate,
X •
ChlH formed by the first quantum. According to this picture, when
•
Z,
photosynthesis proceeds most efficiently, the rate of light absorption by
ChlH must be equal to that by Chi (because only under these conditions
can all quanta be utilized). If this assumption is made, we must postulate
a close similarity between the spectra of the compounds Chi and ChlH.
This, too, is not impossible, but more remarkable than a spectroscopic
similarity between X- Chi -HZ and HX-Chl-HZ.
If the changed form of the chlorophyll complex is green, the question
arises as to the photochemical effect of light absorbed by this form. One
possibility is that the quanta absorbed by the changed form produce no
photochemical effect at all; the other that they cause photochemical back
reactions, such as
HX-Chl-Z ^^X-ChlHZ or HX-ChlHZ + A'HO ^^

HX-Chl-Z + A'HsO
Kinetic considerations show that photochemical back reactions would not

in themselves cause Ught saturation, but merely reduce the quantum yield
by the same factor at Franck and Herzfeld, in one of
all light intensities.
their earlier speculations on the mechanism of photosynthesis (1987), sug-

gested that light saturation may be caused by photochetnically induced chain
back reactions: but they abandoned this hypothesis later (1911) in favor of
the assumption that Hght saturation is usually caused by non-photochemical

hack reactions, which compete with the forward dark reaction catalyzed by
>>
the catalyst "B.'
(f) Analytical Formulation: Effect of "Finishing^^ Dark Reactions
The necessity of considering, in addition to the "preparatory" dark re-

actions and the reversible changes in the chlorophyll complex, the "finish-
ing" dark reactions as possible sources of light saturation phenomena in
photosynthesis arises from several observations. was mentioned above
It
that experiments in flashing light (to be described in chapter 34) demon-
strated directly the existence of a "finishing" catalyst with a "working
period" of the order of lO^''^ sec. at room temperature. The maximum
yield obtainable per single flash shows that this catalyst is present in a con-
centration equivalent to 0.05%
of that of chlorophyll. Consequently, as
indicated above (page 1031 imposes a "ceiling" on the over-all rate of
it
photosynthesis of about one molecule carbon dioxide reduced per chloro-

phyll molecule every 40 seconds —
which is close to the actually observed
maximum rate of photosynthesis at room temperature. A second relevant
observation is made by comparing the light curves of photosynthesis of
various plants with the light curves of their fluorescence (cf. part B of this
chapter). If the light saturation of photosynthesis were due to a slow
supply reaction (i. e., to the depletion of one of the reactants, ACO2 or
A'Il20, in intense light), or to slow regeneration of the photosensitive form
of the chlorophyll complex, in both cases, the light saturation would be
associated with accumulation of the photosensitive complex in a chemically
changed form (such as HX-Chl-Z or HX-Chl-HZ), and should therefore
reveal itself by simultaneous changes in the fluorescence yield of the com-
plex. This is actually the case sometimes, but not always. Figure 28.24,
for example, shows light saturation of photosjoithesis of Chlorella without
any change in the yield of fluorescence; and even in figure 28.26, satura-
tion is almost completed before fluorescence begins to change its yield.
In all such cases, saturation must be due to the failure of a finishing dark
reaction to keep pace with the primary photochemical process a failure —
that produces no change in the composition of the photosensitive complex,
but leads to the loss of a large part of primary photoproducts by back reac-
tions.
It was stated before that the distinction between preparatory and
finishing dark reactions is not so clear-cut in Franck's theory of "narcotic
regulation" of photosynthesis. According to this theory, accumulation of
primary oxidation products ("photoperoxides") leads not (or not only) to
back reactions between these peroxides and the primary reduction products
(as was assumed above), but also to the oxidation of certain metabolites
(sugars?) which produces a "narcotic" (a fatty acid?). The latter settles
on the chlorophyll complex, prevents any further acceleration of the pri-
mary photochemical reaction, and causes a strong increase in fluorescence.
In the case of purple bacteria the following two alternative descriptions
of the rate-limiting and fluorescence-enhancing effect of a limited supply
of the reductants (H2, H2S2O3 ) are possible Either one considers this
. . . :
supply as a preparatory reaction, whose slowness causes the photosensitive

complex to accumulate in a changed (more strongly fluorescent) form,
such as X-Chl-Z; alternatively, (using Franck's theory) one can treat
thissupply as part of a finishing reaction ("disposal of photoperoxides"),
and explain its effect on fluorescence as a consequence of the production of
the "narcotic" by accumulated peroxides. In both cases, the light curve
of photosynthesis will approach, with increasing light intensity, a limit de-
termined by the maximum rate of supply of the reductants (either by dif-
fusion or by a preliminary enzymic transformation).

Another argument against general attribution of the assimilation num-
bers of green plants to the rate-limiting influence of preparatory reactions
— specifically, those on the "carbon dioxide side" —was mentioned in Chap-
on photosynthesis.
ter 12 (Vol. I) in connection with the influence of cyanide
It was stated there that the difference in the amounts of cyanide required
to reduce by a certain factor the rate of photosynthesis in different plant
species is most easily understood if one assumes that the cyanide-sensitive
catalyst (which, in all probability, is the "carboxylase" Ea) is not rate-
limiting in strong light in the absence of the poison, and becomes limiting
only when a considerable part of it is inactivated. If a different ratio
Ea/Eb prevails in various species and strains, the fraction of Ea that has
to be inactivated in order to make this catalyst "limiting" also must change
from case to case.
What kind of back reactions can compete with finishing catalytic re-
actions in photosynthesis? In section d of this chapter, we considered the

"primary" back reaction, HX-Chl-Z -^ X- Chi -HZ, which can be called
"detautomerization" of the chlorophyll complex. In equations (28.20) and
competes with the secondary photochemical forward
(28.21), this reaction
reaction, e. g., We noted on page 1024 that this
(28.20b) or (28.21b).
competition can cause carbon dioxide saturation, but not light saturation,
because the proportion of quanta lost by this kind of back reaction is inde-
pendent of one would treat (28.20b) or (28.21b) as a
light intensity. If
catalytic reaction, assigning to it a catalyst with the concentration and

working speed attributed above to "catalyst B," this would produce light
saturation, but the latter will again be associated with the accumulation of
the form X •
Chi Z or
•
HX •
Chi •
HZ and thus with a change in the intensity
:
EFFECT OF FINISHING DARK REACTIONS 1035
of fluorescence. explain saturation not accompanied by changes in

To
fluorescence intensity, the deficient catalyst must be placed, not between
one of the two reactants (ACO2 or A'HsO) and the photosansitive complex,
but between the primary and the finished products of photosynthesis. In
other words, the back reactions caused by the catalytic deficiency must be
"secondary" rather than "primary." A back reaction of this kind was
therefore added as reaction d in mechanisms (28.20) and (28.21). can We
postulate, e. g., that reaction (28.20d) comes into play because the transfor-
mation of the first AHCO2, into a more stable intermediate

photoproduct,
requires a catalyst, Eb (perhaps a "mutase"), which is present in limited
quantity. A similar postulate could be made for the effect of the catalyst
Ec, which is required for the first step in the conversion of A'HO into free
oxygen. Because of the symmetry we have assumed between the right and
left sides in schemes 28.IA and B, a limitation in the utilization of the oxida-
tion products will have the same effect on the kinetics of the process as a
whole as a limitation of the utilization of the reduction products. In the
first case, the secondary back reaction will be accelerated by the accumula-
tion of the primary oxidation product, A'HO; in the second case, by the ac-
cumulation of the primary reduction product, AHCO2.
Using reactions (28.20) we can tentatively assume that Eb acts on the
first reduction product, AHCO2, according to the scheme;
(28.38a) AHCO2 + Eb ^ ^
^ EbAHCO.
(28.38b) EBAHCO2 > Eb +A+ {HCO,}
where { HCO2 } designates a stabilized reduction product.

(If reaction 28.38b is a dismutation, it may require the participation of
two AHCO2 radicals, but we ^nll use here the simplest possible mechanism.)
The rate of photosynthesis is, according to this scheme
(28.39) P = nfcelEBAHCOo]
and the "absolute maximum rate" is:
(28.40) PZ^: = nkeEl
where Eg the total available quantity of the "limiting" enzyme.

is
An equation for P as a function of / and [CO2] can be derived from this

mechanism; but it is complicated, even if all the possible simplifying
assumptions are made, and little could be achieved by writing it do^^^l here.
The situation is simplified if Franck and Herzf eld's

we again make use of
mechanism (scheme 7.VA, Vol. I), in which the oxidant, ACO2, and the re-
ductant, A'H20, belong to the photosensitive complex, and take part in
the primary photochemical process, e. g., in the way indicated in equation
(28.15). In this case, the primary back reaction (detautomerization) itself

becomes a competitor to finisliing dark reactions, and it can therefore be
assumed that the role of the catalyst Eb is to prevent this reaction from de-
stroying the photoproducts. This point of view was used in the elaboration
of kinetic equations of photosynthesis by Franck and Herzfeld (1937).
Their derivations are comphcated by the assumption of four successive
(different) photochemical steps "on the reduction side," alternating with
four (identical) photochemical steps "on the oxidation side"; the product
of each of these steps was supposed to require stabilization by the same
"catalyst B," in order to prevent this step from being reversed by a dark
reaction. Instead of trying to present here the derivations of Franck and
Herzfeld we will use a simpler mechanism embodying the same basic con-
cept of primary back reaction as cause of Ught saturation. This mechanism
is similar to the one given in equation (7.13) in Volume I, but is further
simplified by the substitution of a single primary photochemical step for

the two steps (7.13a) and (7.13b). The reaction scheme is:
k*I
(28.41a, a') ACOs-Chl-A'H.O ^=± AHCOa-Chl-A'HO
k'
Primary forward and
back reaction
k Catalytic
(28.41b) AHCOa-Chl-A'HO + Eb ^— EbHCO. + A-Chl-A'HO
> stabiliza-
(28.41c) EbHCOo
k'
'-
—>Eb + {HC021
\ tion of re-
duction
product
(28.41d) A-Chl-A'HO + H2O > A-Clil-A'HjO + (HO) V'Reloading" of
(28.41e) A-Chl-A'HaO + CO2 > ACO.-Chl-A'HoO Jchlorophyll
We assume that Eb is needed only for "stabilization" of the reduction

product, AHCO2, and that this stabilization is achieved, in reaction
(28.41b), by taking the reduced group, HCO2, away from the chlorophyll
complex and thus preventing its back reaction with the oxidized group,
HO. not suggested that the radicals HCO2 or OH occur in the free
(It is
state; these symbols can stand for corresponding functional groups in

larger molecules, as indicated by braces in 28.41c and d.)
In order to simplify the derivations still more and to elaborate only the
effect due to the back reaction, we further assume that the reactions (28.41d
and e), by which the photosensitive complex is supplied with fresh oxi-
dant and fresh reductant, respectively, are practically instantaneous.
Under these conditions, only two factors can cause light saturation: (a)
accumulation of the photosensitive complex in the tautomeric form
AHCOg-Chl- A'HO; and (5) accumulation of the catalyst Eg in the bound
:
form, EbHC02. The equation for P which takes into account these two
effects is quadratic; its one significant sohition is:
/n\^^<=^*^Chlo + Kk*I + KkÊl

A- 'A-.' 4-
(28 42) P = I'"
2A-e
j-Â.A-/Chlo + A-'A,^+A-;A-/ + A-;A-.E;^y

_ ,,j,o,*,chlo]'^^^
\
where n, as before, means the number of elementary photochemical steps

(28.41a) required for the reduction of one molecule of carbon dioxide.
AC02-Chl-A'H20
(28.41a) kl k' (28.41a')
AHCOa-Chl-A' HO
(28.41b) +£b K
_yv_
EbHCOz
r A-Chl-A'HO
kzoo + HjO + COj

k.
Jk.
f (28.41 d^^) ^
Eb+(hCG2} ACGj-Chl-A'HjO (hoJ
I
jCHzO} 02
Scheme 28.11. PhotoyyuUiesis according to mechanism (28. -11).
Equation (28.42) describes a hyperbolic light curve with an initial slope
(28 43) {dP/dI)o = nA:eE"BA:*Ch]o/(A;' + /ceE")
If A;' <C /iêEB (so that back reactions play no important part in weak light)
we have:
(28.44) {dP/dI\ = n/j*Chlo
In other words, in weak quanta absorbed are used for photo-

light, all light
synthesis with the maximum quantum yield, n. If, however, k'

possible
is not <C A'eEe, the quantum yield will remain < n even if extrapolated to
zero illiunination.
The maximum absolute yield obtainable in strong light, according to
equation (28.42), is:
:
(28.45) PZl: = nhCh\ok:El/(hCh]o + K)

(the lower index being justified by the assumption that reaction with
carbon dioxide is practically instantaneous). If k^ <^ fcgChlo, equation
(28.45) is reduced to:
(28.46) PSS: = nk:El
which obviously is the maximum possible rate of restoration of the free

catalystEb by reaction (28.41c). It will be noted that, if A^gChlo is not
•C kl, this rate is not reached even in saturating light intensity; in other
words, the catalj^st Eb is never utilized to the maximum of its capacity.
In the opposite extreme case, Aê ^
AêChlo, the saturation value becomes:
(28.47) PTsl: = nkeChloEl
in other words, the limiting rate is the maximum rate of reaction (28.41b),
reached when, in the photostationary state, practically all chlorophyll
complexes are in the "tautomeric" form, AHC02-Chl-A'H0.
This last conclusion reminds us of the fact that the Franck-Herzfeld
variant of the mechanism of EB-limitation of photosynthesis does not
strictly belong under the heading of "limitation by finishing dark reac-
tions," since we have defined finishing reactions as those whose slowness
does not affect the composition of the photosensitive complex (and thus
the rate of the primary photochemical process) The reason why the reac-
.
tion catalyzed by catalyst Eb in scheme (28.41) does not qualify as finish-

ing reaction is obvious: it is the assumed stable association of the sub-
strate of this reaction, { AHCOo}, with chlorophyll. As long as this photo-
chemical product has not reacted with Eb, it "blocks" the return of the
chlorophyll complex into the photosensitive form, AC02-Chl-A'H20.
The half-saturating light-intensity according to equation (28.42) is:
(28.47A) v/
,_«(*' + « Wili^')
k*WTWm
(It will be recalled that equation 28.42 was derived by assuming complete
and instantaneous saturation of the acceptor with carbon dioxide; there-
fore no [CO2] -proportional factor appears in equation 28.47A.)
The hyperbola represented by equation (28.42) is not rectangular; it
therefore cannot be represented in the form F/(Pmax. — P) = const. X /•
The degree of its deviation from the shape of a rectangular hyperbola, with
the same initial slope and same saturation value, can be seen from a com-
parison of the half-saturating intensity (28.47 A) with the half-saturating
intensity derived from (28.43) and (28.45) by means of equation (28.48C)
: :
The two expressions become identical if A"e ^ A-gChlo ; in which case we write
(28.47Ba) :// = (k' + hEl)/k*
This was stated before to mean that the bottleneck reaction is (28.41b),
while the catalyst, Eb, is present in excess. According to (28.47) it also
means that the maximum yield Pniax! is proportional to Chlo; and the
observâtions on aurea leaves, quoted above on page 1031, indicate that this
condition is not realized in nature. The reverse relation, AgChlo ^ A"e,
which allows Eb to be fully utilized and the maximum rate (equation 28.46)
to be independent of chlorophyll concentration, appears the more likely ap-
proximation to natural conditions. This inequality reduces (28.47A) to:
(28.47C) v/ = ^"'^^'l^teCht^"^
^^^' << ^"^^^^"^
Furthermore, we have seen above that, for the utilization of practically

allthe absorbed light quanta at low light intensities, k' must be >C A-eEs;
we thus have approximately
(28.47D) ./J - ^'°2iiichl^° (A-; <C A-eChlo; k' <^ k.El)
(nonrectangular hyperbola), and:
(28.47E) vJ-^ (A-; » A-eChJo; k'<^k,El)
(rectangular hyperbola). It thus appears that the hyperbola (28.42)

ascends more steeply and reaches saturation more suddenly than a rec-
tangular hyperbola with the same parameters n and Pmax.'- This deviation
from the shape of a rectangular hyperbola is in the direction actually noted
in many experimental light curves (c/. the speculations of Brackett and
Smith in the next section).
It was stated above that the empirical relation between the parameters
n, i/J, and Pmll'. is approximately that required for a rectangular hyperbola.
However, the calculation which led to this conclusion was one of order of
magnitude only more precise determinations of the light curves may show
;
whether a deviation from the rectangular hyperbola by a factor of 1/2 in

the i//-value is compatible with the facts. Such a determination should not
be beyond the precision with which kinetic measurements of photosynthesis
can be carried out at the present time (although the persistent disagreement
about a factor of about 1/2 in the value of n, to be described in the next
chapter, indicates the difficulty of such measurements).
It is, combine the above derivations with the as-

of course, possible to
sumption carbon dioxide supply (in consequence of slow diffusion,
of slow
slow carboxylation or low content of the enzyme Ea), in other words, to
drop the assumption that equation (28.41e) is an instantaneous reaction.
In this case, one would have to take into consideration the accumulation
of the chlorophyll complex also in the "carbon dioxide-denuded" form,
A-Chl-A'HoO. Such derivations were actually carried out by Franck and
Hcrzfeld, using their eight-step mechanism.
It may be noted that in the case of mechanism (28.41), as in that of the
previously considered mechanisms, the result would be formally the same
if the limiting finishing catalyst Eb would be assigned to the stabilization
of the oxidation product {HO} instead of the reduction product, JHCO2I,

i. e., if this catalyst would be needed to make possible reaction (28.41d),
rather than reaction (28.4 le).
The reason Franck and Herzfeld had to assume that the catalyst En works on
all seven mtermediate products (and does not merely create a single "bottleneck,"
e. g., after the fourth, or seventh photochemical step) is as follows:
If one assumes eight
photochemical steps and only one bottleneck, then, in strong light, the intermediate
just before the bottleneck will accumulate at the expense of all the others. If now the
light intensity suddenly reduced, a certain time must elapse until the distribution of
is
intermediates can become uniform. Uniform distribution is, however, necessary to ob-
tain the maximum quantum yield (since for this, all eight photochemical steps must occur
with equal frequencies). It follows that sudden reduction of light intensity from the
saturation range to the linear range should cause photosynthesis to drop "too low," and
then gradually recover to the normal value. Franck and Herzfeld took it for granted that
no such effect exists, and this caused them to assume equal proportions of back reactions
equal role of the catalyst Eb) for all eight intermediates.
(i. e.,
However, Steemann-Nielsen (1942, 1949) has described experiments in which "in-

duction losses" following a transition from stronger to weaker light have in fact been ob-
served in the algae, Fucus serratus (1942), and Cladophora insignis (1949). (These ob-
servations will be described in chapter 33, dealing with induction phenomena.) It is,
however, not at all certain that the induction effects observed by Steem.ann-Nielsen are
actually caused by unequal distribution of intermediates in strong light; the observed
duration of the induction period (up to 30 niin.) seems much too long for this origin. It
seems more likely that these induction losses are related to "narcotization," photoxida-
tion, or some other type of inhibition or ]iartial destruction of chlorophyll in strong
light.
This loss of active chlorophyll is not noticeable when the over-all rate is limited by a
chlorophyll-independent catalyst, such as Eb; consequently, in strong light, even if
only a fraction of chlorophyll is active, its activity may suffice to keep all Eb occupied.
In the light-limited state, on the other hand, absorption of light by "narcotized" (or
otherwise inhibited) chlorophyll must reveal itself in a proportionally decreased rate of
photosynthesis.
Obviously, questions about the distribution of intermediates become unnecessary if
one postulates only one photochemical step followed by dark dismutations or coupled
oxidoreductions.
SELF-NARCOTIZATION 1041
Another consequence of the assumption of an eight times repeated action of the

catalyst Eb is that the estimate of the ratio E°/Chlo, must be reduced from 1 2000 to :
1:250 (cf. chapter 34).
(/) Analytical Formulation: Narcotization
Franck has evolved the concept — repeatedly referred to above — of in-
ternal regulation of photosynthesis by "self-narcotization." This self-

narcotization occurs, according to Franck, whenever excess light, or lack of
reactants, or presence of poisons, make the photochemical apparatus de-
ficient in normal substrate, and creates the danger of this apparatus "run-
ning amok" and destroying valuable cell components (including itself).
The specific form of this protective mechanism, envisaged by Franck,
consists in the formation of a "half-oxidized" metabolite (a plant acid?),
which is adsorbed on chlorophyll, blanketing all or part of it, preventing its
photosensitizing activity and incidentally enhancing itS fluorescence. We
will derive kinetic equations for a simple "servomechanism" of this type,
to see whether it can introduce fundamental changes in the shape of the
light curves of photosjmthesis (and fluorescence).
We consider the following mechanism:
X-Chl-HZ _±^ XH-ChlZ

XH-Chl-Z(+{C02l) J^Z^ [HCOo} + X-Chl-Z
X-Chl-Z (+{H201) JÎZ:^ X-Chl-HZ + {OH}
(28-47Fa) X-Chl-HZ (+ ICO2! + {H2O!) > JHCO2! + tOH} +

X-Chl-HZ
K
(28-47Fb) {OHl +E > EOH
A-e'
(28.47Fc) EOH > E(+ O2)
K
(28-47Fd) I
OH} + X-Chl-HZ (+ H,M) > X-Chl-HZ-HM -|- H2O
(= {Chi} + H2O)
K
(28.47Fe) X-Chl-HZ-HM (+ 10.) > X-Chl-HZ (+ M + IHaO)
where {Chi} is an abbreviated expression for the "narcotized chlorophyll,"
XChl-HZ-HM.
The components in parentheses are supposed, for the sake of simplifica-
tion, to be present in excess and to react practically instantaneously, so
that their concentrations do not appear in kinetic equations. The summa-
tion of the first three equations gives equation (28.47Fa) for the primary
photochemical process. It is further assumed that the reduction product.
:
{HCO2}, is removed practically instantaneously, but that the oxidation

product, {OH}, has to undergo an enzymatic transformation (28.47Fb, c),
which includes a "bottleneck" reaction imposing an absolute ceiling

(28.47H) on the over-all rate of photosynthesis.
If reaction (28.47Fa) runs too fast for the reactions (28.47Fb,c) to re-
move the oxidation products immediately, the "narcotization" mechanism
(28.47Fd, e) comes into play; accumulated {OH} reacts with ametaboHte,
H2M (supposed to be present in excess in the cells), and oxidizes it to a
"narcotic," HM, which is adsorbed on chlorophyll and inactivates it
(supposedly without affecting the absorption spectrum, but increasing the
yield of fluorescence). The narcotization is removed by reaction (28.47Fe),
which completes the oxidation of the "half-oxidized" product. (At a con-
stant concentration of oxygen, this reaction can be treated as monomolecu-
lar.)
The rate of photosynthesis is, in this scheme, equal to the rate of the
limiting reaction (28.47Fc)
(28.47G) P = »ê[EOH]
the maximum possible rate is (the subscript max. being justified by the
above-made assumption of practically instantaneous supply of carbon
dioxide)
(28.47H) PZl: = nkeEo
The factor n designates, as before, the number of elementary photo-

chemical steps of type (28.47Fa) required for the production of one molecule
of oxygen.
We introduce the abbreviation:
(28.47Ha) k = ^
An equation for the rate of photosynthesis can be obtained by deter-
mining the photostationary concentration [EOH] and then applying
equation (28.47G). The result is:
(28.471) - - 2(fc - 1)
Lv 2(fc - 1) ) k-i \
(One can easily ascertain that this equation gives correctly, P = for / =
0, and P'"^'^- = nA-eEo for 7 = oo .)
The light curves (28.471) are nonrectangular hyperbolae with the initial
slope:
nkji* nkjcnk*
(28.47J)
\dl Ji = o kekEo + k:Ch\o kUko + knCUo)
:
SELF-NARCOTIZATION 1043
and the half-saturating intensity:
k,E,{k + 1) + 2A-,:Clilo
(28.48) V/ = 2k*kCh]Q
{g) Are Light Curves Hyperholicf
It will be noted that all the kinetic models used in this chapter lead to
quadratic equations for the rate of photosynthesis, P, as function of incident
light intensity, /; in other words, to hyperbolic light curves, P = /(/).
The imposition of a rate "ceiling," caused by limited supply of a reactant
or limited amount of an enzyme (in addition to a rate "roof," imposed by
the minimum number of quanta required to bring about the reaction)
changes the appearance of the light curves for example, it can convert a—
rectangular into a nonrectangular hyperbola—but in the mechanisms
discussed so far, general hyperbolical shape is preserved.
A hyperbola is defined by three parameters. In our presentation of
light curves, the axis of abscissae, I, was parallel to one asymptote of the
hyperbola (the equation of the latter being P = p"''^^-)^ while the axis of
ordinates, P, was chosen so as to make P = at / = 0. One convenient
form of writing the equation of a hyperbola in this system of co-ordinates is:
(28.48A) '"^ pmax. _ p \

'
pmax. /
The three parameters in this equation are 70, the initial slope of the
curve, which is proportional to the maximum ciuantum yield of photo-
synthesis; i/J, the half-saturating light intensity; and P'"'^''-^ the limiting
rate in strong light. If the second term in parentheses vanishes, the equation
becomes:
(28.48B) p^£:ip = p~J = coiist- X /
which is the equation of a rectangular hyperbola. For the quadratic term

in (28. 48 A) to vanish, the following relation between the three parameters
must be fulfilled
E>max,
(28.48C) 70
W
Several equations derived in chapters 27 and 28 for the light curves of
photosynthesis- — —
such as (28.29) actually had the simplified form
(28.48B) however that was not always the case, as shown, e. g., by equa-
;
tion (28.42), which cannot be represented in the form (28.48B).

Since 70 is not so easily measured as the other two parameters (in fact,
determination of 70 may be one aim of analytical representation of the light
: :
1044 THE LIGHT FACTOR. I. INTENSITY CHAr. 2S
curves, cf. chapter 29, p. 1132 ff.), another parameter may be chosen
instead, such as //, the Hght intensity at which photosynthesis reaches
one-quarter of its saturation vahie. This choice leads to a very simple
relation
pmax.
(28.48D) 70
and gives, as equation of the light curve:
p r
- 4P
- "I
(28.48E) / = p^,,. _ p |_6./,
I ,// + p^. (,//
3,//)J
For the hyperbola (28.48E) to be i-ectangular, it must satisfy the very

simple condition:
(28.48F) ,/./ = 3,//
Its equation is then:
(28.48G) / = pî^^^zrp (6 v/ - v/)' "^
]\Iore generally, a hyperbola could be represented in our chosen co-

ordinates, using any three of its points (Pi,/i; PiJ-i; and P3,h) as param-
eters; for a rectangular hyperbola, two points suffice.
These derivations should be kept in mind in evaluating papers, in
which failure to represent empirical data by equations of type (28.48B)
has been taken to mean that the light curves were ''not hyperbolic" {cf
Smith 1936, 1937, 1938).
It was stated on page 1020 that all our derivations of light curve equa-
tions were based on the assumption of uniform light absorption, and are
therefore strictly applicable only to optically thin layers.
The question arises to what extent the considerable optical density of
most actually studied plant objects distorts the shape of light curves —for
example, whether the observed "integral" light curves will be nonhyper-
bolic if the "differential" light curves for each thin layer, with practically
uniform light absorption, are hyperbolae. For monochromatic light, for
which the absorption coefficient is a, the total light flux absorbed in a layer
I (for the sake of simplicity, we assume uniform pigment distribution and
absence of scattering) is
(28.481) /a = /(I - lO-«tci>'io
(assuming chlorophyll to be the absorbing pigment).

If the quantum yield of the over-all process is n, and if /a is expressed
in einsteins per cm.^ per sec, the total rate of photosynthesis is:
:
ARE THE LIGHT CURVES HYPERBOLIC? 1045
(28.48J) P = n/a = n/(l - 10-«icw]i)
(mole CO2 reduced per second per cm.^ of illuminated area).

If the illuminating light is nonmonochromatic, the first part of this rela-
tion remains valid, but 7a is now expressed by an integral:
(28.48K) /a = / - r
Xi
'\^'hen the incident light intensity increases so that light saturation sets
in, most exposed pigment lajêr, the light curve of the integral
in the
yield bends, and does not become horizontal until saturation has become
complete in the deepest lajêr. Consequently (as discussed before, cf. p.
1007 and fig. 28.20), the qualitative effect of inhomogeneous light absorp-
tion must be to broaden the transitional region connecting the linearly
ascending and the horizontal part of the light curves. AVhat we want to
know is how the shape of the light curves is changed by this integration.
Let us assume for the sake of simplicity that the "differential" light cuives
are rectangular hyperbolae:
p.
pmax. _ = k*Ii
p^
jL*r)inax. riQ-Q:(Chl](
(28.48L) Pt = _|_ ^*j jQ-a[chi]i
I
The integrated rate (per unit area) is then
(28.48M) P = =
J^
^'^^
U
U[Chll
[Chi] In 10
^"
1 + ^•*n0^lChlJ^
or:
m dSV'^
^zs.is.n; — ^_ p =
p^^^ ^^^[(^yj
In
jj^ jQ
[( 1 +
_
A-*/)/(l
jj^ j^ ^
+ fc*/10-'^[ch']'.)
A;*7)/(l + fc*/10-«Ichi](o) ]
an ctiuation which does not represent a rectangular hyperbola. It can he

developed into a series:
P
(28.480) pmax. p
^^^ (^ _ «[Chl]/oln
]l 10
"^
(«[Chl]lo In 10) = (2 + k*I)
2! 3!2! (1 + k*I)
(«[Chl]Zoln 10)3 (3 + k*I){2 + k*I)

"*"
..,)
4!3! (1 + k*IY
This series shows that the integral light curve remains practically a
rectangular hyperbola until the third term in the development ceases to be
small compared to 1; then, it looses the hyperbolic shape (because of a
third degree term, containing the product PP). Since the second factor
in the third term decreases from 2 to 1 with increasing light intensity, the
maximum value of this term is a[Chll/o In 10/6. This means that for the
maximum deviation of the integral light curve from hyperbolical shape to
exceed 5%, the integral absorption must exceed 50%:
(7o// = 2; log/o// = a[Chl]ioln 10 = 0.30; a[Chl]/oln 10/6 = 0.05)
At 75% absorption, the maximum deviation will reach 10%. This indi-
cates that to obtain experimental light curves which will permit one to
judge the true shape of the function P = f{I), objects with an optical den-
sity of up to log h/I = 0.5 can be used.
A corresponding derivation for a general hyperbola is much more in-
volved, but conclusions are likely to be not too different from those obtained
here for a rectangular hyperbola.
In the literature one can find many attempts to derive analytical ex-
pressions for the light curves of photosynthesis, partly by using very simple
kinetic models, and partly by empirical approximation.
Among the simplified kinetic equations, reference may be made to those
of Ghosh (1928), Emerson and Green (1934), Baly (1935) and Burk and
Lineweaver (1935); while among the empirical approximations, we may
mention those of Brackett (1935) and Smith (1936, 1937, 1938).
•P CD
-4
o 2
O
O
o
o 16
-8—
-5
12
3 4 5
LOG I, meter candles
Are photosynthesis curves hyperbolae? (after Smith

Fig. 28.23.
1938). SoHd Hnes are derived from nonhyperbolic functions (27.77)
and (28.48P); dashed line from rectangular hyperbolic function.
See text page 1047.
Brackett sought to expres.s the sudden approach of the light curves to

saturation by an exponential curve, while Smith suggested an algebraic
function of a higher order:
LIGHT CURVES OF FLUORESCENCE 1047
(28.48P) p/pmax. = C//V1 + C2/2
or:
(28.48Q) p/V(pmax.)2 _ p2 = CI
To compare the usefulness of functions (27.77) or (28.48P) with that of

the rectangular hyperbola, Smith derived, for both tj-pes of functions,
equations determining the combinations of the parameters [CO2] and 7 for
which the yield P has a certain constant value. Figure 28.23 shows the
results: For the lowest value of P used (log P = 0.8), the dashed curve
derived from the hyperbolic function gave the better fit; but for three
higher values of P (log P = 1.2, 1.6, and 2.0), a very good fit
was obtained by means of equations (27.77) and (28.48P).
B. Light Curves of Fluorescence*

1. Relation between Light Curves of Photosynthesis and Fluorescence
In vitro the intensity of fluorescence usually is proportional to the

intensity of illumination. This is so because both fluorescence and the
"quenching" processes that compete with it (i. e., energy dissipation, and
photochemical reactions) usually are "first order" or "monomolecular"
processes with respect to the concentration of excited pigment molecules.
In other words, each excited molecule has a certain probability of fluores-
cing, a certain probability of being deactivated by energy dissipation and
a certain probability of undergoing a photochemical reaction; all these
probabilities are independent of the concentration of the excited molecules,
and consequently do not depend on the rate of their production and thus
also on light intensity. Under these conditions, the rate of photochemical
reactions too must be proportional to light intensity. Thus, both the quan-
tum yield of fluorescence, <p {= const. X F/I, where F is the intensity of
fluorescent light), and the quantum yield of the photochemical change, 7
(= const. X P/I, where P is the rate of photochemical change), usually are
independent of the intensity of the incident light, 7.
In photosynthesis, we know already that 7 is approximately constant
only within a limited range of low intensities (corresponding to the linear
part of the light curves, cf. table 28.11), and then declines gradually. The
question arises whether, in this case,<p, too, changes with light intensity.
At experiments appeared to indicate that the intensity of fluorescence

first,
continues to increase proportionally with light intensity (i. e., the yield,
(p, remains constant) long after photosynthesis has begun to show light
saturation (i. e., 7 has begun to decHne). For example, Wassink, Vermeu-
len, Reman and Katz (1938), using a suspension of Chlorella vulgaris, found
for F and P the two curves shown in figvire 28.24: The fluorescence curve
remained Hnear up to 16 kerg/cm.^ sec, while photosynthesis became Ught-
saturated at about 10 kerg. Subsequent investigations have sho^vn, how-
ever, that such an absence of correlation between tp and 7 is by no means
—
the general rule. More often, a definite relation usually, antiparallelism
— is found between the two jnelds {i. e., the jaeld of fluorescence increases
12 3 4 5 6 200 400 600

INCIDENT INTENSITY, arbitrary units LIGHT INTENSITY, kerg /cm'^ sec.
Fig. 28.24. Photosynthesis light Fig. 28.25. Yield of fluorescence in-

saturated, fluorescence unchanged in wheat at high light intensity
creases in
Chlorella suspension (after Wassink, when CO2 supply is low and remains con-
Vermeulen, Reman and Katz 1938). stant when [CO2] is high (after McAlister
and Myers 1940).
when the yield of photosynthesis becomes smaller, and vice versa). An in-
crease in the yield of fluorescence at high light intensities was first observed
by McAlister and Myers (1940) in young wheat plants. It began at about
200 kerg/cm.2 sec. However, this increase occurred only when the carbon
dioxide supply was low (0.03%) no change of <p was noticeable even up to
;
700 kerg/cm.2 sec, when this supply was ample (in 4% CO2) (c/. fig.
28.25). It will be noted that McAlister and Myers used much higher light
intensities than Wassink and co-workers; but in their experiments {of.
Table 28.1), saturation was not quite reached, in the presence of 4% CO2,
even at 600 kerg/cm.^ sec.
Franck, French and Puck (1941), working with Hydrangea leaves, found
an increase in fluorescence yield above 20 kerg/cm.^ sec, even with ample
LIGHT CURVES OP FLUORESCENCE 1049
supply of carbon dioxide (1% CO2). Measurements up to 130 kerg/cm.^

sec. indicated 28.26) that, at very high Ught intensities, the yield
(c/. fig.
of fluorescence again becomes stabilized. In other words the yield in-

creases from an initial constant level, (pi, to approach a final, also constant
level,<p2 (— 1-7 <j?i). The transition occurred, in Hydrangea, approxi-
mately in the same intensity region in which photosynthesis became light
saturated (cf. lower curve in fig. 28.26).
LIGHT INTENSITY, kerg/cm' sec
Fig. 28.26. Rate of photosynthesis (P, lower scale) and fluorescence (<p, upper
scale) yield ofHydrangea leaves as function of light intensity (after Franck, et al.
1941). Note that upper curve represents yield, ip, while the two preceding fig-
ures show absolute rates, i.e., intensities, of fluorescence, F.
measurements with Chlorella, as well as with Scenedestnus, were

New
carried out by Shiau and Franck (1947), and an increase in the yield of
fluorescence at high light intensities was now found also in these unicellular
—
algae în Chlorella above 20 kerg/cm.^ sec. and in Scenedesmus above 15
kerg/cm.^ sec.
Extensive measurements of the fluorescence of Chronialimn (strain
D) were described by Katz, Wassink and Dorrestein (1942) and by Was-
sink, Katz and Dorrestein (1942), who found that the yield of fluoresence
of these bacteria increased, at the higher light intensities, even more pro-
nouncedly than that of green plants. This is illustrated by figures 28.30
to 28.33. These figures also show the influence that the supply of the re-
ductants has on the shape of the light curves of fluorescence. In the
presence of abundant reductant (either hydrogen or thiosulfate), the in-
crease of <p in Chromaiium first begins at 15 kerg/cm.^ sec, while witliout

1050 THE LIGHT FACTOR. I. IISTTENSITY CHAP. 28
the reductant the yield is high even at Hght intensities as low as 1 kerg/cm.^
sec.
Figure 28.27 shows the transition from the low intensity yield, (pi, to
the high intensity yield, ipi- The yield in the upper figure becomes prac-
tically constant (v?
= ^2) when the difference between the angles <p and <p2
in the lower figure ceases to be significant.
ChL
Chl
LIGHT CURVES OP FLUORESCENCE 1051
28.28) French and Koski (1951) reported that the chlorophyll fluorescence
of a red alga declined when the exciting green light was raised to 4 kerg/cm. 2
sec. The phj^coerythrin fluorescence remained proportional to I.
2. Efifect of Various Factors on Light Curves of Fluorescence
We have seen above that the yield of fluorescence, <p, is often afi"ected
by changes in the yield of photosjTithesis, 7, when the latter are produced
by variations of light intensity. We therefore expect ^ to be affected also
by other, external or internal factors that influence 7.
(a) Carbon Dioxide
The effect of the factor [CO2] on fluorescence was already mentioned

before. First, we noted the results of McAlister and Myers (fig. 28.25),
who found that, in wheat, (p rises at high light intensity in 0.03% CO2,
while no such change occurs in 4% CO2. In qualitative agreement wdth
McAlister's results, Franck, French and Puck (1941) found that at 17 kerg/
cm. 2 sec. the yield of fluorescence of Hydrangea was about 20% higher in
the absence of carbon dioxide than in the presence of 5% CO2.
As mentioned above, Wassink and Kersten (1945) found a decrease
rather than increase in the yield of fluorescence of diatoms at high light
intensities in the presence of carbon dioxide; in the carbon dioxide-free
suspension (p remained constant up to 100 kerg/cm. ^ sec. {cf. fig. 28.28).
In other words, in this type of plant, as in Hydrangea or Triticum, the jdeld,

<P2, in strong light was
higher without than with carbon dioxide; but this
difference was caused by a decline of <p in the C02-supplied plant, rather
than, as in the other species,by an increase of (p in the starved cells.
it must be borne in mind that according to
In evaluating figure 28.28,
figure 28.5 the photosynthesis of Nitzschia was not completely inhibited in
C02-free air. This probably means that these cells produced, by dark
metabolism, so much carbon dioxide (or intermediates which could be used
directly for photos\Tithesis) that their photosynthesis could not be stopped
by bubbling C02-free through the suspension. Whether this is the cor-
air
rect explanation of the continued oxygen production by Nitzschia in "CO2-

free" air or not, the notation in figure 28.28 certainly means "no
"no CO2"
extemal CO2 supplied," and not "photosynthesis totally inhibited by ab-
sence of CO2."
Comparison of figure 28.28 with the effects of low temperature (fig.
28.39) and cyanide inhibition (fig. 28.44) in the same species shows that the
results would be more plausible and consistent if the designations of the
two curvês in figure 28.28 were reversed.
A still different effect of carbon dioxide on the Ught curves of fluores-
cence was observed by Wassink, Katz and Dorrestein (1942) in purple

bacteria. In the first place, they found no effect of carbon dioxide defi-
ciency at all when reductants were absent (fig. 28.29) —
a condition that
cannot be duplicated in green plants. In the presence of thiosulfate or hy-
drogen, the effect of carbon dioxide deprivation was small but noticeable.
Figure 28.30A obtained in the presence of thiosulfate could be interpreted
as analogous to the findings of Franck and McAlister; here, too,
14
with CO2
12-
o with CO2
A without COz
UJ
o
z
UJ
o
to
UJ
cr
o 6-
INCIDENT INTENSITY, erg/cm'' sec.
Fig. 28.28. Fluorescence and CO2

supply in diatoms (after Wassink and
Kersten 1945).
LIGHT CURVES OF FLUORESCEXCE 1053
influence of the reductant is much stronger than that of carbon dioxide.
Figure 28.34 shows the "critical" intensity, /<-, (fig. 28.27) in relation to the
thiosulfate concentration. It rises from 2 kerg without thiosulfate to 10
kerg in 0.5% thiosulfate, and then becomes more or less constant. Figure
27.13 indicated that thiosulfate "saturation" of photosynthesis occurs in
about the same concentration region.
Wassink and co-workers noted, however, that, with hydrogen as reduc-
tant, at pH 7.6, the transition point of fluorescence was markedly higher
than the saturation point of the gas exchange.
14
1054 THE LIGHT FACTOR. INTENSITY CHAP, 28
JNCIDENT INTENSITY, erg/cm. sec.
Fig. 28.31. Fluorescence of pur- Fig. 28.32. Influence of con-

ple bacteria as function of the concentration of thiosulfate on in-
centration of reductants (after tensity of fluorescence (Wassink
Wassink et al. 1942). 5% CO2, et al. 1942). 5% CO2, pH 6.3,
pH6.3, 29°C. 29° C.
14
without hydrogen
Q 2 % hydrogen
12 A 2.9% hydrogen
o 15% hydrogen
10
UJ
o
8
o
</)
a:
o 6
3
4
INCIDENT INTENSITY, erg/cm' sec.
Fig. 28.33. Influence of concentration of H2 on intensity of fluores-

cence (after Wassink ei al. 1942). 5% CO2, pH 7.6, 29° C.
1.4
1.2
1.0
0.8
0.6
<u
0.4
0.2
0.2 0.4 6 0.8 1.0 1.2
THIOSULFATE CONCENTRATION,?;
Fig. 28.34. Fluorescence transition point I^

as function of thiosulfate concentration (after
Wassink et al. 1942) (cf. fig. 28.27 for meaning
of /.).
2 3
INCIDENT INTENSITY, erg/cm.^ sec.
Fig. 28.35. Influence of excess reductants on

intensity of fluorescence in absence of CO2 (after
Wassink et al. 1942). pH 6.3, 29 ° C.
(c) Effect of Temperature
Kautsky and Spohn (1934) noticed that the fluorescence of leaves was
stronger at than at 30° G. but Wassink, Vermeulen, Reman and Katz
;
(1938) found no such effect on the light curves of fluorescence in Chlorella

suspensions (10-30°) {cf. fig. 28.36 on page 1056).
Franck, French and Puck (1941) found that a sudden cooHng of Hij-
drangea leaves from 23 to 0° C. produced a "burst" of fluorescence, which
subsided after 10 or 20 minutes {cf. fig. 28.37). We will return to this ob-
O 30° C
110
A 12° C
• 28° C
100
A 14° C
90
80
.t: 70
o
LU
O 60
O 50
in
'J
<r
o
3 40
30
20-
- 8
oK * 116x10^
INTENSITY, erg/cm^ sec.
Fig. 28.36. Fluorescence of chlorophyll in suspensions of

Chlorella at different temperatures (after Wassink, Ver-
meulen, Reman and Katz 1938).
servation in chapter 33, in dealing with "induction" phenomena; here we

note that, even after the burst was over, the steady yield of fluorescence
remained greater at 0° C. than at room temperature (fig. 28.37c, levels A
andE).
This temperature effect could be observed only in a certain medium
range of intensities (e. g., at 5 and 27 kerg/cm.^ sec, cf. figs.28.37b and
c); but not in high light (e. g., 220 kerg/cm.^ sec, cf.
fig. 28.37a); or low
light (e. g., 1.8 kerg/cm.^ sec, cf. fig. 28.37c). In other words, a decline
in temperature appeared to have no effect on the steady fluorescence levels,
(Pi and (p2 {cf. fig. 28.27), but caused the transition from (pi to ^2 to occur at
lower light intensities (fig. 28.38). (Figs. 28.37 to 28.45 are on pages
1058-1061.)
The effect of temperature on the light curves of fluorescence was also
observed by Wassink and Kersten (1945) with diatoms. Figure 28.39
shows that the peculiarity noted in the fluorescence curves of the same
species with and without external CO2 (fig. 28.28) is repeated in the fluores-
cence curves at low and high temperature: The yield of fluorescence
either remains constant, or declines at high light intensity. An additional
peculiarity is that the curve obtained at low temperature resembles that
found with carbon dioxide, and the curve found at high temperature re-
sembles that found without external CO2; usually, the reverse relation
prevails, the efiêct of loA\ering tlie temperature being similar to that of re-
moving carbon dioxide. In figure 28.39 the yield at 25° C. remains con-
stant up to 100 kerg/cm.' sec, while the saturation of photosynthesis be-
gins at this temperature at about 20, and is complete at about 45 kerg/em.-
sec.
A temperature effect similar to that observed in green plants was foimd

in purple bacteria by Wassink, Katz and Dorrestein (1942). At 16° C. the
I^, was at about 10 kerg and, at 29°, at about 20 kerg/cm.^
transition point,
sec. more detail, observations by Wassink, Katz and Dorrestein are
In
summarized in figure 28.40.
{d) Cyanide
A shift of the fluorescence transition point toward lower light intensities

can be produced also by the addition of cyanide. A "stimulating" effect
of cyanide on the yield of steady fluorescence was first observed by Kautsky
and Hirsch (1935) in experiments with leaves. Wassink, Vermeulen,
Reman and Katz (1938) found no such effect in Chlorella suspensions (fig.
28.41); but Wassink and Katz (1939) later proved that cyanide stimula-
tion does occur in this organism as well, although only when the cyanide
concentration is high enough to cause complete inhibition of photosynthe-
sis. This concentration completely inhibits respiration also, which is an
important factor from the point of view of the "narcotization" theory of
Franck (since this theory assumes that the "narcotic", which protects the
"idhng" photosjoithetic apparatus, undergoes rapid oxidation when respira-
tion is strong, but can be preserved for a considerable length of time if
respiration is weak) Figure 28.42 shows that the cyanide effect on steady
.
fluorescence of Chlorella is caused by the disappearance of the decline of

fluorescence otherwise observed after the first half-minute of illumination.
This decline is entirely eliminated in 2.5 X 10~^ Mpotassium cyanide
solution (p = 0.33 in fig. 28.42). The effect of cyanide on the stationary
intensity of fluorescence increases at first with light intensity, as shown by
figure 28.43; but Franck, French and Puck (1941) found that it disappears
again if the light intensity is increased still further. For example, 2% gase-
ous HCN in the atmosphere increased the yield of fluorescence of Hydrangea
leaves (in 1% CO2) by 12% when the incident light intensity was 2 kerg.
>
NCIDENT INTENSITY, erg/cm'= sec.
Fig. 28.39. Fluorescence intensity of diatoms

as function of light intensit}' at two temperatures
(Wassink and Kersten 1945).
14
1% Thiosulfate, pH 6 3 15% Hydrogen, pH 7.6
12 o 29° C. 12 V 35° C
A 195° o 29°
a 10 5° A 23°
§ '0 10 a 16°
uj 8
o
UJ
o 6-
UJ
cc
o
3 4 -
2 -
^p*'
Fig. 28.40. P'luorescence curves of Chromatium at different temperatures (after

Wassink, Katz and Dorrestein 1942). (5% CO2.)
1060 THE LIGHT FACTOR. 1. INTENSITY CHAP. 28
100
o not inhibited
A 005 ml.0.03%KCN/ml.
i TO
o
5 60
S 50
^ 40
u
g 30
I'll
1.6x10*
INTENSITY, erg/cm.2 sec.
Fig. 28.41. Influence of cyanide on fluorescence of

chlorophyll in Chlorella suspensions (after Wassink,
Vermeulen, Reman and Katz 1938).
TIME, mm.
Fig. 28.42. Fluorescence-time relation in air as function of inhibition of photo-

synthesis by cyanide (after Wassink and Katz 1939). Parameters are perceotage
KCN in solution from which 0.1 ml. was added to 2 ml. of cell suspension (29° C).
LTOHT CTTRVES OF FLUORESCENCE 1061
20 10
o totolly inhibited o without cyonide
18 1- • not inhibited 9 • 0.003% cyanide
20 40 60 80 100
Fig. 28.43. Stationiiiy values of fluor- Fig. 28.44. Cyanide effect on fluores-
escence of Chlorella as function of light cence of diatoms (25° C.) (after Wassink
and without cyanide (after
intensity with and Kersten 1945).
Wassink and Katz 1939). (Gas phase air,
29° C.)
14 14
% Thiosulfote 15% Hydrogen
o without cyanide o without cyanide
12-
A 0.0167% cyanide A 0,00125% Cyanide
10
4-
INCIDENT INTENSITY, erg /cm." sec.
Fig. 28.45. Effect of cyanide on fluorescence of Chroinatiiim (after Wassink,

Katz and Dorresteiu 1942). 5% COj, pH 0.3, 29° C.
and by 6% when it was 4.7 kerg/cm.- sec, but left it unchanged at 70

kerg/cm.2 sec. In other words, the effect of cyanide was in this case simi-
lar to that of low temperature (or carbon dioxide deficiency); all three
caused a shift of the critical intensity, /„ toward lower light intensities,
but did not affect the two "hmiting" yields, (pi and (p2. Working with dia-
toms, Wassink and Kersten (1945) found the cyanide effect similar to that
of lowering of temperature (fig. 28.33) and of addition (not removal!) of
carbon dioxide. Up to about 6 kerg/cm.^ sec, the yield of fluorescence (at
25° C.) was slightly increased by 0.003% KCN
but, above this intensity,
the yield became much smaller in the presence of the poison (fig. 28.44).
Here again, we note the contradiction to other observations in the apparent
similarity between the curve obtained with carbon dioxide and that ob-
served with hydrogen cyanide (usually addition of hydrogen cjânide has
been found to produce the same effect as deprivation of carbon dioxide).
Furthermore, one notes that in figure 28.44 the noninhibited sample showed
a marked increase in <p at high light intensities in contradiction to the per-
fectly straight line given for similar conditions in figure 28.39).
At 6° C, the yield, (p, remained constant in the presence of cyanide,
up to 130 kerg; it looked as if in this case the low yield, (p2, prevailed down
to the lowest light intensities used.
Katz, Wassink and Dorrestein (1941) stated that in purple bacteria, as in
Chlorella, the effect of cyanide on the transition intensity, 7^, is similar to
that of a decrease in the concentration of carbon dioxide. Wassink, Katz
and Dorrestein (1942) gave several fluorescence curves for potassium cy-
anide-poisoned bacteria. In the absence of reductants, the addition of up
to 0.0167% KCN had no effect on fluorescence —a result analogous to that
obtained in carbon dioxide starvation experiments, and thus in agreement
with the assumption that hydrogen cyanide is primarily a poison for carbon
dioxide fixation. Very high cyanide concentration, on the other hand, had
a strong depressing effect on fluorescence of Chromatiiim, even in the ab-
sence of hydrogen donors (>50% depression in 0.05% KCN). In the
presence of reductants, fluorescence curves with and without cyanide
(figs. 28.45) showed small but distinct differences, again somewhat similar
to those found with varying carbon dioxide supply (cf. fig. 28.30).
(e) Hydroxylamine and Azide
Observations of the effect of these two poisons on fluorescence were

made by Wassink and co-workers (1942) in purple bacteria. They are
illustrated by figs. 28.46-28.47 (p. 1064). The influence of hydroxylamine
appears similar to that of potassium cyanide no effect up to 0.05% (a—
concentration at which photosynthesis is about 50% inhibited), then (at
0.1%) a strong stimulation of fluorescence. The effect of sodium azide

differs from that of either potassium cyanide or hydroxylamine in two
respects. Fluorescence is affected (c/. fig. 28.47) even in the absence of
reductants; and the typical effect is a decline, rather than a rise of the
yield of fluorescence.
(/) Ion Concentration
In Chromatium, the yield of fluorescence can be affected by changes in

pH. According to Wassink and co-workers (1942) the sign of this effect
depends on whether molecular hydrogen or thiosulfate is used as hydrogen
donor. (We saw on page 952 that the same is true of the influence of pH
on the yield of carbon dioxide reduction by these two reductants.) This is
illustrated by figure 28.48, page 1065.
No experiments are available on the effect of other cations or anions on
the yield of fluorescence of green plants or purple bacteria.
{g) Narcotics
Narcotics were found by Kautsky and Hirsch (1935) to increase the

steady fluorescence of aquatic plants; this result was confirmed by Was-
sink, Vermeulen, Reman and Katz (1938), cf. fig. 28.49. According to
Franck, French and Puck (1941), very high concentrations of carbon di-
oxide (e. g., 20%) produce a similar effect. (It was stated in chapter 13
that the effect of excessive concentrations of carbon dioxide on photosynthe-

sis resembles narcotization.)
The phenomenon was also studied in purple bacteria, by Wassink, Katz
and Dorrestein (1942). Figure 28.50 shows typical results obtained in the
presence and in the absence of reductants. The picture is similar to that
with sodium azide: In the absence of reductants, the addition of increas-
ing amounts of ethylurethan causes a progressive quenching (rather than
stimulation) of fluorescence (although the effect is reversed at > 1.5% ure-
than, at least below 15 kerg/cm.^ sec). In the presence of reductants,
moderate quantities of urethan have little if any effect on the fluorescence
curve, while quantities > 2% have a strong enhancing effect.
(h) Oxygen
Kautsky, whose theory of an exclusive transfer of excitation energy to

oxygen was described in Volume I, (page 514), was naturally interested in
the quenching of the fluorescence of leaves by oxygen. However, neither
Kautsky, Hirsch and Davidshofer (1932), Kautsky and Hirsch (1935) nor
Wassink, Vermeulen, Katz and Reman (1938) could find any distinct in-
fluence of changes in the external oxygen concentration (between 1 and
% Hydroxylamine
10
o
UJ
o
CD
UJ
cr
o
3
Effect of />H on fluorescence of Chrornatium (after Wassink,

Katz
Fig. 28.48.
and Dorrestein 1942). 5%
CO2, 29° C.
120
o not inhibited
110 A 005 ml 60% urethon/ml
I
6.
V 1.6 X 10"*
INTENSITY, erg/cm^ sec
Fig. 28.49. Influence of ethylurethan on fluorescence of

chlorophyll in suspension of Chlorella (after Wassink, Ver-
meulen, Reman and Katz 1938).
No Reductant
o
in
LlI
INCIDENT INTENSITY, erg/cm sec. B

Fig. 28.50. Effect of ethylurethau on fluorescence of Chromatium in the ab-
sence and pressence of reductants (after Wassink, Katz and Dorrestein 1942).
5%C0-,, pH7.6, 29° C.
100
Photosynthesis
Fluorescence
CO
<
O
O
U.
o
I-
<
a:
10 20 30 40 X 10*
LIGHT INTENSITY, erg/cm.^ sec.
Fig. 28.51. CO2 assimilation and fluorescence vs. incident light intensity (after
McAlister and Myers 1940). Solid symbols, intensity of fluorescence, F; open
symbols, rate of CO2 uptake, P.
;
INTERPRETATION OF LIGHT CURVES OF FLUORESCENCE 1067
100%) on the yield of steady fluorescence of leaves and algae. McAlister

and Mjêrs (1940) found that an increase in oxygen concentration from 0.5
to 20% resulted in a marked increase in fluorescence (c/. fig. 28.51) an —
effect opposite to quenching, and probably associated with the inhibiting
influence of oxygen on photosynthesis, described in Volume I (chapter 13).
Shiau and Franck (1947) noted that, at low light intensities, fluorescence
of green algae was stronger in nitrogen than in air, but that the increase
of (p with increasing light intensity began earlier in air. In some cases the
two curves even crossed each other, so that in strong light the aerated sus-
pension fluoresced stronger than the nonaerated one.
We have spoken in this chapter only of changes in chlorophyll fluores-
cence caused by internal chemical transformations associated with photo-
synthesis —a relation that reveals itself indirectly, by comparison of the
influences of light intensity, temperature and poisons on the yields of
photosynthesis and fluorescence. In vitro, quenching of chlorophyll fluores-
cence can be produced directly, by the addition
of certain substances under-
going autoxidation, as well as of many oxidants, including free oxygen
(chapter 23, section A6). No observations have been made on the
quenching of chlorophyll fluorescence m vivo by amines (or other possible
substrates of sensitized photoxidation), while the effect of oxygen was
described above as complex and probably mostly indirect.
Shiau and Franck (1947) found that quinone depresses fluorescence in
Chlorella, if added in the dark or in light after long anaerobic incubation.
3. Interpretation of Light Curves of Fluorescence
In absence of positive information to the contrary, we have assumed,

throughout the preceding sections, that all the observed changes of fluores-
cence were increases and decreases in the fluorescence yield, tp, without
significant shifts in the position of the fluorescence bands. This point
could, however, profit by exact investigation. Changes in the structure
of the chlorophyll complex (e. g., conversion of X- Chi -HZ to HX-Chl-Z,
not to speak of reversible hydrogenation, Chi ^ rChl) could well find ex-
pression in the variation of the position and shape of the fluorescence bands
and the selective spectral sensitivity of the photometric devices used could
convert these changes into apparent variations in the intensity of fluores-
cence. While it is extremely unlikely that such spectral effects were re-
sponsible for all or even a large fraction of the described intensity changes,
it might be unwise to ignore their possibility.
True changes in the intensity of fluorescence can be caused by two fac-
tors: alterations in the relative probabilities of fluorescence and energy
dissipation in the light-absorbing complex, and changes in the probability
:
of the primary photochemical process. As described previously (c/. Vol.

I, page 546, chapter 23. A8, and chapter 24, the three processes—energy
dissipation (rate constant, h; quantum yield, 5), chemical transformation
(rate constant, kt] quantum yield, 7) and fluorescence (rate constant,
kf-, quantum yield, <p) — compete for the absorbed light energy. If all
three competing processes obey the law of monomolecular reactions, their
quantum yields are determined by equations of the type

(28.49) 'P = A-/ /(A-/ + ki + ki)
{cf. equation 19.8). the primary photochemical process requires en-

If
counters with a kinetically independent reaction partner, A, the quantum

yield equation becomes:
(28.50) f = kj/(kf + ^'1[A] + ki)
where fcl is a bimolecular rate constant {cf. equation 23.18, p. 797). In

photosynthesis, we assumed the primary photochemical process to be a
tautomerization (such as X Chi HZ ^ HX Chi Z, or ACO2 Chi A'HaO
^
• • • • •
^
•
AHC02-Chl-A'H0, or AC02-Chl-A'H20 AC02-ChlH-A'H0

^^ AHC02-Chl-A'H0). Since tautomerization is a monomolecular
process, equation (28.49) can be used; changes in tp are thus indicative of
variations in the composition or structure of the photosensitive complex,
which affect the rates kt and ki. (The fluorescence rate constant, kf, itself
remains practically unchanged as long as the intensity of the absorption
band is not changed significantly, since both are determined by the transi-
tion probability between the ground state and the excited state.) One
could, of course, also consider the possibility of fluorescence quenching by
collisions with alien molecules (i. e., the addition of "bimolecular" terms in
the denominator is often observed in fluorescent
of eq. 28.49), since this
gases and seems
solutions; however, plausible that changes in fluorescence
it
associated with photosynthesis are due to changes within the chlorophyll

complex, rather than to the formation or disappearance of new kinetically
independent quenching substances. The "natural" life-time of the excited
state of the chlorophyll molecule has been estimated (cf. page 534) as of the
order of 8 X
lO^^ sec; and the low yield of fluorescence in vivo (order
0.1%) indicates that the actual life-time is about one hundred times shorter,
or ~8 X 10-^'' sec. produce, under these conditions, a marked effect
To
on the intensity of fluorescence by kinetic encounters the quenching mole-
cules must occur in concentrations high enough for the encounter intervals
to be not much longer than 10"'" sec; and this requires concentrations
of the order of at least 0.01 and more prol)ably 0.1 mole per liter. It seems
unlikely that such high concentrations of freely moving molecules of reac-
tion products should actually arise and disappear during photosynthesis.

. : —
The relationship between the yield of photosynthesis and the yield of

fluorescence has often been presented as a simple either-or; if this were
correct, then whenever P increases, F should decrease and vice versa. In
fact, however, (p is much too small (^^ 0.01) to be an effective competitor
of 7 (^:^ 1 primaiy process, although as many as 4 or 8 primary
for the
processes be needed to reduce one molecule of carbon dioxide). The
may
actual competition is between cp and 5, between the primary photochemical
reaction and the dissipation of energy. The yield of fluorescence can be
considered an index of the value to which these two much more efficient
processes together have reduced the life-time of the excited chlorophyll
molecules. In other words, we can write, in good approximation, instead
of (28.48)
(28.51) <p=^ kf/{k, + kd) and also y ^ kt/ikt + kd); 8 ^ kd/{kt + kd)
Equation (28.51) shows that (p does not necessarily increase by the same
amount by which y decreases (or vice versa), since simultaneous changes
in 5 can change not only the absolute magnitude of the effect but even
its sign. If kt decreases e. g., if the primary photochemical reaction be-
comes altogether impossible, while ka remains more or less unchanged,

fluorescence must of course increase; but if simultaneously with the de-
cline of ki, kd is strongly increased, in other words, the dissipation of light
energy is strongly accelerated by the change in the structure of the pig-
ment complex, the yield of fluorescence, cp, may decline parallel with the
jdeld of the photochemical transformation, 8.
In discussing the theory of light curves of photosynthesis in section A7

of this chapter, we assumed that normally the photosensitive chlorophyll
complex has the composition X • Chi • HZ (or ACO2 Chi A'HR, if we postu-
• •
late direct association of chlorophyll with bound carbon dioxide and the
reductant). In inten.se light or when one or both of the reactants, CO2
and HR, are absent, or when the preparatory catalysts are poisoned, the
chlorophyll complex may go over predominantly into a tautomerized or
chemically changed state. If the normal state is X- Chi -HZ, the likely
changed states are HX-Chl-Z (tautomeric), HX-Chl-HZ (reduced state)
and X-Chl-Z (oxidized state); if the normal state is AC02-Chl-A'RH,
the possibilities include, in addition to the tautomeric, oxidized and reduced
states, also three "starved" states, namely A-Chl-A'R (carbon dioxide-
starved), AC02-Chl-A' (reductant-starved) and A -Chi -A' (totally starvêd
.state)
Furthermore, one can envisage states in which CO2 or are not merely RH
missing, but are replaced by other molecules either also suitable to serve —
as hydrogen acceptors or donors (such as oxygen, or the "substitute oxi-
dants" and "substitute reductants" considered in chapter 8; see, for ex-

ample, page 543, Vol. I) or inert, such as various "narcotics." The latter
may perhaps displace from the chlorophyll complex not only the reactants,
CO2 or R,H, but even their "carriers," A and A'. Franck and co-workers
(1941-1950) concluded, mainly from studying induction phenomena (chap.
33), that narcotizing substances are formed within the cell, e. g., in the
dark, by fermentation, particularly under anaerobic conditions, and in
light, by photoxidation, or by the mechanism which we have repeatedly de-
scribed previously —the reaction of the accumulated oxygen precursors,
"photoperoxides," with oxidizable cellular constituents, a reaction that oc-
curs whenever the removal of these peroxides is too slow, e. g., in conse-
quence of insufficient concentration of the enzyme Eg. This "self-narcoti-
zation" is considered by Franck the main cause of the most striking changes
in fluorescence —when <p increases by a
two or more (c/. fig. 28.50).
factor of
Many instances of an increase in the yield of fluorescence
by adsorption of
"protective" substances have been observed in vitro, although it is by no
means a general rule that all adsorption increases fluorescence. In chapter
23 (page 776) we have seen that association with such substances as lecithin
or oleic acid protects the fluorescence of chlorophyll, while adsorption on
starch, alumina or proteins quenches it more or less completely. It seems
likely that it is the nature and orientation of the forces between the pigment
molecule and the adsorbent that determine whether the effect of these
forces is to permit the excitation energy to spread over a larger number of
degrees of freedom, including those of the associate molecules, and thus
facilitate its conversion into heat, or whether their effect is to orient and
some otherwise freely vibrating or rotating parts in the pigment mole-
stiffen
cule thus making the dissipation of energy within it more difficult.
itself,
Another way in which complexing or adsorption may delay internal dis-

sipation of excitation energy, offers itself
if dissipation occurs only after
the excited molecule assumes a certain configuration (corresponding to the

crossing point of two potential energy curves in the diatomic model).
Temporary dissipation of the excitation energy over a larger number of
degrees of freedom (which becomes possible when the molecule is com-
plexed or adsorbed) can lengthen the average time needed by the molecule
to reach the "critical" configuration.
With chlorophyll fluorescence in vivo, the effect of a typical narcotic
ethylurethan was found to consist in an increase in yield of fluorescence in
ChloreUa by about 25% (fig. 28.49), in a quenching of fluorescence of
Chromatium in the absence of reductants (fig. 28.50A), and a considerable
enhancing effect (up to about -f 40%) on the same fluorescence in the pres-
ence of a reductant (fig. 28.50B). (The yield of fluorescence in the presence
of 3% urethan is the same with and without the reductant about halfway —
:
between the two yields without the narcotic.) Franck suggested that
"self-narcotization" may be a protective device of major importance for the
preservation of plants from destructive photochemical reactions (such as
photoxidations) that are likely to occur whenever the photosynthetic ap-
paratus is for some reason or another prevented from working on its normal
substrates.
obvious from this discussion that we should not be astonished to
It is
find rather complicated changes in the yield of fluorescence when we accel-
erate or retard photosynthesis by changing external factors such as hght
intensity or the concentrations [CO2] or [RH], or by adding various inhibi-
tors.
As discussed previously on page 1013, one would expect, in general,
that, whenever photosynthesis is limited or slowed down by "starvation"

{i. e., by the slo\vness of a preparatory dark reaction), the composition
of
the chlorophyll complex will change, the photosensitive form will be used
up, ki will therefore go down and ^ will increase correspondingly—unless
ka increases simultaneously and so strongly that its increase overcompen-
sates the decrease of k^ equation 28.51). On the other hand, if photo-
{c.J.
synthesis is limited or slowed down by "constipation" i. e., by the slowness

of a "finishing" dark reaction, the composition of the chlorophyll complex
will remain unaffected and fluorescence yield will show no change, unless
one produced indirectly by the "self-narcotization" postulated by Franck.
is
should be recalled here that, according to the criterion we have es-

It
tablished to distinguish preparatory from finishing dark reactions (page
1013), slow return of the photochemically changed form of the chlorophyll
complex into the normal photosensitive form must be considered a prepara-
tonj reaction (since its slowness reduces the rate of the primary photochemi-
cal process) This remains true whether this restoration occurs through a
.
"forward" reaction with the oxidant ACO2} or the reductant ({ A'H20| or

{ ,
H2R in purple bacteria); or by reaction with an intermediary catalyst

such as (28.41b), or by "primary back reaction" such as (28.41a'). Many
of the mechanisms which we have considered involved
of light saturation
the transition of the chlorophyll complex in strong light into a changed
(tautomerized, oxidized, reduced, denuded or narcotized) form. The ac-
—
cumulation of this form which we assume to be photochemically inert
(-y = 0) —
held responsible for changes in the fluorescence yield observed
is
in strong light. We will designate this inactive form generally as {Chl|.

If the quantum yield of fluorescence of the photosensitive form of the chloro-
phyll complex of the inactive form {Chl| is <pi, and the ab-
is ^1, and that
sorption coefficients (and the spatial distribution) of the two forms are the
same, then the observed yield of fluorescence {<p = F/I; number of quanta
emitted per quantum absorbed) is
.
— [IChU]
(28.51A) <p = <Pi + {'Pi <fi)
Chlo
The fluorescence yield light curves <p — f(Ia) are thus linear derivatives
of the function [ { Chi } ]
= /(/«) . In practice, we use as independent vari-
able, the incident light intensity /; since we assume uniform light ab-
sorption (z. e., an optically thin system), we have (c/. equation 28.33) la —
fc*7Chlo. The relation of the fluorescence intensity curve F = f{Ia) to the
fluorescence yield curve <p — /(/„) is:
1'^^'^^!^='
(28.51B) F = ^h = <fj, + if, - î)
Chlo
(la = absorbed flux; F = emitted flux; I is measured in number of ein-

steins per sec. per cm.'^; Chlo in moles/liter.)
It is easily possible to derive fluorescence yield curves for those of the
kinetic models discussed in section A7 in which light saturation is associated
with the slowness of a preparatory dark reaction (including in this definition
the reactions which restore the chloroph3dl complex after its photochemical
change)
We begin with the case in which strong light causes accumulation of
the chlorophyll complex in the carbon dioxide denuded form (such as
A-Chl-A'H20), or in the totafly reduced form (such as HX-Chl-HZ), be-
cause of slow rate of reaction of carbon dioxide (with the acceptor. A, or
with the reduced intermediate, HX). We can use here the simplest mech-
anism of this type (rather than the more elaborate mechanisms discussed
in chapter 27), namely:
k*I
(28.51Ca) ACO.-Chl-A'HzO
+ H2O
> A-Chl-A'H.O + {OH} + {HCO2!
(28.51Cb) A • Chi A'HoO

•
+ CO2 ^ ACO2 Chi A'HaO
•
•
or:
k*I
(28.51Da) X- Chi -HZ
+ H2O
> HX-Chl-HZ + [OH]
^
'
(28.51Db) HX-Chl-HZ + CO2 — > jHCOo) + X- Chi -HZ

The stationary concentration [{Chi}] (= [A-Chl-A'HaO] or fHX--
Chl.HZ]) is:
^*'^Chlo
(28.51E) [{Chl}]=
k'lCO-i] + k*I
For the light intensity at which the yield of fluorescence, <p, is equal to
the arithmetic mean of <pi and ^2 we obtain:
^'^^^''^
(28.51F) /,I =
-
V^^ k*
In this special case, photosynthesis reaches half -saturation at the same in-
tensity as fluorescence The rate of photosynthesis according to schemes
.
(28.51CorD)is:
P = k'k*IC\xU/{k'{C02] + k*I)
and the saturation rate is P"*''- = A;'Chlo, which gives for !}, I the value
(28.51F).
can next consider the case discussed on page 1028, in which light
We
saturation is due to the accumulation of chlorophyll in the reduced form,
HX-Chl-HZ, because of slow primary hack reaction. (In other words, we
assume mechanism 28.21, with the specific assumption that reaction 28.21b
is practically instantaneous.) This leads us (for the simplest case when the
carboxylation equilibrium is undisturbed by photosynthesis) to equations
(28.28) for the rate P, and equations (28.30) and (28.31) for the half-
saturating light intensity of photosynthesis. The concentration of chloro-
phyll complexes in the inactive form is given in this case by equation
(28.26) the midpoint of fluorescence transition again coincides with the
;
half-saturation of photosynthesis.
We
can further consider reaction mechanism (28.41) (with the simpli-
fying assumption of instantaneous "reloading" with CO2 and H2O), in
which light saturation is ascribed to the combined effects of slow restora-
tion of the catalyst Eb (assumed to "stabilize" the first reduction product,
HCO2), and primary back reaction (28.41a'). The equation for
of the slow
the concentration of "inactivated" chlorophyll complexes is in this case
quadratic, and its one significant solution is:
(28.51G) [{Chl|](= [AHCOo-Chl-A'HO]) =

k'k: + kxî - u-*7Chio + Kk*! ,
r/ k'k: + kXEi - kek*ich\o +k k*i \
2k.{k' + k*I) ^ LV 2fce(A;' + k*I) J
+ k,{k'
k:k*ICh]o
+ r
k*I)
The midpoint of the fluorescence transition is:
p - k'K + fceA:e'EE + A:efc'Chlo/2

^^^^^^) •A-' k*{k: + A-eChlo/2)
As could be expected, this midpoint coincides with the half-saturation

of photosynthesis (28.47 A) only in the case K>
A-eChlo, when (28.5 IH)
reduces itself to (28.47Ba). (This extreme case is practically identical
with the one derived above from mechanism 28.21 in both of them, half- ;
saturation is reached when one-half of all chlorophyll complexes are in the

changed state— catalyst Eb not being fully utilized even in the light-
saturated state.) 1/, / differs from ./, / when the EB-Hmitation is significant
(_/. e., when A'g is not 1^ /fgChlo).

:
In the other extreme case, when k^ <C fceChlo, we have:
^^^^^^) V2^ - ;fc*fceChlo/2
Comparison with (28.47C) shows that in this case
^^^•^^^^ 'h^ - V2^ 1,1 + A:;A'eE»B + 2k'K )
in other words, half-saturation of fluorescence occurs later (in fact, 7nuch

later, because fceChlo ^ 2k') than the half-saturation of photosynthesis.
Finally, we can derive the equations for tp and i^J for the "narcotiza-
tion" mechanism (28. 47 A). The photostationary concentration of "nar-
cotized" chlorophyll [{Chi}] is:
/oo r:1T^^ rsriv^ni = kkeEj — {k — 2)fc'^/Chlo + A:X'hIo

+
(28.51K)
,
[{Chi! ]
2(yt - m: + k*i)
rf kkÊj - (k - 2)A-*7Chlo + 1
=
A-^Chlp X k*ICh]l 'A
Lv 2{k - i)(a: + k*i) ) ^ {k- \w^ -I- k*i)\
where k has the meaning defined in eq. (28.47Ha).

This expression reduces itself, as required, to [{Chi}] = at / = 0,
and to [{Chi}] = Chlo at / = oo.
The equation for the midpoint of fluorescence transition, [ { Chi } ]
=
3^ Chlo, is:
.OgriT^
^^^^^^)
. F J _
- C\-iW„{k + 1) + 2AAeEo
V2^ A;*(/t + DChlo
Comparison with equation (28.48) shows that fluorescence can be half-
saturated either earlier or later than photosynthesis, depending on whether
(28.51M) ^ (= F f) is < 1 o^" > 1
(fluorescence is half -saturated when Chi = 3^ Chlo, photosynthesis is

[
{
} ]
half-saturated in this model when [EbOH] = 3^ Eb; the two conditions

are satisfied at the same light intensity only when = 1). A;
The curves representing fluorescence intensity, F, as function of light

intensity can be derived from the various expressions we have obtained for
[ { Chi } ] by inserting them into equation (28.51B) . The resulting equations
are either second or third degree, indicating that the curves F = /(/)
are either hyperbolae or third order curves. They begin at 7 = with the
slope <p, and approach at high /-values the slope ^2 (lower part of fig.
28.27).
A number of experimental fluorescence curves of this type are repro-
duced earlier in this chapter (e.g., figs. 28.29, 43-48, 50). We are more
interested, however, in fluorescence yield curves, tp = /(/) ; a plot of this
kind is shown in the upper part of figure 28.27. This figure indicates that
the "critical" intensity, Ic, as defined by Wassink and co-workers (fig.
28.27) is quite different from the "midpoint" i// —

the latter is situated at
much higher hght intensities. The ^-curves too, often are hyperbolae;
this is, for example true for the kinetic models leading to equations (28.51E)
(slow reaction with CO2), and (28.22) (slow primary back reaction). On
the other hand, equations (28.51G) (Ee-limitation) and (28.51K) ("narco-
tization") mdicate cvbic equations for <p = /(/). This is of interest in con-
nection with Franck's idea of "self-regulation" of photosynthesis: Franck

envisages a narcotization mechanism which would not affect photosynthesis
(and fluorescence) at low light intensities, but would become operative
rather suddenly when a finishing dark reaction ceases to be able to cope
with the photoperoxides produced by the primary photochemical process,
and would shut off a part of the chlorophyll apparatus sufficient to reduce
the formation of photoperoxides to the amount which the limiting reaction
can handle. The assumption of a self-regulating mechanism of this kind is
a tempting hypothesis, because of the general importance which "feed-
backs" and "servomechanisms" have acquired in mechanical interpreta-
tions and imitations of life processes. If such a mechanism were operative,
the curves [{Chi} ]
= /(/) (and with this, also the curves, <p = /(/)) would
have to have sigmoid shapes as indicated by dotted line in figure 28.27.
It was shown above that half-saturation of fluorescence wîll occur simul-
taneously with the half-saturation of photosynthesis whenever the latter
corresponds to one-half of all chlorophyll complexes being in the inactive
state. This will be the case, e. g., when saturation is caused by insufficient
supply of carbon dioxide (or other reactants) . On the other hand, if satura-
tion is caused by slow removal of photoproducts from chlorophyll (e. g.,
by EB-limitation in mechanism 28.41), we can expect half-saturation of
fluorescence to require stronger light than half-saturation of photosynthesis.
This seems to be the case in figure 28.26 (Hydrangea leaf) where half -satura-
tion of <p occurs at about 60 kerg, and that of P at about 30 kerg.
We will now review briefly the experimental results described in sections
1 and 2 {cf. figures 28.24-28.51) in the light of these theoretical concepts.
The occasionally observed Hght saturation of photosjmthesis unaccom-
panied by changes in the yield of fluorescence (illustrated most strikingly
by fig. 28.24) must indicate that saturation was caused by a finishing dark
reaction that did not affect the composition of the chlorophyll complex,
either directly or indirectly (through the formation of a "narcotic"). This
may be an example of pure "catalyst B" limitation, and saturation by
secondary back reactions. It can be asked whether, under these conditions,
a change in be observed at some higher intensity in the saturation
<p will
region, process becomes too fast for some preparatory

when the primary
reaction to keep pace with it. A\Tiether this is the case might depend on
whether the back reactions give products suitable for direct use in the pri-
mary photochemical process, or products that have to undergo again the
slow preparatory catalytic reactions. For example, if the back reaction is:
: :
107G THE LIGHT FACTOR. I. INTENSITY CHAP. 28
(28.52) AHCO2 + A'lIO > ACO2 + A'11,0
or:
(28.53) AHCOa-Chl-A'HO— —^ ACOo-Chl-A'H.O

and if no exhaustion of the photosensitive form has occurred at the time
hght saturation has been produced by Eb deficiency, there is no reason why

such exhaustion should occur if the hght intensity is stepped up still further,
since the products of the back reaction are wiuly to participate again in the
primary photochemical reaction. Experimentally, an increase of fluores-
cence at "supersaturating" light intensities has been noted in several cases
discussed above. This could be explained by assuming, with Franck, that
the back reaction liberates so much energy as to cause the reversal not only
of the first oxidation-reduction step, but also of the carboxylation reaction
(28.54) AHCO, + A'HO > A + CO. + A'HÔ (or A + COo + A' + H.O)
or
(28.55) AHC02- Chi -A'HO > AChlA'HaO + CO2 (or A-ChlA' + CO2 + H.O)
In this way, the products of the back reaction are added to the pool of
free carbon dioxide and water rather than to the immediately available
substrates of the primary photochemical process, ACO2 and A'H20.
We recall that this hypothesis was first suggested by Franck to ex-
plain an entirely different observation —
the "carbon dioxide burst" some-
times observed in the first minutes of illumination {cf. Vol. I, page 207, and
chapter 29, page 1093).
When the yield of fluorescence goes up with increasing light intensity,
as in figure 28.25, and reaches a new steady value, ^2, in the region of the
light saturation of photosynthesis (fig. 28.26), this can be taken as a sign
that saturation is it is thus under-
due to a preparatory dark reaction;
standable why, in McAlister's experiment represented in figure 28.25, this
change was observed in a C02-deficient medium and not in 5% CO2.
The results obtained by Wassink and Kersten with Nitzschia (fig. 28.28)
are puzzling. The above 50 kerg/cm.^ sec. tp decreases rather than
fact that
increases with light intensity could be formally explained by assuming
that, in this organism, the form of the chlorophyll complex that accumu-
lates during intense photosynthesis has a higher value of ki {i. e., dissipates
energy more rapidly), so that the sum h -\- h increases in strong light even
if kt declines to zero. AVhat is more difficult to explain is that in the
absence of carbon dioxide the diatoms retain the high yield of fluorescence
{(Pi) in strong light, while one would offhand expect that, in this case,
the
lower value (^2) would prevail from the very beginning. (It was mentioned
on page 1051 that the curves would be easier to understand if the designa-
tions "with carbon dioxide" and "without carbon dioxide" were exchanged!)
In the case of purple bacteria, several states seem to be needed to ex-
plain the light curves of fluorescence. First of all, the low value of if in weak
hght (the sigmoid shape) needs interpretation. It is probably associated
with the substitution of intercellular hydrogen donors for the external re-
ductants, which occurs while photosynthesis is slow. The coincidence of
the two curves in figure 28.29 seems to indicate that, when photosynthesis
is prevented by the absence of reductants, either chlorophyll accumulates
in one and the same form in the presence and in the absence of carbon
dioxide, or the two forms (e. g., HX-BChl-Z and X-BChl-Z) accumu-
lated under these conditions have a practically identical rate of energy
dissipation, h. In the presence of reductants, the two forms accumulated
with and without carbon dioxide (perhaps X-BChl-HZ and HX-BChl--
HZ) possess, to the contrary, a very different fluorescence capacity. How-
ever, as the light intensity is increased, a further change in the composi-
tion of the complex occurs, leading to the crossing of the curves with and
without carbon dioxide. Figures 28.31-28.35 confirm that the absence of
reductants causes (in the presence as well as in the absence of carbon di-
oxide), the accumulation of a form with considerably increased capacity
for fluorescence (which may be X-BChl-Z, or AC02-BChl-A').
alternative explanation of the effect of reductants on fluorescence
An
can be given on the basis of Franck's concept of "self-narcotization."
Franck assumes that reductants such as hydrogen or thiosulfate intervene
in bacterial photosynthesis by reducing the "photoperoxides" formed by
the primary photochemical process. If the reductants are deficient, the
peroxides accumulate and produce the "narcotic," that blankets the
chlorophyll and causes fluorescence to become stronger. The absence of
C'Oa has less effect in bacteria because they are studied under anaerobic
conditions, permitting no photoxidation. Wassink, Katz, et al. (1938,
1942,1949) explained the effect of reductants by assuming that an "energy
acceptor," capable of taking light energy over from bacteriochlorophyll,
thus quenching its fluorescence, can be formed exclusively by enzymatic
transformation of the rc(hictants. They followed that CO2 must have no
effect on fluorescence at all —whii-h is not true.
The effects of cyanide and on fluorescence (and photo-
of loio temperature
synthesis) can often be explained by assuming that the primary effect
of both is the retardation of the carbon dioxide supply processes. However,
we have seen, in part A, that not all tlie experimental results on cyanide
inhibition agree with this simple explanation; the same is true of the
fluorescence measurements. It is, for example, not clear why the concen-
trations of cyanide needed to markedl}^ affect fluorescence are so much
higher than those needed to inhibit photosynthesis. These and the results
obtained with several other poisons and narcotics require so many ad hoc
explanations that we do not want to attempt them here. Parallel measure-
ment of gas exchange and fluorescence in the presence of various inhibitors
seems to be a very promising approach to the unravelling of the complex
happenings in the photosensitive chlorophyll complex; but the presently
available results are hardly sufficient to warrant a detailed attempt.
—
Franck to whom we owe both the fundamental concepts of fluorescence
and the demonstration of how these concepts can be usefully applied to the
—
study of photosynthesis has WTitten two reviews of this subject (1949,
1951), which contain many observations and interpretations that could not
be covered in the above presentation.
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1080 THE LIGHT FACTOR. I. INTENSITY CHAP. 2R
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,
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B. Light Curves of Fluorescence
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Iowa, 1949, p. 293.
Katz, E., "Chlorophyll Fluorescence as Energy Flowmeter for Photosyn-
thesis," ibid., p. 287.
1950 Franck, J., A7in. Rev. Biochem. (in press).
1951 French, C. S., and Koski, V. M., Proc. Sac. Exptl. Biol, (in press).
Chapter 29
THE LIGHT FACTOR. II. MAXIMUM QUANTUM YIELD OF

PHOTOSYNTHESIS*
In analyzing the light curves of photosynthesis in chapter 28, we did

not discuss the slope of the initial linear part. This is a particularly im-
portant quantity, since it determines the maximuni quantum yield of photo-
synthesis and the maximum conversion of light into chemical energy achieved
in this process. The present chapter will deal with these two subjects.
The definition of maximum quantum yield as the limiting slope of the light curve
at low light intensities implies that this curve has no inflection. An inflection has often
been observed in the light curves of purple bacteria, but it has usually been assumed that
the light curves of algae and higher plants show no such complication. Certain recent
observations (Kok, van der Veen) lead, however, to new doubts concerning the shape of
the light ciu-ves below the compensation point; we will discuss these observations and
their possible significance for the determination of the maximum quantum yield later in
this chapter (page 1113).
If the yield of photosynthesis is expressed in moles of reduced carbon

dioxide (or liberated oxygen), P, and the light absorption, la, is given in
einsteins of absorbed photons, the ratio y = P/Ia is the quantum yield.
(In many papers on photosynthesis, and photochemistry in general, the
quantum yield is designated by the letter cp, which we reserved — (c/. Vol. I,
page 546) —for the quantum yield oi fluorescence.

If the yield is measured by the energy content (heat of combustion) of
the produced carbohydrates, — AHc, and /„ is given in calories, the ratio
e = —AHc/Ia can be called the energy conversion factor. The relation be-
tween 7 and e is:
(29.1) 6 = -AH,ny/NAhv = 3.9() X IQ-'' X„^7 — 4 X lO'^ X,„^7
where AH„, is the molar heat of combustion of one {CH2OI group in carbo-
hydrates (approximately 112 kcal, or 4.69 X 10^^ erg); Na, Avogadro's
number (6.02 X lO^^); h, Planck's constant (6.55 X 10""); and p, the
frequency of light (3.00 X 10^'' /K J-
The concept of the "quantum yield" of a photochemical process arose
from Einstein's application of quantum theory to photochemistry in 1913.
Einstein suggested, in elaboration of Planck's concept of vibrational energy
quanta of electrons in atoms and molecules, that light energy, too, consists
1083
—
1084 THE LIGHT FACTOR. II. QUANTUM YIEL CHAP. 29
of finite quanta (photons); from this, he deduced that the number of

molecules, N, changed photochemically by the absorption of a certain
amount of light, must be equal to the number of absorbed photons, N^^
(Einstein's law of photochemical equivalency). In the following years,
rapidly accumulating rate measurements of photochemical reactions made
it primary photochemical
clear that Einstein's principle applies only to the
process, while the observed over-all rates of photochemical reactions usu-
ally depend on the efficiency of secondary reactions, which follow the pri-
mary photochemical step. Over-all rate measurements therefore only
seldom lead to straightforward confirmation of the equivalency law (in
other words, the empirical quantum yields usually are smaller — or larger
than unity).
Photosynthesis, as the most important photochemical process in na-
ture, naturally came under scrutiny from the point of view of its quantum
yield. The problem appeared particularly intriguing because of the
strongly endothermal character of the photosynthetic reaction. It could
easily be calculated that one quantum of visible light (energy available:
40-60 kcal/einstein) is insufficient to convert one molecule of carbon di-
oxide (and one molecule of water) into a link in the carbohydrate chain
and a molecule of oxygen (energy needed: about 112 kcal/mole). It was
obvious that several quanta must cooperate in the reduction of one mole-
cule of carbon dioxide. The question was: how many (or rather: how
—
few since, from the point of view of reaction mechanism, we are above all
interested in the maximum quantum yield obtainable under the most fav-
orable conditions).
According to equation (29.1), the answer to this question meant also
the determination of the maximum efficiency of plants as converters of
light energy into chemical energy. Over a century ago, in 1845, Robert
Mayer recognized that storage of light energy by conversion into chemical
energy is a most important aspect of plant activity on earth (c/. Vol. I,
chapter 2). We have described in the preceding chapter several investiga-

tions in which the yield of this conversion was measured over considerable
periods of time, and concluded that under natural conditions it is rather
low — of the order of 2-5%.
It was known, however, since Reinke's investigation in 1883 (c/. page
964) that the light curves of photosynthesis are convex; the curvature
sometimes becomes apparent even at very low light intensities (c/. chapter
28, section A2). This means that the energy conversion efficiency and
the quantum yield increase as light intensity decreases. Warburg and
Negelein (1922) set out to determine the maximum quantum yield by
measuring the yield in very Ioav light. Their work marked the beginning
of a new stage in the quantitative study of photosynthesis,
QUANTUM YIELD MEASUREMENTS BY THE MANOMETRIC METHOD 1085
The inverse of the quantum yield (or quantum efficiency, which is the
same thing), is called the quantum requirement. Kegrettably, the first
—
term is often used when the second one would be appropriate for example,
it is said that "the quantum yield of photosynthesis is 4" (or 8, or some
other number, where n > 1), instead of saying that it is V4 (or Vs, or, gen-
erally, 1/w).
1. Quantum Yield Measurements by the Manometric Method

INIost quantum yield determinations of photos>aithesis were carried out
by the manometric method described in chapter 25 (see fig. 25. 3 A). In
this method, the change of gas pressure is measui'cd first above a dai-kened,
and then al)ove an illuminated cell suspension. The net effects obser\'ed
are the result of pressure changes due to the production and consumption
of both carbon dioxide and oxygen. If both the respiratory quotient and
the photosynthetic quotient are unity, the net pressure changes are different
from zero only because of the greater solubility of carbon dioxide in water
(or, still more, in alkaline buffers), as compared with that of oxygen. To
obtain a check on the two quotients, the measurement can be repeated, in
darkness and in light, with a different ratio of gas-filled and liquid-filled
volumes (cf. fig. 25. 3H).
{a) Investigations of Warburg and Negelein
Warburg and Negelein (1922, 1923) were the first to apply the mano-
metric method. They worked with suspensions of the unicellular green
alga Chlorella. (The species was described by them as Chlorella vulgaris,
but subsequent experience makes it uncertain whether it was this species,
or C. pyrenoidosa.) To avoid the difficulties of the measurement of light
absorption in plants caused by scattering (cf. chapter 22), they used dense
suspensions, absorbing practically all the incident light. Consequently,
at any given moment, most of the cells were shaded, and their contribution
to photosynthesis was small; on the other hand, all cells contributed
equally to respiration. For this reason and because of the low light in-
tensities used (of the order of 1000 erg/cm. ^ sec), the total volume of
respiration was larger than that of photosynthesis. (In other words,
Warburg and Negelein worked below the compensation point.) They
noted that the respiration of Chlorella was markedly stimulated by pro-
longed exposure to light(cf. chapter 20, page 564) In order to avoid such
.
changes in respiration during the experiment, Warburg used illumination

periods of not more than 10 minutes, separated by equal or longer periods
of darkness. Because of the sluggish response of the manometer to changes
of gas concentration in the liquid, the determination of the gas exchange
1086 THE LIGHT FACTOR. II. QUANTUM YIELD CHAP. 29
generally requires an interpolation, illustrated by figure 29.1. The uncer-

tainty caused by this interpolation is unimportant for extended illumina-
tion periods, but can markedly affect the results obtained in short experi-
ments, for example, experiments of 5 or 10 minutes. Furthermore, short
illumination periods increase the importance of induction phenomena.
Warburg knew from his earlier work (c/. chapter 33) that an "induction
loss" {i. e., delayed onset of photosynthetic activity) can occiir after dark
periods of the order of several minutes, but he also knew that this effect
disappears if the light intensity is reduced considerably below the satura-
tion region {cf. fig. 33.8). Since in the quantum yield work very low
or, in the notation used in chapter 28 (pp. 1032, 1036, etc.):
,TT ^
, .
forward reaction back reaction
(29.2a) ACOa-Chl-A'HjO > AHCO.Chl-A'OH >
light
^'''^'''"''°"
A'H,0*Chl.ACOf A-Chl-A' + HÔ +A + CO^
Here, asterisks indicate that the compounds formed by back reactions contain consider-
able excess energy and therefore tend to decompose into their constituents. These back
reactions normally occur only in saturating light (they are, in fact, supposed to be re-
sponsible for saturation); but in the first moment of illumination, practically all
AHCO2 formed (even the small amounts produced in weak light) undergoes back reac-
tion,because during this "induction period," certain catalysts have not yet been "re-
activated," and are unable to take care of the products of the first photochemical reac-
tion.
A difficulty of this hypothesis even with a 100% yield of the back reaction,
is that,
the rate of production of ACO2 in must be small compared with the same
weak light
rate in strongly oversaturating light. In the latter case, all intermediates formed in ex-
cess of the saturating rate are supposed to undergo back reactions; and yet, under
appropriate supply conditions, no carbon dioxide limitation is observed, indicating that
either theACO2 complexes formed by back reactions do not dissociate, or the recombina-
tion of A and CO2 is so fast as to prevent any exhaustion of ACO2 (in other words, the
rate ceiling imposedby the formation of ACO2 must be high compared with the full rate
of theprimary photochemical process and not only compared with the rate of the finish-
ing dark reaction).
—
The total volume of the gush which is about equivalent to the quantity of chloro-
—
phyll present in the cells is in agreement with Franck's hypothesis; but the slow re-
absorption of carbon dioxide in the dark (c/. fig. 29.3B, p. 1092) requires an explana-
tion, since thetime course of the "pick-up" (c/. Vol. I, fig. 22) indicates that the car-
boxylation equilibrium C'Oo +
A -^ ACO2 usually is established in a few seconds. It
may be noted that a similar difficulty was encountered in the attempt to attribute the
uptake of radioactive carbon dioxide in the dark (fig. 21, Vol. I) to the same carboxyla-
tion process. Another problem presented by the necessity of a high carbon dioxide
is
concentration (>o%) shape of the carbon

for the "saturation" of the gush, since the
dioxide curves of photosynthesis indicates that the acceptor must be saturated with
carbon dioxide even below 0.1% CO2.
These discrepancies suggest that perhaps the gush and its reversal in the dark are
manifestations of a carbon dioxide metabolism related to respiration and fermentation
rather than to the first step of photosynthesis; but this hypothesis, in turn, fails to ex-
plain the apparent close relation of the carbon dioxide liberated in the gush to the
chlorophyll complex (without such a relationship, a photochemical liberation of carbon
dioxide with a high quantum yield would be difficult to understand).
The "cross-linking" of respiration and photosynthesis at an intermediate reduction
level, between CO2 (L = 0) and carbohydrate (L = 1), e.g., on the level of oxalacetic
or malic acids (L = 0.625 and 0.75, respectively), hypothesized by Calvin and co-workers
(chapter 36), if confirmed, could explain how respiratory decarboxylations might be af-
fected by reactions in the photochemical reaction sequence.
A carbon dioxide burst of the volume observed by Emerson and Lewis
would be largely absorbed in carbonate buffers. As it was, Warburg and
Negelein had decided that acid solutions (e. g., water equilibrated with an
^
atmosphere containing 5% CO2) offer a better promise of full photosynthe-

tic efficiency, and chose to use them instead of the "unphysiological" alkal-
ine buffers. Since only a small part of the carbon dioxide liberated in the
burst can be caught in pure water, the larger part must escape into the gas
space, and the consequent increase of pressure will be interpreted as in-
creased photosynthesis, unless a check on the photosynthetic quotient,
Qp, reveals that the liberated gas is mostly carbon dioxide and not oxygen.
It will be noted (c/. Vol. I, page 31) that we designate the photosynthetic
quotient as Qp and define it as the (positive) ratio — AO2/ ACO2, while
Warburg (and many others) designate the photosynthetic quotient as 7
(using the symbol ip for the quantum yield), and define it as the (negative)
ratio +ACO2/AO2.
The discovery of a carbon dioxide gush by Emerson and Lewis has made
the interpretation of the results of Warburg and Negelein uncertain. The
latter's quantum yield values, calculated from net pressure changes in 10
minute exposures, with the assumption Qp = 1 (or more exactly, 1.09),
turned out to be close to 0.25 or V4- Offhand, this result seemed eminently
satisfactory in consideration of the fact that the reduction of carbon di-
oxide by water involves the transfer of four hydrogen atoms {cf. chapters
3 and 7, Vol. I).
If the value M
had not been so plausible chemically, the fact that this
high yield could be obtained only by following a specific schedule of experi-
ments, combined with special methods of cultivation of the algae, would
perhaps have attracted more attention. At first, using Chlorella cells
grown in full light, Warburg and Negelein obtained only quantum yields
of <0.06. Later they found much higher yields are obtainable with sus-
pensions adapted to weak light. These experiments were carried out in
yellow -f orange light; high values of 7 (up to 0.3) were obtained by extra-
polation to 7 = 0, since the light curves bent markedly even below 1000
erg/cm.2 sec. In a second paper (1923), in which monochromatic fight
was used, the curvature was less pronounced and Warburg and Negelein
calculated, without recourse to extrapolation, quantum yields ranging from

0.20 in blue, to 0.23 in red fight, corresponding to energy conversion factors
from 0.34 to 0.59. The least number of quanta of red fight containing suf-
ficient energy to cover the energy expenditure of the reaction CO2 + H2O
—
O2 + H2CO is three the lowest plausible number of elementary reaction
{ I ;
steps is four (corresponding to the transfer of four hydrogen atoms from

water to carbon dioxide). From Warburg and Negelein's results, it ap-
peared that plants are able to achieve photosynthesis with not more than
four photochemical steps, leaving only a small margin to cover losses of
energy by dissipation into heat, wh'n-h appear inovitaljle in a complicated
chemical process.
When theorists tried to devise a detailed mechanism of photosynthesis,

from that of
plausible not only from the point of view of chemistry, but also
energetics, they found themselves badly hampered by the straight-jacket
into which the limitation to four quanta had put them.
Franck and Herzfeld (1941), in particular, pointed out that, if the reduction of
carbon dioxide has to be brought about by several consecutive photochemical steps (as
in scheme 7.VA), the intermediates must have a certain degree of stabiUty in order to
avoid reoxidatiou while waiting for the stipply of another quantum of light energy. Con-
K(>quently, the ".stabilization energy" of several intermediate products must be added to
the energj' requirements of photosynthesis; and a further allowance must be made for
the heat of formation of the {CO2} complex and the heat of decomposition of a per-
oxide (which is the probable precursor of free oxygen evolved in photosynthesis).
2 [HjOo] + (H; CO}
Lhp I ^,5 kcal
— Oo + (CH2O} + 3H2O
AW. ^ 10 kcal
hHj ^ 10 kcal AHr= 112 kccl/mole
AHj^ a 10 kcal
CO2 + H2O 1
^^•(co^r^-"'^^'^'
(CO2} + H
Fig. 29.2, Energy requirements of a linear four-stage mechanism of

photosynthesis (after Franck 1941). Total energy required Ai? + A/fc +
MI[C02\ + Ai/ii + Ai7i2 + A//13 + Ai/p = 210 kcal. /mole.
The energy relations in photosynthesis, according to Franck and Herzfeld, are illus-
trated by figure 29.2, in which

cOs) (the energy of formation of the carbon dioxide-
Afl'i
acceptor complex), A///j, Aff/j, Aff^/^ (the "stabilization energies" of three intermedi-
ates) and A/7p, the (>nergy of stabilization of the end products (which includes the de-
composition energy of the peroxide, {II2O2}) are shown as additional energy terms, which
together with the accumulated chemical energy, A^c, must be supplied by light.
The stabilization energy required for the prevention of "backsliding" of intermedi-
ates in periods of several minutes (which must pass, in weak light, in a dense suspension
of green cells, between the absorption of two light quanta by one and the same chloro-
phyll molecule) must be of the order of 10 kcal /mole; the heat of formation of the CO2I {
complex was estimated in chapter 27 as >20 kcal/mole. Altogether, three intermediates,

the ICO2} complex and the primary peroxide, must increase the energy requirements of
photosynthesis from 112 kcal/mole to probably as much as 210 kcal/mole whereas 4 —
einsteins of red light (X = 660 m^) provide only 170 kcal.
The energy requirements are somewhat different if a "pyramidal" mechanism is
postulated instead of the "linear" reaction sequence represented in figure 29.2 {i.e., if it
is assumed that four light quanta produce four identical pairs of intermediates, which
then undergo dismutation by dark reactions, finally giving one pair of finished products,
[CH2O] + O2; cf. Vol. I, pages 156, 158 and 164). In this case, the four quanta re-
quired to reduce one molecule of carbon dioxide can be absorbed by four different chloro-
phyll molecules. (The same result can be achieved by other physical or chemical mech-
anisms permitting a "collection of quanta" absorbed by several pigment molecules in
one "reaction center." These "photosynthetic unit" theories will be presented in
chapter 32.) In this case, the total energy requirement is obtained by adding to the
accumulated energy (112 kcal/mole) approximately 20 kcal liberated in the formation
of the [CO2] complex, and the energy amounts liberated in the several dismutations (or
other "quanta-collecting" processes). It may be noted that one dismutation reaction
(dismutation of a peroxide, yielding an oxide and free oxygen) was included also in the
"linear" scheme. The dismutation of hydrogen peroxide liberates as much as 46 kcal
per mole O2 (Table ll.I, Vol. I); but dismutations of organic compounds, such as the
Cannizzaro reaction, are less exothermal (about 10 kcal/mole; cf. Table 9.III, Vol. I).
Even so, three such dismutations, together with one dismutation of a peroxide, will
bring the total energy requirement of the "pyramidal" reaction scheme up to the same
210 kcal, which were estimated above for the "linear" reaction sequence.
For 16 years, the "4 quanta mechanism" of photosynthesis ^vas the ob-
ject of admiration and the source of headaches for those who approached
the problem of photosynthesis from the point of view of energy conversion.
During this time, no serious attempts were made to check the experimental
foundations of this mechanism, and the results of Warburg and Negelein
were considered final.
We will see in the next section that even during this time some measure-
ments were made with the higher plants that gave considerably lower quan-
tum yields; but because of less suitable objects and less precise methods,
they were not considered to throw doubt on the validity of Warburg and
Negelein's results. Beginning in 1938, however, a series of investigations
appeared, in which the photosynthesis of the same algae as used by War-
burg was studied by several methods (gas analysis, polarography and calori-
metry) in the laboratories of the University of Wisconsin these measure- ;
ments gave rather widely scattered results, but the yields were invariably
much lower than 0.25. The maximum quantum yields observed in this
work (to be described in some detail in section 2) were of the order of 0.1.
These publications induced several investigators to repeat Warburg and
Negelein's determinations, adhering as closely as possible to the original
technique.
Rieke (1949) used monochromatic light (mercury lines 546 and 578
m^) and an "integrating box" for the determination of light absorption.
The pretreatment of the algae (adaptation to weak light), the light intens-
ity (about 1000 erg/cm.- sec.) and the illumination periods (10 minutes)
—
were the same as in Warburg's work. The quantum yields calculated from
ten experiments at 578 m/u ranged from 0.18 to 0.24, and those calculated
from six measurements at 546 mn, from 0.17 to 0.20.
Thus, Rieke found Warburg and Negelein's results reproducible, but
only by strict adherence, not merely to the original method of culturing
of the algae, but also to Warburg's schedule of illumination. Wassink,
Vermeulen, Reman and Katz (1938) noted another peculiarity—the quan-
tum yields determined according to the procedure of Warburg and Nege-
lein, —
were affected by temperature they increased from 0.11 to 0.20 when
the temperature was changed from 0° to 29° C.
Another peculiar observation was made by Rieke, who found that the
kind of water used in the preparation of the algal culture had an effect on
the quantum yield. Emerson and Lewis (1938, 1939) confirmed this.
They used water from seven different sources, and obtained, under other-
wise identical conditions, variations in the quantum yield from 0.16 to
0.27.
The lowest values were obtained in glass-distilled water; addition of a stock niixture
of "microelements" (B, Zn, Co, Mn, Mo, Cr, Ni, Co, W, Ti, V) increased it markedly;
a similar result was achieved by an increase in the amount of ferrous sulfate in the nu-
trient mixture (apparently, this compound contained all the micronutrients as
impuri-
ties).
Emerson and Lewis found that, for securing the highest yields, the cells
had to be grown for 5 or 10 days at 15-20° C, 20 cm. from four grouped 60
watt lamps followed by 3 days 30 cm. from a single 100 watt lamp. In
agreement with Wassink and co-workers, they found a temperature effect
the highest quantum yield was observed at 10° C. At least 5% CO2 had
to be present during the quantum yield measurement, and the light in-
tensity could not exceed 350 erg/cm. ^ sec.
These experiments could have been interpreted as indicating possible
reasons for the low yields observed at Wisconsin, and thus supporting the
validity of the results of Warburg and Negelein, if a new difficulty had not
appeared. Emerson and Lewis found that, by combining all the favorable
factors, 7 values could be obtained that were considerably above Warburg
and Negelein's value of 0.25. With 10 min. illumination periods, 7 values
up to 0.31 were obtained; and these were further increased by making
the illumination periods even shorter-. Since even a quantum yield of M
presented grave difficulties from the point of view of thermochemistry,
yields of one third or higher were clearly incompatible with the accepted
over-all reaction of photosynthesis.
Emerson and Lewis suspected (1938, 1939) that the oxygen production
in the first minutes of illumination may occur by reduction of accumulated
intermediates of dark metabolism, rather than of carbon dioxide, and thus

require less energy. This caused them to inquire into the value of the
photosynthetic quotient in the first minutes of illumination. It was in this
12
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inquiry that they noticed the carbon dioxide "burst" (1040, 1041), and
it could have been responsible for the
suggested that failure to recognize
large pressure changes observed, and the high quantum yields calculated by
Warburg and Negelein.
Figure 20.3, taken from Emerson and Lewis (1041), shows the rate of
pressure changes as observed in minute-to-minute measurements in two
vessels with different liquid gas ratio. : It indicates that upon illumination,
an gush occurs, which lasts, in the light of (ho particular intensity
initial
used, for about throe minutes, and then gives place to a more or less steady
rate of pressure change. In dark, too, the steady rate of gas consumption
is not established at once; after rapid, irregular variations, a rather ex-
tended, slow decrease in the rate is observed; subsequent experiments
have shown that it takes about one hour for the rate of gas uptake to be-
come quite steady.
The quantum yields of Warburg and Negelein were obtained by averag-
ing the pressure changes over ten minute periods, which included the time
during which the gas burst could have occurred, according to figure 20.3A.
Obviously, values of the quantum yield calculated in this way could be de-
ceptive, and higher averages could result from the use of periods shorter
than ten minutes.
By comparing the curves obtained with two vessels, Emerson and Lewis
(1040, 1041) sought information as to the relative role of oxygen and car-
bon dioxide in the gas burst, and in the extra gas consumption in darkness.
They found (fig. 20. 3B) that both were due to carbon dioxide and not to
oxygen; the absorption and liberation of the latter (solid line in fig. 20. 3B)
showed only minor disturbances, which could perhaps be attributed to
uncertainties in the evaluation of the measurements.
The factors that Warburg and Negelein, Rieke and Emerson and Lewis
have described previously as indispensable for the realization of the highest
quantum yield were found by Emerson and Lewis to affect mainly or ex-
clusively the carbon dioxide gush.
The carbon dioxide concentration in the medium affected the quantity of carbon di-
oxide taken up in the dark, and therefore also the amount of the gas released in the light.
This offered an explanation of the observation that large concentrations of carbon di-
oxide (such as 5%) were needed to obtain high quantum yields. (The light curves for
different values of the parameter [COa]— c/. figures 28.1 to 28.5— indicate that the latter
should be without influence at such low light intensities.)
A similar consideration applies to the role of temperature. It is known {cf. Figs.
28.6-28.8, and chapter 31) that at low light intensities, when the photochemical process
proper limits the over-all rate of photosynthesis, changes in temperature have no in-
fluence on the maximum quantum yield. Thi' observed effect of temperature contra-
dicted this experience. Now, it became likely that the effect of temperature was due to
its influence on the carbon dioxide uptake in the dark and its subsequent disengagement
in the light, and not on photosynthesis itself.
L
The pretreatment of the algae also seems to be much more important for the carbon
dioxide gush than for the steady rate of photosynthesis. Cells having a high rate of
respiration produced a greater carbon dioxide gush than cells with a low rate of
respiration; by culturing Chlorella cells in dim light throughout, cells with low respira-
tion, showing almost no gush, could be obtained.
When the carbon dioxide giish was neutralized by substituting carbon-

ate buffers for carbonic acid solution, Emerson and Lewis obtained quan-
tum yields of the order of 0.1, independently of the previous illumination
and the kind of water used in its prepara-
of the cells, the age of the culture
tion. —
(However, the presence of certain microelements ^particularly man-
—
ganese still appeared to be important.)
The conclusions of Emerson and Lewis agree with the findings of Daniels and co-
workers (1939), who grew Chlorella with the addition of soil extracts, of a nutrient solu-
tion of 28 elements, or of sea water, in lake water, well water and distilled water, with-
out appreciable changes in the quantum jdeld.
Emerson and Lewis did not calculate quantum jaelds from measurements of the
type illustrated by figure 29.3B, but merely drew from the evaluation of these experi-
ments the conclusion that with Chlorella the quantum yield of oxygen liberation (and of
carbon dioxide uptake, once the burst is completely over) does not differ significantly
in acid phosphate buffer and in alkaline carbonate media. If this is so, then the abso-
lute determination of the quantum yield is better carried out in carbonate buffer, where
all effects caused by carbon dioxide exchange are eliminated and therefore no need arises
for the use of the two-vessel method. The latter depends on comparatively small
differences, and has a correspondingly low precision.
The quantum yield measurements made by Emerson and Lewis with Chlorella in
alkaline buffers consistently gave values between 3^ and }{i; and we will see below
that Warburg, Burk and co-workers have since confirmed this result (leaving
Eichhoff as the only investigator to have claimed that quantum yields of the order of
34 can be obtained with Chlorella in carbonate buffer).
A set manometric quantum yield determinations with Chlorella

of
pyrenoidosa was made by French and Rabideau (1945) as a check on their
measurements with isolated chloroplasts (cf. section 4). Using carbonate
buffer No. 9 as medium they found in red light (660-720 m/x) y values be-
tween 0.063 and 0.113, or from 9-16 quanta per reduced carbon dioxide
molecule, with no significant variations between 1.4 and 8 kerg/cm^ sec.
According to Emerson and Lewis, the true maximum yield of photo-
synthesis does not change much from species to species. This is indicated
by the similarity between the above-mentioned results with Chlorella and
Gabrielsen's obsei-vations on the leaves of the higher plants (page 1118),
and confirmed by Emerson and Lewis' measurements listed in table 29.
These measurements were made in carbonate buffers after preliminary —
tests had shown that in none of the organisms used was the yield in phos-
phate medium significantly higher than in carbonate buffer.
y (at ca
: 8
2
6
7
in yellowsodium light, the yields were approximately independent of the

carbon dioxide concentration (1-9%), and of temperature (17-25°
initial
C). The light curves remained fairly straight up to 10 or 20 kerg/cm.^

sec. The quantum yields were calculated from the rates observed at 10
kerg/cm.2 sec. The resulting 7 values are given in Table 29.11. The
smallest I/7 values measured were of the order of 11, corresponding to
7 < 0.09.
Table 29.11
Quantum Yields of Oxygen Production by Horticultural Plants"
(after Wassink 1946)
Nunaber of
Plant 1/7 mea'surements
Strawberry 12 1-17 .8
. 23
Kohlrabi 13.5-15.4 5
Chinese cabbage 10 8-14
. . 8
White succory 13 6-16
. . 4
Tomato 16.0-43.5 17
Cucumber 13 8-24
. . 5
Endive 21.8 2
Asparagus 14 6-18
. . 4
" At 10 kerg/cm.2 sec.
The significance of the variations of the quantum yield of Chlorella

with wave length will be discussed in chapter 30, primarily from the point
of view of the function of carotenoids in photosynthesis. Most experi-
ments on the quantum yield of photosynthesis of ''colored" algae (brown,
red or blue-green) were carried out with a similar aim in mind elucidation —
of the role of phycobilins and carotenoids. As far as the absolute value
of the maximum quantum yield is concerned, there seems to be no funda-
mental difference between these algae and the green plants, as is shown by
the following observations
Emerson and Lewis measured manometrically the quan-
(1941, 1942)
tum by the blue-green unicellular alga Chroococcus,
yield of photosynthesis
and found a maximum 7 value of about 0.08 in red Hght. Working with a
suspension of diatoms {Nitzschia closterium) Button and Manning (1941)
,
found an average quantum yield of 0.063, with single 7 values up to 0.1.

Wassink and Kersten (1944) used another diatom, Nitzschia dissipata, and
calculated the quantum yields from manometric measurements, with il-
lumination periods of 30-60 min., in sodium light of about 10 kerg/cm.^ sec.
(The light curves obtained by these authors showed only small deviations
from linearity at these comparatively high intensities; of. fig. 28.5).
From one third to one half of the incident light was absorbed in the vessel.
In five experiments at 25° C, and one at 6° C, I/7 values from 11.4 to
13.9 \ver(; obtained, witli an average of 12.9, or 7 = 0.078, and 7,„ax =
0.09.
Tanada (1951) made a systematic study of the quantum yield of the di-
atom Nayicula minima in dependence on ^va^"e length. This Mork will be
described in chapter 30 (p. 1173) what is significant for the purpose of the
;
present discussion is that the maximum yield obtained in carbonate buffer

No. 9 was 7o = 0.11(0.05), corresponding to a quantum requirement of
I/to = 9; and that the yield in acid phosphate buffer appeared to be,
at all light intensities, 5-10% lowei^ (and not 10-20% higher, as in ChloreUa)
than that in alkaline carbonate buffer. In this case, Warbui'g's attribution
of a ciuantum requirement of ^^ 10 to a ''nonphj'siological" pll does not
seem plausible.
Similarly to Kok Tanada found the quantum yield to depend
(p. 1113),
on the age Both the quantum yield in weak light and the
of the culture.
maximum rate in strong light dropped sharply from their original values
(7o = 0.115, P™^" = 3.0 mmÔ./mm^ cells hr), between the sixth and the
eighth day of cultivation (70 = 0.07; P"'^" = 1.0 on the eighth day); the
decline continued steadily but more slowly for the next 12 days until, on
the 20th day, 7u was down to 0.0() and P""*^ was down to 0.75.
We thus see that a whole series of manometric determinations of the
maximum quantum yield, applied to green higher plants, green algae,
blue-green algae and diatoms, and made in acid solutions as well as in car-
bonate buffers, gave ± 0.02. With the exception of
7„iax values of 0.1
Emerson and Lewis' measurements, which have revealed the carbon di-
oxide burst, they were all carried out under conditions when the burst,
if it did occur at all, was either absorbed by the medium or minimized by
the averaging of results over an extended period of time. Many of the

experiments were performed in somewhat stronger light (up to ten times
that used by Warburg), and this, too, is Hkely to minimize the effects of
the burst. The experiments of Emerson and Lewis, and of Rieke, carried
out under exact adherence to the Warburg-Negelein procedure, showed
that Warburg's results can be duplicated, if the carbon dioxide burst is
treated as part of normal gas liberation by photosynthesis.

We now turn to the other side in the controversy — investigations in
which Warburg and Negelein's results were confirmed imder conditions
which did not seem to admit of the interpretation suggested by Emerson
and Lewis.
First, we have to mention the investigation by Eichhoft' (1939), carried
out in Noddack's lalioratory. Iilichhoft" suspended Chlorclla cells in car-
bonate-bicarbonate buffers and measured the quantum yield after a prelimi-
nary illumination for 15 or 30 min. (obviously these two precautions should
have prevented the carbon dioxide burst from affecting the results) Eich- .
hoff worked either with a "dense" suspension of ChloreUa pyrenoidosa,

absorbing 67% band 10 mju wide at 650 ran, isolated by a

of red light (a
Christiansen dispersion and 39% of green light (567.5 mû), or with a
filter)
"thin" suspension absorbing 22% of red light; the absorption was deter-
mined by means of the "ellipsoid photometer" (c/. chapter 25, page 844).
Each run included (a) a "dark adaptation" period, (6) a period in which
respiration was measured, (c) a "light adaptation" period, (d) an illumina-
tion period, (e) a second "dark adaptation" period and (/) a final period of
respiration measurement —
each period lasting from 15 to 30 min. The
illumination intensity was high enough for photosynthesis to exceed respi-
ration (from 500 to 5000 erg/cm. ^ sec). The quantum yields obtained
varied between 0.25 and 0.19, for both dense and thin suspension, with the
lower value (0.19 to 0.22) observed only at the higher light intensities and
interpreted as indications of an incipient light saturation. Similar y
values were found at 567.5 m/x. The quantum yield was found to be con-
stant over a wide spectral region, including the near infrared (where other
observers found no photosynthesis at all; cf. chapter 30, page 1155).
According to a review by Franck and Gaffron (1941), Emerson and

Lewds, as well as Rieke, have tried to imitate Eichhoff's experiments (par-
ticularly with respect to the method of cultivation of the algae), but were
unable to obtain the high yields claimed by him.
One peculiar feature of Eichhoff's light curves {cf. fig. 30.7) is the early saturation
in red light. 70% of maximum photosynthesis is reached, according to these curves,
with an incident intensity of only 3 kerg/cm.^ sec. Eichhoff's figures suggest that as
little as 5 "energetic meter candles" of red light (he calls a monochromatic energy flux
equal to the total "white" flux from a Heffner candle an "energetic meter candle") are
equivalent, as far as photosynthesis is concerned, to 15 klux of white light from a 500
watt incandescent lamp! The latter, according to page 838, corresponds to a flux of at
least 60 kerg/cm.^ sec, counting only the photosynthetically active region (400-700 m/i),
while 5 "energetic meter candles" are equivalent (using Gerlach's value for the radiation
of a Heffner candle) to only 4.7 kerg/cm.^ sec. Even though the suspension may absorb
red light three or four times more efficiently than the (infrared- free) white light, the dif-
ference between the amounts of red light and white light required to bring about the
same rate of photosynthesis remains striking. It suggests that the absolute intensity
of the red light might have been underestimated by Eichhoff by as much as a factor of
three or five. If this was the case, all quantum yields calculated by Eichhoff must have
been in error by the same factor (since no quantum yield determinations were made in
white light).
More recently, Warburg (1946, 1948) undertook, with Kubowitz, to

repeat the original experiments of Warburg and Negelein, taking into con-
sideration Emerson's criticism. Warburg calculated that, in order to ac-
count for his 1923 results in the way suggested by Emerson, the average
photosynthetic quotient during the 10 min. exposure must have been Qp =
AO2/— ACO2 = —0.26. (In other words, four volumes of carbon dioxide
must have been produced in light for each volume of oxygen liberated.)
The value used by Warburg and Negelein, Qp = +1.09, was determined

by gas analysis, in much stronger light than was used for quantum yield
determinations. Warburg now redetermined the Qp value in less intense
light, manometrically, by means of two sets of measurements in one vessel
filled with two different volumes of liquid (5 and 8 ml.). He found a Qp

value of +1.07, practically identical with the quotient used by Warburg
and Negelein.
Furthermore, Warburg and Kubowitz could find no evidence of a "gas
burst" in the first few minutes of illumination, except for a comparatively
small effect, observed at the higher light intensities, especially when foam-
ing occurred in the reaction vessel (c/. fig. 29. 4A).
With the belief in the validity of the original experimental procedure
thus strengthened, Warburg and Kubowitz proceeded to make new de-
terminations of the quantum yield by the The vessel
one-vessel method.
was not silvered to better observe bubble formation (which Warburg con-
sidered the most serious source of error) readings were made without inter-
;
ruption of shaking. The light used was mostly the yelloAV mercury lines
(578 m^) with an intensity of 325-2920 erg/cm. ^ sec.
To minimize caused by sluggish gas exchange, a smaller fluid
effects
volume was used than in 1923 and two glass beads were put into each vessel
to act as stirrers. Curves such as those in figure 29.4B and C were con-
sidered by Warburg as confirmation of the interpretation of pressure dis-
turbances at the beginning of the light and dark periods, as consequences
of this sluggishness. He described these disturbances as "symmetric,"
meaning that the two disturbances cancelled each other and the y values
therefore were the same, whether they were determined from the steady
rates, omitting the measurements in the first few minutes of light e., (?'.
from slope AB), or by interpolation, as in figure 29.1 {i. e., from slope CD).
This procedure is equivalent to integration of the gas exchange over the
whole period of the experiment, including the two transition intervals.
This obviously does not apply to the curve in figure 29. 4A, where the pressure in-
crease in the first minute is faster than afterward. Calculation from the steady*
state (slope AB) gives in this case a I/7 value 25% higher than that obtained by inte-
gration (slope CJ)).
Table 29. Ill summarize.-^ the results. Warburg concluded from these
experiments that the limiting quantum yield in weak light is 0.25 and that
it declines to about 0.20 at 1500 erg/cm.^ sec. He suggested, as general
explanation of the smaller values found bj^ other observers, failure to cul-
ture algae of the highest efficiency; but his description of the methods of
culture revealed no significant difference from those used by Emerson or
Rieke.
Table 29.III
Quantum Yields after Warburg (1946, 1948)"
significant carbon dioxide burst could have occurred during the iUumina-
tion period. However, this procedure would only be permissible if the gas
exchange in light were the result of the superposition of a photochemical
process (photosynthesis, with possible addition of a carbon dioxide burst)
upon a dark process whose rate is the same in darkness and in light. This is
usually assumed to be true of respiration (although some doubts exist even
here) ; but it is not true of the reahsorption of the carbon dioxide burst (since
this process occurs only in the dark). By neglecting this component of the
gas exchange in the dark after a period of illumination, one automatically
eliminates from the calculated "light effect" a part, if not practically all,
of the carbon dioxide burst — in the same way in which the effects of the
sluggishness of the manometer are eliminated in the procedure illustrated
by figure 29.1; no wonder that the ratios Qp for the calculated "light
effect" prove to be close to unity. (Whether the elimination of the burst is
practically complete or only partial, depends on what fraction of the burst
is reabsorbed during the dark period utilized in the calculation of the "light
effect.")
To decide whether a significant carbon dioxide burst does occur in light
(and is reabsorbed in darkness), the ratios AO/ACO2 should be calculated
for the illumination and the dark period separately instead of calculating
them directly for the "light effect." The ratios Qdark and Qught might
—
each be quite different from 1 and yet, the ratio "Qp" for the "light
effect" might show no significant deviation from unity. Thus, the method
of calculating Qp used by Warburg (1948) to prove the absence (or at least,
practical insignificance) of the carbon dioxide burst is inappropriate for this
—
purpose even if the experimental data used had been adequate.
Emerson and co-workers argued, however, that the experiment itself
was open to criticism. They pointed out that the value Qp = 1.07 was
derived from measurements lasting for about 40 minutes. The plot given
by Warburg shows that if only the first 10 minutes of these measurements,
i.e., the period of quantum yield measurements, were taken into considera-
tion, much smaller values of Qp would have been obtained. Furthermore,

the Qp measurements were made in light of 3780 erg/cm.^ sec. (ten times as
strong as that at which quantum yields close to 0.25 were obtained) and
in red light, while the 7 measurements were made in yellow^ light, Avhich is
considerably less strongly absorbed.
The conditions of Qp measurement differed from those of 7 measure-
ment also in temperature (20° C. instead of 10° C), carbon dioxide con-
centration (8% CO2 in O2, as compared with 5% CO2 in air) and volume of
respiration (twice as high in quantum yield measurements as in Qp meas-
urements, indicating different culture conditions) It was mentioned before
.
;
that the voUime of the carbon dioxide burst depends on all these conditions
the .statement that theQp measurements were made "under the conditions
of quantum yield determinations" was therefore not justified.
Rabino witch (1947) pointed out that experiments show the integrated
volume of the "burst" to change only little with light intensity, the main
effect of the latter being on the suddenness of the burst. This relation is
to be expected for photochemical emptying, with a high quantum yield

(perhaps as high as 7 =1) of a "carbon dioxide reservoir," containing a
finite volume of carbon dioxide (the exact volume being dependent on con-
ditions that prevailed prior to illumination) . If the volume of the reservoir
corresponds to about one molecule carbon dioxide per molecule chlorophyll,
the time required for complete emptying must be of the order of the time
required for each chlorophyll molecule in the suspension to absorb a quan-
tum of light; in the dense suspensions and in the low light used for the
quantum yield determinations, this time is of the order of ten minutes (c/.
chap. 32) ; this is then the expected duration of the burst. In stronger
light, the burst will be proportionally shorter.
If the volume of the burst increases only little or not at all with light
intensity, its importance at 3780 erg/cm.^ sec. would be much smaller

than at 320 erg. /cm. ^ sec. (where the value 7 = 0.25 was found).
Emerson and Nishimura (1949) criticized also other experimental
aspects of Warburg's work. They pointed out that the use of equal liquid
volumes and different gas volumes in the two-vessel method (Emerson and
Lewis, cf. fig. 25. 3B) assured better comparability of the gas exchange than
Warburg's use of a single vessel filled with different amounts of liquid (since
the efficiency of gas exchange between the two phases depends on the
volume of the liquid). Objection was raised also to Warburg's time
schedule, which involved consecutive runs first with the more concentrated
and then with the more dilute suspension. Because of continuous change
in the rate of respiration of cell suspensions, only simultaneous exposure
and darkening of tAvo aliquots of the same cell material could vouchsafe the
required high degree of their physiological comparability. Emerson and
Lewis themselves did not quite meet this requirement: they, too, worked
first with one, and then with the other vessel, but in contrast to Warbvu'g,
they used a fresh aliquot of the stock suspension for each experiment (con-
sidering this a less objectionablecompromise than the use of a single sample)
first measurements in a smaller volume of liquid and, after
for a series of
dilution, for a second series of measurements in a larger volume.
The discussion of these apparently minor details points to the great
practical difficulty of the (theoretically so simple) two-vessel method: it
rests on the assumption that the obsei-ved difference between the two
pressure changes is due entirely to different relative volumes of liquid
and gas. Even a very small difference in the actual amounts of gas pro-
duced (or consumed) in the two vessels, or in the speed with which this gas
is transferred into the manometer, can lead to large errors in calculation.
Several reasons for such variations can be anticipated. Small physio-

logical differences may exist between samples taken at different times from
the same stock solution or may arise in the course of the experiment.
C'Onsiderable discrepancies of light absorption can bo caused by different
position of the two vessels in the light beam or by dilforcnces in their
shape and wall material. Shaking thins out the liquid layer in the middle
of the vessel, and the consequent incompleteness of absorption depends on
the total amount of the liquid present and on the shape of the vessel.
In 1948-1949, an unsuccessful attempt was made to settle the quantum
yield controversy by a combined Warburg and Emerson in the
effort of
latter's laboratory. Subsequently, in the summer of 1949, Warburg, Burk
and co-workers (1949''^, 1950^-^) carried out quantum yield measurements
by the two vessel method (this time with equal liquid volumes) at the Na-
tional Cancer Institute in Bethesda and at the Woods Hole Marine Bio-
logical Laboratory. In these experiments, single yields as high as I/70 =
0.44 (70 = 2.3) were observed; even the average yield was markedly above
0.25. The conditions under which these high jnelds have been obtained
were quite different from and, in some respects, opposite to those which had
been recommended bj'^ Warburg and Negelein in 1923.
Cell Culture. The reduction of light intensity in the last day of culti-
vation, recommended by Warburg and Negelein to adapt Chlorella cells
to weak light, was discarded by Warburg and Burk. Another precaution,
called unimportant by Warburg and Negelein, was now found to be es-
sential: fast bubbhng of the carbon dioxide-bearing gas through the
culture bottle, preventing the cells from settling out, and thus assuring
adequate supply of oxygen and carbon dioxide to all of them.
Very concentrated suspensions were used: 0.3 cc. cells in 7 cc. culture
medium (phosphate buffer, p}l 4.9, saturated with 5% CO2 in air). (In
1948, Warburg used only 0.1 cc. cells in 5 or 9 cc. solution.) This was done
to ensure complete light absorption, despite an increased rate of shaking,
which created a more pronounced "hole" in the center of the reaction vessel;
—
but the respiration correction the main source of uncertainty in measure-
—
ments of this type was thus made even larger than before.
Response of the Manometer. Compared to the 1923 experiments,
the efficiency of shaking was increased (horizontal, back-and-forth motion
of a rectangular vessel with an amplitude of 2 cm., 150 times per min.).
It was asserted that stirring was thus made so effective that the response
of the manometer to gas production (or absorption) in the liquid was
l)ractically instantaneous. No correction (of the type illustrated in fig.
29.1) was therefore used to account for diffusion through the liciuid and
the exchange between the two phases. Instead, the yields were now cal-
culated from manometer readings made at the very moment of changing
from darkness to light, or from light to darkness. Errors caused by
"physical lag" had been considered of prime importance in 1923 and 1948;
in some examples given in these earlier papers the calculated quantum
yields would have been quite different without correction for this lag.
Time Schedule. The two vessels were filled simultaneously with ali-
quots of the same culture, and exposed alternatively to the same beam
of light (e.g., 10 min. light on vessel I, then 10 min. light on vessel II,
then again 10 min. light on vessel I, and so on). Both vessels (total
volumes 14 and 18 cc, respectively) contained the same amount of liquid
(7 cc). It was argued that whatever physiological differences may have
existed between the cells in the two vessels during the first exposure
(because of a "phase difference" of 10 min.), must have disappeared after
several light-dark cycles. This is plausible; however, Emerson and co-
workers found that at least five or six (10 min. light + 10 min. dark)
cycles may be needed to eliminate the initial difference, while in many of
Warburg and Burk's published experiments (cf. table 29. IV) only 2 or 3
cycles were used. The alternate exposure schedule was altogether aban-
—
doned in almost one half of all experiments namely those in which
"background" illumination was used to compensate respiration.
Light Measurement. No physical determination of light intensity
was made by Warburg and Burk. (Bolometers had been used in earlier
experiments, both by Warburg and by Emerson.) Instead, light intensity
was determined by means of the ethyl chlorophyllide - thiourea actinom-
eter, for which a quantum yield of 1.0 was previously found (bolo-
metrically) by Warburg and Schocken in Emerson's laboratory (cf.
chap. 35). The quantum yield of the actinometer is known to decline
with increasing light flux, particularly >0.1 /zeinstein/min. Many runs of
Warburg and Burk were carried out in stronger light; the intensity of
the beam was reduced in these experiments to about 0.1 êinstein/min.
by means of calibrated wire screens before it was directed on the actinom-
eter.
In Warl)urg's 1923 and 1940 measurements, the
Light Intensity.
use of very weak incident, light was considered important, since the
quantum yield was found to decline significantly (cf. fig. 29.8) with in-
creasing light intensity, beginning as early as at 1000 erg/cm. ^ sec. (about
0.03 )ueinstein/cm.2 min.). In the Warburg-Burk work, much higher
incident light intensities werc^ uschI: instead of uniform illumination (jf
almosi the whole boltoin aiea, as used in earlier experiments (1923, 1948),
— —
a sharp beam was now thrown on the bottom of the vessel (cross section
of the beam, about 3 cm. 2; bottom area, 8.3 cm. 2). The total light flux
(red light, 630-650 m/x) was 0.2-0.6 Meinstein/min., i.e. 0.07-0.2 /xeinstein
per cm. 2 min. ca. ten times higher than the intensity at which quantum
yields of 0.25 had been obtained in 1948. Because of the extremely high
density of the suspension, practically all this light was absorbed within a
1 mm. thick bottom layer (0.3 cc.) of the suspension; thus, at any given
time, >95% of the cells were in darkness, while <5% were exposed to
light, the incident intensity of which was close to the saturating value
(the photosynthesis of light-adapted Chlorella is saturated, in red light,
in a flux of about 0.5 jueinstein/cm.^ min.).
Intermittency Effect. The finding of the highest quantum yields
ever observed when the was
almost saturating intensity
incident light of
appears startling. Warburg, Burk and co-workers explained this paradox
by the intermittency of illumination because of fast shaking, individual
:
cells remain only for a very short while in the illuminated zone, and then
plunge into darkness. (Assuming uniform stirring, each cell must spend
>95% of the total "illuminationtime" in darkness, and less than 5% in
Hght). Warburg and Burk proclaimed as a "new principle" that this
type of intermittency of illumination permits maximum light utilization.
This assertion is not easily reconciled with the results of experiments in
flashing light, to be discussed in chapter 34:
According to these experiments, intermittent illumination cannot in-
crease light utilization above the maximiun value possible in steady low
light. All that intermittency can do is to bring the quantum yield in parti-
ally oreven completely saturating light close to— but never quite up to
the quantum yield in low steady light.
For the quantum yield increase caused by intermittency to be at all
significant, the light periods must not be longer than the "Emerson-Arnold
period" (0.01 sec. at 20° C, c/. chapter 34), allowing the limiting catalyst to
work in the dark, after the flash is over, for a period of time which is sig-
nificant compared to the duration of the flash itself. It is doubtful, however,

whether shaking at the rate of 2.5 swings per second could lead to illumina-
tion flashes of 0.01 sec, or shorter.
Certainly, the "dark periods" in Warburg and Burk's experiments
must have been :»0.01 sec; therefore, the assumption of Warburg and
Burk, that under the conditions of their experiments all cells are engaged
uniformly in photosynthesis throughout the "illumination period," requires
revision of the major conclusions derived from experiments in flashing light.
However, this assumption is not necessary for the validity of their argument
(while the duration of the light period, mentioned in the preceding para-
graph, is of crucial importance).
QUANTUM YIELD MEASUREMENTS BY THE MANOMETRIC METHOD 1 107
Background Light. Because only 5% of all cells were illuminated at

any given moment, even the very high incident intensity of the red light
used in the Warburg-Burk measurements did not prevent the respiration
correction from being equal to or larger than the photochemical gas ex-
change. It has been suggested (this hypothesis will be discussed later in
this chapter), that the effect of light below the compensation point could
consist in reversing respiration midway (and not after it has led to the
ultimate products, CO2 and H2O). To check this hypothesis, Warburg,
Burk and co-workers made experiments in which the net gas exchange in
light was made positive by substituting for dark periods, periods of dif-
fuse illumination of the reaction vessels by white "background light" of
such intensity that photosynthesis equalled or exceeded respiration.
This background illumination was maintained also during the "light"
period (when a measured beam of red light was added to it), so that the
"light effect," from which the quantum yield was calculated, was the
increment of gas exchange caused by an increment of illumination. War-
burg and Burk argued that the quantum yields obtained in this way must
be those of true photosynthesis, with the storage of 112 kcal. chemical
energy per mole of liberated oxygen, and could not be those of a partial
reversal of respiration (with an unknown, and possibly small, conversion
of light energy into chemical energy), since this reversal, if at all possible,
should be accomplished already by the background illumination.
It will be noted that this argument is tied up with the assumption of
—
uniform photosynthetic activity of all cells those that are momentarily
illuminated by the flash as well as those that are momentarily in darkness.
If only the actually illuminated cells (or cells <0.01 sec. out of the illumina-
tion zone) can contribute significantly to photosynthesis, then only the
part of the background light that falls on these particular im-
cells is of
portance. This part is insignificant if the background light from above
falls
and is absorbed in the top layer of the suspension — while measured red light
enters the vessel from below and absorbed in a thin bottom layer of the
is
suspension. Warburg and Burk (1950) described a single experiment in

which the background light, similarly to the measured light, was thrown
on the vessel from below. This light was so strong as to overcompensate
respiration about fivefold; nevertheless, the addition of the measured light
produced an increment of oxygen production equivalent to a quantum re-
quirement as low as 2.8. It is unfortunate that this particularly important
experiment was carried out with a particularly unsatisfactory time schedule
— three 5-min. light-dark cycles in one vessel, followed by two 10-min.
light-dark cycles in the other vessel.
Quantum Yield in Carbonate Bufifers. The same cells which gave, in
Warburg and Burk's experiment, high quantum yields at pH 5 (culture
1108 THK LIGHT FACTOR. IT. QUANTUM YIELD CHAP. 29
medium) gave 2-3 times lower yields (7 = 0.10-0.09) in carbonate buffers

(pH ^^ 9), both with and without compensating white light (last section
of table 29. IV). Yields of 0.12 or less were obtained also in bicarbonate
solutions equilibrated with 5% carbon dioxide in air (pH 7-8). It thus
appears that for Warburg and Burk's cells (grown in acid medium) even
neutral solutions were "unphysiological."
Respiration in Light. The question whether respiration is affected
by light is measurement of the rale of photosynthesis in
crucial for the
weak light. methods (Vol. I, Chapter 20, and Chapter
Different indirect
36) have been used to answer it, and have given contradictory answers
(including all three alternatives: "no change," "stimulation," and "in-
hibition"). The simplest approach to this prol)lem is to remove (e.g.,
by absorption in alkali) all carbon dioxide (including that produced by
respiration) and to measure the oxygen consumption in light unobscured
by photosynthesis. This procedure was attempted repeatedly, but with-
out success, because immediate photosynthetic reutilization of respiratory
carbon dioxide competed too effectively with its absorption by the com-
paratively remote external absorber. In fact, it proved difficult to reduce
photosynthesis in this way much below the compensation point. War-
burg et.al. (1949^) reported, however, that Avith the increased frequency
of shaking, now able to absorb respiratory carbon dioxide
they were
in an arm of the reaction vessel so effectively that the
alkali-filled side
rate of oxygen uptake by a Chlorella suspension in light was exactly the
same as in the dark. They saw in this experiment the proof that respira-
tion as such is quite unaffected by (red) light, and refutation of all hy-
potheses which postulate an exchange of intermediates between photosyn-
thesis and respiration.
Complete prevention of photosynthetic reutilization of respiratoiy
carbon dioxide by absorption of the latter in an external absorber (although
reutilization must be possible even before the carbon dioxide had escaped
from the cell into the medium), is a remarkable achievement. A possible
reason why Warburg and co-workers were successful where others have
failed is intermittent illumination. For 95% of the "light period" each in-
dividual cell is practically in darkness. Respiration goes on during all this
time; all, or at least a large part of the carbon dioxide produced while the
cell is in the shade may be able to escape into the medium before the cell
had moved into the illuminated zone. Once a carbon dioxide molecule is
in the medium, it may have a much greater chance to diffuse into the gas
space than to diffuse into the small illuminated volume. In this way, 80
or 90% of respiratory carbon dioxide
produced during the "light period"
could perhaps escape re-utilization by the cells and reach the external ab-
sorber.
It will be noted that this explanation could not be used if Warburg and
Burk's concept of all cells being uniformly engaged in photosynthesis
throughout the "light period" were correct. More specifically, this ex-
planation requires that not only the photocatalytic mechanism responsible
enzymatic mechanism responsible
for the liberation of oxygen, but also the
for the uptake of carbon dioxide, should cease operating within <0.1 sec.
after the cells are darkened. This seems to contradict the assumptions
which we used on p. 207 in the explanation of the "pick up" of carbon
medium, on p. 308 in the
dioxide after intense illumination in C02-deficient
explanation of the effect of cyanide on yield of photosynthesis in flashing
light,and will use in chapter 36 in accounting for C*02 uptake by preillu-
minated cells. In all these cases, we have assumed that the capacity to
take up carbon dioxide survives, in preilluminated cells, for several seconds
(or even minutes) after the cells had been darkened. However, as in many
such cases, apparent contradictions may arise from the use of a qualitative,
"yes or no" approach, where a quantitative, "more or less" analysis is re-
quired.
Summary of Warburg and Burk's Quantum Yield Measurements.
Table 29. IV gives a summary of the quantum efficiencies reported by War-
burg and Burk (1950); several of the experiments in this table have al-
ready been discussed above.
Whittingham, Nishimura and Emerson (1951) were able to reproduce
Warburg and Burk's results by strict adherence to the same experimental
arrangement and schedule of operations. However, they concluded that
these results were affected by a sj'-stematic error. Following are the major
points of their criticism.
1. The two-vessel method
is very sensitive to slight errors in mano-
metric determinations. Thus, a difference of 0.3 mm. in the pressure

change registered in one of the two vessels over a 10-min. period may
change the calculated oxygen yield by a factor of two. Such a difference is
well within the limits of experimental error of the method of Warburg and
Burk (as contrasted to the much more precise measurements with the
differential manometer, emplojêd by Emerson and Lewis.)
2. This low precision of the method leads to random scattering of re-
sults, (for example, in experiment No. 7, the I/7 values derived from in-
di\'idual cycles scattered from 2.3 to 14). This can be corrected by averag-
ing over a sufficiently large number of cycles. However, only in a few
experiments of Warburg and Burk, as many as five or six 10 min cycles were
used; in most others, only two or three. This explains why even the
averaged I/7 values scattered from 2.3 to 4.9.
3. H,andom errors can explain the scattering of the results, but only a
systematic error can explain the consistent finding of I/7 values considerably
Table 29.IV
Quantum Requirements of Oxygen Production (I/7) and of Carbon Dioxide
Consumption (Qp/7) Calculated by Warburg and Burk (1950) for Chlorella
pyrenoidosa
Number, duration and order of cycles

Expt. 1 = light d = dark
No. Description (or background light) 1/7 Qpi
A. Experiments at pH 5 (two vessels)
1
QUA NTUM YIELD MEASUREMENTS BY THE MANOMETRIC METHOD 1111
B. Experiments at pH 9.2 (Single vessel)
2 Same material as in
—
6. While Emerson and co-workers were able to closely reproduce the

measurements of Warburg and Burk (I/7 — 3-4), the results became quite
different if light and dark periods were lengthened to 30 minutes or if
the vessel shape h2 (fig. 29. 4A) was substituted for hi in the two-vessel
combination. Either change led to 7""^ > 9, with the same cells which
gave values of 3-4 by following Warburg and Burk's specifications. These
changes should diminish errors due to different physical lag, though per-
haps not eliminate them, nor overcome all the disadvantages in the tech-
nique of Warburg and Burk. However, it is significant that the yield was
found to be dependent on both timing and vessel shape.
h, H Hj
Fig. 29. 4A. Manometric vessels for two-vessel method of quantum yield
measurements. H, vessel with small gas space; hi hi, vessels with large
gas volume.
7. Warburg and Burk have calculated — AO2/ACO2 values of the

order of 1(±0.2) for the ''light effect" in the two-vessel experiments.
However, this does not prove that no significant carbon dioxide burst and
—
gulp had occurred in their experiments, but as already was explained on
—
page 1101 merely that the "gulp" in the dark period compensated more
or less completely for the burst in the light period. This compensation is
inevitable in a series of light-dark cycles in which approximately stationary
conditions are established after a few cycles. Separate calculation of
— AO2/ACO2 in light and in darkness (suggested on p. 1102) gives în the —
few cases where the necessary data are provided by Warburg and Burk
values quite different from 1, with deviations in the direction required by
the burst-and-gulp hj^pothesis.
8. Warburg and Burk's experiments Avith white (or red) background
light have additional uncertainty because they were carried out by consecu-
tive, and not alternate, measurements in the two vessels. Earlier experi-
ments of Emerson and Lewis had indicated that the burst occurs not only
upon change from light to dark, but also upon change from one light in-
tensity to a higher one; thus, the interpretation of experiments with light-
compensated overcompensated respiration as "base line" can be the
(or
same above for the light-dark experiments.
as suggested
9. To sum up the conclusions of Emerson and co-workers, the experi-
ments of Warburg and Burk can be duplicated by strict adherence to their

specifications. However, the results obtained in this way are not only of
low precision (as revealed by wide scattering) but, what is more important,
,
contain a systematic error.

The differential Emerson and Lewis (1941)
manometer experiments of
had been far more Warburg, Burk
precise than either the experiments of
et al. (1948-1950), or the experiments which Emerson and co-workers made
in 1949-1950 under conditions closely imitating those of Warburg and

Burk. It seems that the most reliable of the presently available data on the
quantum yield remain those derived from these older measurements.*
Manometric quantum yield measurements have also been reported by
Kok (1948, 1949). He considered the loss of light by scattering in thin
suspensions as a lesser experimental difficulty than the large respiration,
the wide variation of local light intensity, and the intermittency of illumi-
nation inevitable in strongly agitated, dense suspensions. He therefore
worked with Chlorella suspensions that absorbed only 30-40% of the in-
cident light (yellow sodium light), and used an Ulbricht sphere for the meas-
urement of absorption. He found practically linear light curves up to re-
—
markably high incident intensities sometimes as high as 20 times the
respiration-compensating light! (Compare chapter 28, section A2).
Quantum yield deteraiinations were made by Kok in four different ways:
(1) by measuring the carbon dioxide exchange only, oxygen being absorbed
by chromous chloride in the side arm of the Warburg vessel (2) by measur- ;
ing the oxygen exchange only, carbon dioxide being absorbed, in the usual
way, in carbonate buffer (3) by measuring both the carbon dioxide and the
;
oxygen exchange by the two vessel method, and (4) by measuring the net
exchange in a single vessel, and assuming Qp = 1.09.
The quantum requirements, I/7, were calculated from the slope of the
straight, ascending section of the light curves, thus avoiding explicit use of a
respiration correction. [The underlying assumption is, of course, that the
respiration, R, is the same at all light intensities at which a straight line is
obtained for the function P — R = /(/)]. Kok found the so-calculated
efficiencies todepend on the age of the suspension (c/. fig. 28.13). (This
probably means, primarily, dependence on the freshness of the culture
medium.) The yields were almost independent of the temperature and the
light intensity used in the cultivation of the algae. They were about 20%
higher in acid media (water, cultvu'e liquid, or phosphate buffer) than in
alkaline carbonate buffers. Lowering the oxygen pressure to 0.25% had
no on the quantum yield, and the same seemed to be true of changing
effect
the temperature from 10 to 20 or 30°C.
The 1/7-values obtained by the four methods ranged (apart from a
* New results by Warburg and Burk (1951 '2) pertain not so much to the question
of thequantum mechanism of utilization of the
yield of photosynthesis, as to that of the
quanta. They will be described in chapters 36 and 37.
few exceptionally high figures) from 6.9 to 12.9; the average for non-
alkaline media (methods 1, 3, and 4) was I/7 = 7.85; for alkaline buffers
(method 2) about 10. Kok estimated I/7 = 6.75 as the most probable
lowest value of the quantum requirement.
The most interesting (and controversial) finding of Kok was that linear
extrapolation of the light curves to 7 = consistently lead to considerably
smaller values of the gas exchange than would have corresponded to the
respiration of the same cells in the dark. Upon closer study, he concluded
that the light curve underwent a sudden change of slope by a factor of
about somewhere near the compensation point (fig. 29. 4B). He took
2,
this to mean
that the quantum efficiency was constant from near the
saturation point down to the compensation region, and then doubled sud-
denly. This shape of the light curve—according to Kok consists of three
it
practically linear segments —has not been found by any of the previous
observers; however, Kok claimed a confirmation of the sharp break in the
P = /(/) curve by new analysis of the data of Kopp and of Gabrielsen.
If the slope of the light curve changes by a factor of two at the com-
pensation point, the rate of respiration in strong light, determined by linear
extrapolation of the light curve from above the compensation point to
7 = 0, must indicate a rate of respiration in light equal to one half of the
rate of respiration in darkness.
Later (1949) using a more precise manometric device (a "differential
volumeter") Kok found, as an average of 50 experiments with Chlorella
cells grown in Knop's medium, Rugu = 0.5 Rdnvk- (To increase /^dark,
3
O
O
1
these measurements were made However, the break in the

at 30°C.)
p = /(/) curve was now found not exactly at the compensating intensity,
but at about twice this intensity (fig. 29.4B(A)). Since iûght was equal to
0.5 Edark, the ratio of the slopes below and above the break is, for these
algae, 1.33 rather than 2.0.
With Chlorella cells grown in glucose solution, the break was below
the compensation point (fig. 29.4B(5)) ; the slope below the break was, in
this case, exactly one half of that above it, which meant that iûght was
greater than 0.5 /^dark— perhaps reflecting enhanced respiration in the cyto-
plasm. A break in the same region {i.e., below the compensation point)
was found also in the Haematococcus pluvialis grown in inorganic medium;
but in this case, the slope below the break was less than twice that above it.
Light curves obtained with Cahomba leaves (floated on carbonate buffer)
indicated that the break was present there too, and that -Rught 0.5 Rdnvk- —
Kok suggested that these experiments indicate the existence of two hght
processes, with the quantum requirement of the "low-light process" (which
he called "light respiration," cf. below) exactly one half that of the "high
light process" (true photosynthesis). When the slope in the low-light
region was less than twice that above it, he interpreted this as indication
that the two light processes were occurring simultaneously. When the slope
in low hght was exactly of that in high light, Kok assumed that the high
H
light process did not begin until the low-light process was saturated.
Kok considered these experiments (which had indicated a probable
lowest 1/7-value of 6.75 above the break), as making plausible a quantum
requirement of 6 for the high light process (true photosynthesis), and 3
for the low light process ("light respiration"). However, according to
Franck (1949), Rieke found in Kok's method of light measurement an
error which might have reduced the calculated quantum requirements by
20%; with this correction, the results become consistent with the assump-
tion of quantum requirements of 8 and 4, respectively.
The sharp breaks in the light curves, found by Kok, are very improbable
(cf. the discussion in chap. 26 of the
impossibility of a sharp break between
the ascending and the horizontal part of the light curve, postulated by
Blackman). However, even if the light curves are smoothly curved rather
than broken lines the possibility remains that they may decline in the
low hght region more steeply than would be expected from their shape in
—
the region of higher light intensities. Once before in the explanation of
the alleged incapacity of cyanide to reduce photosynthesis below the com-
pensation point (Vol. 1, page 308)— we have been led to the hypothesis that
compensation of respiration in light may not requrie complete photosyn-
thesis. We will confront the same situation in the description of the study,
by Calvin and co-workers of respiration in light with the help of tracer
lllfi THE LIGHT FACTOR. II. QUANTUM YIELD CHAP. 29
carbon (Chapter 36). From the latter experiments, Calvin drew the same
conclusion as Kok — that the rate of respiration in strong light is only about
one half of that in darkness. (The remaining one half may represent the
proportion of the total cell respiration taking place outside the chloro-
plasts and therefore not affected by light.)
Kok suggested that photosynthesis substitutes for respiration by produc-

ing energy' carriers (such as high energy phosphates or "HEP" molecules)
which the organism rcfiuires for its metabolic activity, and which it ordi-
narily derives from respiration. More specifically, Kok postulated that
the primary light reaction in photosynthesis has a twofold function: (1)
to produce reducing and oxidizing agents (HX and Z, cf. Vol. I, scheme
7. IV) CO2 to C'HoO and of oxidizing Ho(J
capable, respectively, of reducing
to O2;and (2) to produce HEP-molecules by transphosphorylations coupled
with back reactions between these primary products:
HX + Z + phosphate > HZ + X + HEP

Until respiration is fully compensated — or, rather,suspended as unneces-
sary (at least, in the chloroplasts) —the absorbed light is used only or
mainly to produce HEP molecules. Respiration of one ICH2O} group
has been reported to produce six HEP molecules (Ochoa, Lippman) Kok ;
suggested that the same number can also be obtained by recombination of

six (HX -f Z) pairs, and that these six pairs can themselves be produced
by three cjuanta. Thus, two HEP molecules, containing about 20 cal./mole
disposable energy, are fonned by one quantum of red light (about 40 cal./
einstein).
Above the compensation point, Kok assumed a quantum requirement
of 6; he postulated that here, too, each cûantum produces two (HX + Z)
pairs, and that out of twelve such pairs (produced by six quanta), four
(produced by two quanta) react further to reduce CO2 to CH2O and to { } { }
oxidize H2O to O2, and eight (produced by four quanta) react back, con-
verting eight low energy phosphates into eight HEP molecules (which, in
turn, are utilized as "boosters" in the reduction process). The quantum
requirement would then be 3 for the reversal of respiration and 6 for true
photosynthesis. (No explanation was given by Kok why eight HEP
molecules are needed in the latter case, as against only 6 in the first one.)
This, obviously highly arbitraiy scheme made to fit the (supposedly)
experimentally indicated l/7-values of 3 and 6, is closely related to the
"energy dismutation" schemes (such as scheme 9. HI) proposed (among
other possible reaction schemes of photosynthesis and chemosynthesis) in
chapter 9. The assumption that one third of all quanta are used in photo-
synthesis to provide oxidation and reduction agents, and two thirds for the
formation of energy boosters (HEP molecules), imitates the mechanism of
— —
chemosynthesis postulated for hydrogen bacteria (schemes 9. IV) in which

two hydrogen molecules reduce carbon dioxide, utihzing the energy lib-
erated by the oxidation of four hydrogen molecules by oxygen.
According to Franck (1949) (c/. page 1115), it is permissible to interpret
Kok's results as indicating quantum requirements of 8 (rather than 6) for
tme photosynthesis and 4 (rather than 3) for the "low light process." If
one wants to retain Kok's picture, one can, for example, suggest that four
quanta procUice four oxidation and reduction agents (4HX + 4Z), while
—
the other four produce by back reaction of another four (HX + Z) pairs
eight HEP-molecules. (The numerical analogy with the hydrogen bacteria
would be lost in this way; but it is more plausible that one ciuantum pro-
duces a single + HX
Z pair than that it jjroduces two such pairs, as was
suggested by Kok).
Van der Veen (1949), in the course of a study of induction phenomena
by the thermal conductivity method (chapter 33), found, for tobacco leaves,
a light curve of photosynthesis similar to that recorded by Kok a straight —
line up to 450 lux, and another straight line, with about half of the slope
of the first one, from 450 to 3200 lux. He combined Kok's concept of the
reaction mechanism of photosynthesis with scheme 9. IV, interpreting the
"energy dismutation" postulated in this scheme {cf. pages 164 and 239,
Vol. I), as production of HEP molecules by recombination of a part of the
primary photochemical oxidation and reduction products, and "boosting"
by these HEP molecules of the reductive power of the remaining reduction
products. The specific numbers used in his scheme (eight recombinations
to four oxidation-reductions) taken from Kok, could equally well be re-
placed by others by four recombinations and four oxidation-reduc-
e.g.,
tions, as in scheme 9. Ill; and the same is true of the number of quanta re-
quired (six, or eight, or even twelve).
A somewhat different — —
and perhaps more plausible interpretation of
a comparatively low quantum requirement of "anti-respiration" in weak
light has been suggested (Franck, 1949) : This is the (repeatedly mentioned)
possibility that intermediates of respirationcan be drawn into the photo-
synthetic cycle and reduced back to the carbohydrate level, and that a
smaller number of quanta is required for this process than for complete
photosynthesis. It is important to note that such a half-way interception
of respiration would not cause a deviation of the AO2/ ACO2 ratio from its
—
normal value of (approximately) 1 since the only gas exchange measured
in low be that due to residual normal respiration (e.g., respiration
light will
outside the chloroplasts). Calvin suggested, on the basis of certain C(14)
tracer experiments, that a cross-link between respiration and photosynthe-
sis exists on the level of malic and oxalacetic acid; however, these observa-
tions are still controversial {cf. chapter 36).

Franck (1949), in offering an explanation of Warburg and Biirk's results

in terms of photochemical half-way reversal of respiration, suggested that
the extent to which this process occurs depends on the capacity of respira-
tion intennediates (which probably are organic acids) to penetrate from the
protoplasm into the chloroplasts, and that this capacity is affected by the
physiological state of the cells. It remains to be seen whether this explana-
tion can suffice to explain why many careful experiments have failed to
show the existence of the phenomenon. Thus, Emerson and co-workers
never had observed any curvature of the light curves in the region of the
compensation point, which would indicate a lower quantum requirement in
very low light. Brown and co-workers (1950) found no evidence that
light interferes with respiration in mass-spectrographic experiments: The
uptake 0(16)0(16) from the air continued in light, while 0(16)0(18) was
of
evolved simultaneously by photosynthesis from algae suspended in 0(18)-
enriched water. It was mentioned before (page 1108) that Warburg and
co-workers (1949) arrived at a similar conclusion by observations of the rate
of oxygen consumption in darkness and light under conditions assuring
rapid removal of respiratory carbon dioxide from the medium; it was, how-
ever, suggested that these findings might have been contingent on the inter-
mittency of illumination, which prevented the utilization for photosynthesis
of a large proportion of respiration products.
To sum up, the possibility of photochemical utilization of respiratory
intennediates remains controversial, and the effect of this re-utilization
on the quantum requirement in weak
an open question. Suggestive
light,
experimental evidence is and a positive answer.

available for both a negative
(On the positive side: Warburg's cyanide experiments, Kok's and van der
Veen's broken light curves, Calvin's carbon tracer experiments. On the
negative side: Emerson and Lewis' smooth light curves, Warburg's experi-
ments in C02-free medium. Brown's respiration study with oxygen iso-
topes.") Whether Franck's suggestion, that the chloroplasts sometimes
are and sometimes are not permeable to respiration intermediates formed
in the cytoplasm, can explain these contradictions is uncertain. A knowl-
edge of the relative contribution of chloroplasts and cytoplasm to total
cell respiration in the dark would be useful in this connection; but no esti-
mate of this relation has as yet been made.
2. Nonmanometric Measurements of Quantum Yield
The results of nonmanometric measurements of the quantum yield

on the whole agree with the lower figures (I/7 = 10 ± 2) found by Emer-
son and Lewis, Rieke, and others by manometric studies rather than with
the higher figures (7 = 3^^ to 3^4) claimed by Warburg and Burk.
NONMANOMETRIC MEASUREMENTS OF QUANTUM YIELD 1119
(a) Chemical Methods
Wurmser (1923, 1925, 1926) made a few quantum yield determinations

with the green alga Ulva lactuca. Each experiment lasted for several hours,
and consisted in the measurement of change in oxygen concentration in solu-
tion by Winkler's method. The rate of absorption of Ught was calculated
from comparison of transmission by green and discolored thalli (c/. chapter
22, page 675), using a theoretical equation to take into account scattering
(c/. page 713). Wurmser found, in some of these experiments, energy con-
version factors up to 50%, corresponding to quantum yields up to }4-
However, the calculated yield in the (weakly absorbed) green light turned
out to be so much higher than in the (strongly absorbed) red light, that it
indicated probable grave errors in the calculation of absorption. Warburg
(1925) therefore did not consider these experiments of Wurmser as sig-
nificant confirmation of his own results.
Briggs (1929) obtained, with leaves of Phaseolus vulgaris, at hght in-
tensities 5-10 times stronger than those used by Warburg and Negelein,
yields from 7-17 cc. O2/5OO cal absorbed energy; with yellow elm leaves,
from 5.3 to 8.9 ml.; with green elm leaves, from 12 to 20 ml.; and with
leaves of Samhucus nigra, from 9 to 19 ml. These values correspond to
quantum yields <0.1.
In 1935, Gabrielsen, working with plants of Sinapis alba, calculated,
also from gas-analytical measurements, by extrapolating the light curves
(fig. 30.8A,B) to zero illumination, e values from 0.13 in blue, to 0.36 in red
Hght, corresponding to maximum quantum yields of 0.1 ± 0.02. Gabrielsen
did not question at that time the correctness of Warburg's results, and
thought that his lower yields must have been due to the use of a less ef-
ficient species.
Later (1947), Gabrielsen repeated these experiments with Sinapis,
Corylus and Fraxinus leaves, and found 70-values between 0.082 and 0.078.
In the first of a series of investigations emanating from the University
of Wisconsin, Manning, Stauffer, Duggar and Daniels (1938) determined
the quantum by gas analysis, comparing the composition of a gas
yield
(containing approximately 5% CO2 and 5% O2) conveyed through a Chlo-
rella suspension in the light and in the dark. The suspensions were less
dense than in Warburg and Negelein's work, absorbing only 10-50% of the
incident light; the intensity of the latter (green line from a mercury lamp)
was somewhat higher than in Warburg and Negelein's experiments (1000-
1750 erg/cm. 2 sec.) 60 minute periods of illumination were used. The 7
;
values derived from the absorption of carbon dioxide were not very different
from those calculated from the increase in the concentration of oxygen, thus
indicating that the quotient Qp was close to unity.
The quantum yields obtained in these experiments scattered consider-

ably —from 0.01 to —but never exceeded the
0.1 latter limit. Still lower
quantum yields (0.002 to 0.027) were obtained in experiments in white
light; in this case, however, about ten times higher intensities of incident
lightwere used, so that saturation effects appeared possible. Experiments
with a different technique (closed reaction bottles, no stirring, analytical
determination of the change in [O2] in solution by Winkler's method)
yielded 7 values between 0.02 and 0.065.
In another paper from the same laboratory, Manning, Juday and Wolf (1938) de-
scribed experiments in which bottles containing Chlorella suspensions were deposited at
different depths in a lake, and thus exposed to different intensities of illumination, rang-
ing from full sunlight (600 kerg./cm.'' sec, not counting the infrared)down to 6 kerg/cm.*
sec. The change in color of the light with depth (cf. Table 22. XI) complicated the calcu-
lation of the number of absorbed quanta; the results were therefore less exact than
those of the first paper. However, the approximate magnitude of 7 values was the
same as in other experiments —
about 0.05 at the lowest light intensities (at 10 meter
depth); c/. figure 29.5.
0.07
O
UJ
3
f-
<
3
O
0.1 0.2 0.4 10 20 40 100

LIGHT INTENSITY, (erg/cm^ sec.) x 10"
Fig. 29.5. Quantum efficiencies for Chlorella (after Manning, Juday,

and Wolf, 1938). Curve A, 3.17 hr., cell concn. 3,250,000/ml.; B, 1.03
hr., cell concn. 718,000/ml.; C, 4.00 hr., cell concn. 331,000/ml.; D, 4.00 hr.
cell concn. 718,000/ml.; E, 4.05 hr., cell concn. 1,900,000/ml.; F, 3.30 hr.,
cell concn. 1,210,000/ml.
(6) Polar ographic Method
In a third investigation from the Wisconsin laboratories. Petering,

Duggar and Daniels (1939) applied the polar ogra phi c meOiod {cf. page 850)
because it permitted the determination of respiration immediately before
and after a period of photosynthesis, without the delays (illustrated by fig.
29.1) inherent in the manometric method. Figure 29.(» shows the polaro-
NONMANOMETRIC MEASUREMENTS OF QUANTUM YIELD 1121
graph to respond almost immediately to transitions from respiration (in

darkness) to photosynthesis (in light) and vice versa. In the top and
bottom curves, the illumination is below the compensation point, and
photosynthesis manifests itself in a reduced rate of consumption of oxy-
gen; in four other curves, the oxygen concentration increases during
photosynthesis. The curvatures of the respiration curves show the un-
certainty involved in calculation of the respiration correction. The rate
of oxygen consumption in the five minutes immediately following the cessa-
tion of illuminationwas used by the authors in the calculation of this cor-
rection. (This method gives the highest respiration correction and conse-
quently the highest quantum yield values.)
-
in 41.00
c
T3
O 39.00
•£ 37.00
£
o
c
o 35.00
>
o
33.00 -
z
O
t 31.00-
29.00 -
o 27.00 -
o
> 25.00 -
X
o
20 40 50 30 100
TiME.min
Fig. 29.6. Photosynthesis and respiration of Chlorella measured by a

polarograph (after Petering, Duggar and Daniels 1939).
The experiments were made with white light; from 27 to 48% of the
incident light was absorbed by the suspension. The calculated quantum
yields ranged from 0.045 to 0.100, clustering around 0.07, and showing no
trend with light intensity in the range from 1000 to 6000 erg/cm. ^ sec.
New experiments with the polarograph were conducted by Moore and
Duggar (1949). In these Chlorella cells were first illuminated with light
of one color (intensity, 800-2500 erg/cm. ^ sec.) and then light of another
color was added, and the additional yield determined. The idea behind
this procediu'e was that the uncertainty concerning the amount of respira-
tion in light can be eliminated by subtracting from the total gas exchange
in the two combined beams the gas exchange in one beam alone. (There
seems to be no difference between this method and calculation of 7 from the
difference in yield at two light intensities in light of the same color.) The
results of these measurements are shown in Table 29. V. The 7 values
Table 29.V
POLAROGRAPHIC DETERMINATION OF QUANTUM YiELDS OF Chlorella IN ReD
AND Red plus Blue Light
(after INIoore, and Duggar 1949)
Initial beam Added beam
Abs. Abs.,
% % 1/7
6500 1958 76 0.074 4358 1575 100 0.11 9.1

6500 783 46 0.12
4350 1288 91 0.09 6500
6500 1610 49 0.10
6500 1392 74 0.10
6500 1188 52 0.11
NONMANOMETKIC MEASUREMENTS OF QUANTUM YIELD 1123
3. Although the medium (nutrient solution, pH 5.5) satisfied Warburg's require-

ments of "physiological" conditions, presence of mercury drops introduced a danger of
poisoning. In fact, such poisoning has been observed, but deemed too slow to affect the
measurements.
(c) Calorimetric Method
The basis of the calorimetric determination of the yield of photosynthe-

sis — which is a direct measurement of the energy conversion yield, e, rather
than of the quantum yield, 7 —was described in chapter 25 (page 854).
The first to carry out such measurements was Arnold in 1936-1937; how-
ever, so strong was the beUef at that time in the correctness of Warburg's
value, y = 14, that Arnold took his inability to obtain this yield as indica-
tion of a failure of the method, and did not pubhsh his results until 1949
(reference to them was made by Franck and Gaffron 1941). Arnold used
a modified Callender's radiobalance, originally designed to measure heat
production by radioactive materials. Its period was so small that com-
plete measurements could be made in from 1 to 10 minutes. Between
0.05 and 4 mm.^ of cells, in Knop's solution or carbonate buffer, were used.
One run was made with healthy ChloreUa cells, and one run with the same
cells inhibited by ultraviolet irradiation. Respiration was assumed to be
unaffected by ultraviolet light (c/. Vol. I, page 344). The results are shown
in Table 29.Vl.
Table 29.VI
Calorimetric Determination of Quantum Yield (after Arnold 1949)
Extra heat
evolved in light"
With With 100 Aff^

inhibited healthy 1 '
cells, cells, a
Cells /„ J„ - AH^ AH^ % 1/7
HEAT PRODUCTION IN MICROAVATTS
C. pyrenoidosa 5^08 3^70 Os 27^ 9.5

4.60 3.80 0.80 17.4 14.8
4.46 3.90 0.56 12.5 20.6
2.24 1.90 0.34 15.2 16.9
Avocado leaf 0.786 0.656 0.130 16.5 15.6
C. vulgaris 0.912 0.700 0.212 23.2 11.1
C. pyrenoidosa 1.86 1.34 0.52 27.9 9.2
16.2 12.9 3.3 20.4 12.6
heat PRODUCTION IN ARBITRARY UNITS

:
Measurements with a photocalorimeter {cf. page 854) were carried out

also at Wisconsin,by Magee, DeWitt, Smith and Daniels (1939), and gave
7 values from 0.049 to 0.110 (the average of 17 experiments was 7 = 0.077
or ITy = 13). No pronounced change with light intensity was noted be-
tween 1200 and 8000 erg/cm. ^ sec.
More recently, Tonnelat (1944, 1946), who worked in Wurmser's lab-
oratory in Paris, published similar results of an investigation initiated in
1939. Tonnelat measured the heat developed in an adiabatic microcalorim-
eter under three conditions: (i) when the calorimeter contained an il-
luminated black-bottomed vessel with pure water (heat evolution, £"0);
{2) when it contained the same vessel with an equal volume of an algal
suspension in the dark (heat evolution, Er, due to respiration) and (5) ;
when it contained the same suspension and was illuminated (heat evolu-
tion, E = Eo-{- Er - AHc). The energy yield, «, was then:
(29.3) e = AHc/E = (^0 + Eh - E)/E
and the quantum yield, according to equation (29.1), assuming X = 530

mju (green light isolated by Wratten filter No. 62)
(29.4) 7 = e/(4 X 10-5 X 530) = 6/2.12
Illumination lasted for 16 hours without noticeable deviation from

linearity (t. e., presumably, from the constancy of both R and P); even
2
in the very low Ught used (about 1.5 X lO"* einstein of green light/cm
min., or 550 erg/cm.^ sec), such long duration of the experiments seems
dangerous. The results of Tonnelat's determinations are shown in Table
29.VII.
T.\BLE 29.VII
Efficiency of Photosynthe.sis (after Tonnelat 1944)

Measured with Photocalokimetkr
Chlorella Energy
concn., conversion
Experiment cclls/0.5 cm.^ factor
(1) Reflectingbottom 32 X 106 0.26 (±0.06)

(2) Reflectingbottom 33 0.34(±0.05)
(3) Reflectingbottom 37 0.26 (±0.06)
(6) Black bottom 36 0.12 (±0.07)
(7) Black bottom 51 0.31 (±0.05)
(8) Black bottom" 48 0. 18
" With 0.1% agar.
Experiment 5 shows that the yield became low in very concentrated

suspensions (a result Tonnelat attributed to inhibited gas exchange in the
dense layer of cells on the bottom of the vessel, but which also could be due
QUANTUM YIELD OF BACTERIAL AND ALGAL PHOTOREDUCTION 1125
to "self -inhibition" effects, described on page 880). Experiment 8 shows

decreased efficiency in the presence of agar— an observation that Tonnelat
suggested may explain some of the low values found by McGee, DeWitt
et al. (who used agar to prevent the suspension from
settling).
Experiments were performed in vessels with black or reflecting bottom.

In the first case, values could be too low (if absorption of light by the
t
blackened bottom was counted as absorption by the cells) in the second ;
case, they could be too high (because of possible escape of reflected

radia-
(experi-
tion). Figures in Table 29.VII indicate that the first effect is real
ment No. 6 shows a lower value of y at low cell concentration), but reveal
no due to reflection.
effects
Tonnelat concluded from these experiments that the energy conversion
factor, 6, is about 0.30, and calculated from this a quantum yield of Vg.
Equation (29.1) gives, however, 7 = 0.030/2.12 = 0.U5, or approximately
}i. Tonnelat's error was probably due to the use of an incorrect value,
8.9 X 10-12 instead of 7.9 X lO"^" erg/mole, for the heat effect of photo-
synthesis.
3. Quantum Yield of Bacterial and Algal Photoreduction
French (1937^), who worked in Warburg's laboratory, used the bacterial

species Streptococcus varians to study the quantum yield of the reduction of
carbon dioxide by molecular hydrogen (cf. chapter 5, page 104). The rate
of hydrogen disappearance was determined manometrically. The lines
852 and 894 m^ were isolated by filters from the fight of a cesium lamp;
from 17 to 58% of this light was absorbed by the suspension. The quan-
tum yields calculated by French ranged from 0.07 to 0.23 molecule of carbon
dioxide e., from 0.14 to 0.46 molecule of hydrogen)
{i.
transformed per
quantum, depending on the pretreatment of the bacteria. French con-
sidered these experiments proof that carbon dioxide reduction by Athio-
rhodaceae requires four quanta per molecule of carbon dioxide, similarly to
the assimilation of green plants according to Warburg and Negelein. As
mentioned before, the fight curves obtained by French in this work
were sigmoid: the y,naz- values were derived from the maximum slope of
these curves (reached in the inflection point). This procedure requires
justification. To divide the increase in yield in a certain region of the light
curve by the corresponding increase in light absorption, and to call the quo-
tient "quantum yield" presupposes that the increase in light intensity pro-
duces a certain additional amount of photosynthesis, characterized by its
own quantum yield. Wassink, Katz and Dorrestein (1942) suggested
that this may, in fact, be the case, if the bacteria use, in weak light, mainly
intracellular organic compounds, instead of the externally supplied react-
ants. As light intensity increases, this photochemical process is soon light
saturated (because of supply limitations, or because of the limited amount

of a necessary enzyme) ; the normal reduction of carbon dioxide with ex-
ternal reductants then comes into its own. The manometrically deter-
mined quantum yield of the photochemical process using intercellular sub-
strates can be smaller normal photoreduction, for two reasons:
than that of
(a) When hydrogen is used as external
reductant, any utilization of non-
volatile, intracellular reductants will diminish the rate of gas consumption,
even if the quantum yield of carbon dioxide reduction is the same with both
types of reductants. In French's experiments (1937) with Streptococcus
varians, the light curve had total gas consumption, AH2 + ACO2 as
ordinate; its sigmoid shape may have been due pntirely to an initial de-
ficiency in the consumption of hydrogen alone. In the experiments of the
same author with Spirillum ruhrum (1937^) a nonvolatile reductant was
used, and the curve showing ACO2, as function of light intensity, showed no
inflection. However, in the more recent experiments of Wassink, Katz and
Dorrestein (1942) with Chromatium, sigmoid curves were obtained not only
with hydrogen but often also with thiosulfate as reductant {cf. fig. 28.11).
A different explanation is needed in this case.
(6) It was described in chapter 5 (Vol. I, page 106) how, when exter-
nally supplied organic compounds are utilized by photosynthesizing purple

bacteria, the proportion of "coassimulated" carbon dioxide can vary widely
(or carbon dioxide may even be liberated), depending on whether the or-
ganic compound is utilized mainly or exclusively as hydrogen donor (as in
Foster's experiments with secondary alcohols), or serves also as the source of
carbon. Wassink, Katz and Dorrestein suggested that the same applies to
photochemical utilization of intracellular organic materials; here, too, the
consumption of external carbon dioxide may be more or less completely
suppressed by the utilization of the carbon (in the form of freshly formed
carbon dioxide, or of oxidation intermediates) produced by dehydrogenation
of the organic reductant.
These two considerations provide a plausible explanation of sigmoid
gas exchange curves, but do not fully justify calculation of the maximum
quantum yield of photoreduction from the slope of the steepest section of
the light curve. In the first place, the utilization of internal reductants is
at present merely a hypothesis. In the second place, assuming this hypo-

thesis is correct, it is still possible —and, indeed, likely —that, as light in-
tensity increases, photoreduction replaces (and not merely supplements)
the photochemical transformation of intercellular substrates. This can
occur either because of exhaustion of the intracellular material, or because
enzymatic system (as in the "de-adaptation" of hydrogen-
of changes in the
adapted green algae; cf. Vol. I, chapter 6). More precise measurements
of the photosynthetic ratio A [CO2]/ A [reductant], at ditTerent light intensi-
QUANTUM YIELD OF BACTERIAL AND ALGAL PHOTOREDUCTION 1127
ties, and investigations of the effect of intensity and duration of illumination

on the shape of the light curves, could help to elucidate the situation. Un-
til there is proof that the sigmoid shape of the light curves actually is due to
an internal photochemical process resulting in no (or only little) gas con-

sumption; and until it has been proved that this process, having become
saturated in very low light, continues at a constant rate as the light grows
stronger, the legitimate way to interpret the light curves is the conserva-
tive one: to consider the sigmoid shape as evidence that the average
quantum yield of photoreduction, y, first increases with light intensity and
then decreases again. The measure of j in each point of the curve then is
the slope of the straight line drawn from this point to the origin of the co-
ordinates,and not the slope of the tangent. (Similarly, we do not attri-
bute the convex part of the light curves to a superposition of low-yield
photosynthesis upon persisting high-yield photosynthesis, but to a decrease
in the average yield.)
In this way, we can deduce from the sigmoid light curves only a lower
limit of the maximum quantum yield (this limit being given by the slope
of the tangent to the curve that passes through the origin of the coordi-
nates). This limit, derived from French's light curve of Streptococcus
varians, is about jnm. = 0.11.
In French's study of Spirillum ruhrum (1937^), the yield was measured
by the uptake of carbon dioxide. As mentioned above, the light curves
showed, in this case, no initial curvature; their slope corresponded to a
quantum yield of the order of 0.06 to 0.Q7. These values were termed
"unreliable" by French because of inexact determinations of light absorp-
tion. Subsequently, however, yields of similar magnitude were found in
several investigations by the Dutch group (Wassink, Katz and co-workers).
In their measurements, the initial concavity of the light curves often was
quantum yield.
only slight, and did not affect essentially the calculated
Eymers and Wassink (1938) measured the quantum yield of photosyn-
thesis by Thiorhodaceae, with thiosulfate serving as a reductant and
a cesium or sodium lamp as light source. The results are shown in Table
29. VIII. One notices that these organisms have a very strong dark meta-
Table 29.VIII
Quantum Yields of Cakbon Dioxide Reduction by Purple Bacteria
WITH Thiosulfate as Reductant (after Eymers and Wassink 1938)
Light
source
holism, which makes the exact evaluation of the quantum yield difficvilt.
The largest 7 values ever observed by Eymers and Wassink were about
0.11.
In a subsequent investigation from the same laboratory (Wassink, Katz
and Dorrestein 1942), a summary of additional 7 determinations for the
same species {Chromatium D) was given, which included values obtained
at two different pH values and with hydrogen as well as with thiosulfate
as reductant. They are shown in Table 29. IX. The figures are described
Table 29. IX
Quantum Yields of Chromalium under Different Conditions

(after Wassink, Katz and Dorrestein 1942)
Quantum yields, \/y
Reductant
—
OXYGEN LIBERATION BY ISOLATED CHLOROPLASTS 1129
species. These yields could be measured at light intensities up to 7000

erg/ cm.- sec; at the higher intensities, transition to ordinary photosyn-
thesis occurred too rapidly.
4. Quantum Yield of Oxygen Liberation by Isolated Chloroplasts
French and Rabideau (1945) measured the quantum yield of the "Hill
reaction" (photochemical oxygen production from ferric oxalate solution,
sensitized by a chloroplast suspension). This reaction was described in
chapter 4 (Vol. I) as possibly representing ''one half of photosynthesis"
namely, photoxidation of water, with the ferric salt instead of carbon di-
oxide serving as oxidant. Chloroplast suspensions were obtained from
spinach, or from Tradescantia, by maceration and centrifugation, and ad-
ded to 0.5 M K2C2O4 + 0.01 M FeNH4(S04)2 + 0.02 M K3Fe(CN)G. The
solution also contained 0.20M sucrose and 0.17 M sodium sorbitol borate
buffer. A 10% NaOH solution was present in a side arm of the manome-
tric vessel to absorb carl)on dioxide (which could be produced by respira-
tion). Figure 29.7 shows the course of pressure changes in a Warburg ap-
100
LU
(T
CO
cr
Q-
UJ
o
X
o
1130 THE LIGHT FACTOK. II. QUANTUM YIELD CHAP. 29
Table 29.X
Quantum Yield of Oxygen Evolution by Illuminated Chloroplast Suspensions
FROM Spinach (After French and Rabideau 1945)
MAXIMUM QUANTUM YIELD IN RELATION TO LIGHT CURVES 1131
Table 29. XI
Quantum Requirement of the Hill Reaction (after Ehrmantraut and Rabino-
WITCH 1951)
(Incident Light Flux 1.5 X 10"' Einstein per Min.: t = 10°C.; 200 mL Cells)
Material Oxidant Quantum requirement
Chlorella 0.5 mg. quinone in 3 cc.
Chlorella
Chloroplasts
(from Phytolacca americana)
Same
Same
Chlorella
5. Maximum Quantum Yield in Relation to Light Curves as a Whole
While much time and ingenuity have been invested in measuring the
yield of photosynthesis in very weak light in order to determine directly
the maximum quantum yield, no comparable effort has been made to ex-
tend these measurements to higher light intensities and to connect them
with the determination of the general shape of the light curve, described
in chapter 28. Tliis hiatus is worth filling in.
Pitfalls and corrections that loom large in the interpretation of experi-
mental results obtained in very weak light gradually fade into unimpor-
tance as light intensity increases. If the light curves, P = f(I), are smooth
curves of a comparatively simple and analytically expressible form, it
should be possible to determine the initial slope of these curves (i. e., I/to)
by extrapolation from reliable observations in comparatively strong light.
At least, it should be possible to use, as an additional criterion of reliability
of measurements in very weak light, the requirement that they should be
compatible with the results of measurements further up the light curve.
Reversing the above argument, the reliability of results obtained in
strong light, can sometime be judged by inquiring into their compatibility

with the quantum yield measurements in weak light.
(a) Extrapolation of Maxwium Quantum Yield from Measurements

at Higher Light Intensities
Many empirical light curves appear to be practically straight lines up

to comparatively high light intensities. If this straight line passes through
the zero point of coordinates, it seems safe to assume that its slope actually
represents the maximum quantum yield of photosynthesis under the condi-
tions to which the light curve refers. Probably, a more reliable value of
7n can be derived from this slope than from single points measured, with all
possible accuracy, near the origin of the coordinates, where the per cent
error of measurements is high and the respiration correction is larger than
the total measured gas exchange.
Whether the slope of light curves which appear as straight lines not
passing through the zero point, can be used to determine the quantum
yield, is less certain. Such a determination implies the assumption that
an increment of absorbed light energy produces an increment of true photo-
synthesis, while the photochemical process (or processes) responsible for
the curvature of the light curve near the zero point continue at the same
above the turning point-
rate at all light intensities
Warburg, Burk and co-workers (1949, 1950), and Moore and Duggar
(1949) detemiined the quantum yield of photosynthesis from the ratio of
— —
the increment of oxygen production and the increment of absorption,

in tlie region above the compensation point, and noticed no systematic
difference between the vakies obtained in this way, and these deteimined
in low Hght (in other words, the hght curve appeared, in these experi-
ments, as a straight hue passing through the zero point). Kok (1948,
1949), on the other hand, found for P = f{I), a straight hne passing above
the zero point, and concluded that the quantum yield of true photosyn-
thesis is lower than that of a photochemical process ("photorespiration")
which predominates in low light, is light-saturated in the neighborhood of
the compensation point, and runs at the same saturation speed at all the
higher intensities. Finally, French, and Wassink et at., working with purple
bacteria, found, in moderate light, approximately straight light curves,
whose linear extrapolation passed helow the zero point. He used the slope
in medium light to calculate the "true" quantum yield of bacterial photo-
synthesis —on the assumption that in weak light a photochemical process
occurs which either causes no consumption of hydrogen and carbon dioxide
at all, or does it with a much higher quantum requirement than bacterial
photosynthesis in stronger hght. In this case, too, the "low light process"
is supposed to continue at the same rate at all intensities above its light
saturation.
The upward cur\'ature of the light curves of bacterial photosynthesis
seems to be a generally encountered phenomenon, but the occurrence of an
accentuated downward curvature (or even of a sharp turn) of the light
curves of ordinary photosynthesis near the compensation point, remains
controversial. Equally open to doubt are assertions that the light curves of
photosynthesis (with or Avithout a break in the compensation region) re-
main linear, above this region, almost up to saturation. At least, the
most precise experiments in this field e.g., those by Emerson and Lewis
showed neither of the two phenomena, but indicated that the light curves
gradually bend downward with increasing light intensity, the first signs of
curvature being noticeable even at light fluxes of the order of only 1 kerg/
cm.- sec.
Such curves are most easily interpreted: we recall that all kinetic
mechanisms analyzed in chapter 28 lead to hyperbolic lighl curves. More
complicated kinetic mechanisms may lead to light curves of a higher order;
\)\\i, in any case, these curves will approach the limiting slope asymptoti-
cally, and are unlikely to become straight lines at any finite value of light
intensity. The most precise method for determining the maximiun quan-
tum yield may well be to measure the yields systematically as a function
of light intensity' in the region where deviations from linearity are small,
then to find an equation representing 7 adeciuatelj' as a function of /, and
use it for extrapolation to / = 0.
:
A more ambitious and significant undertaking would be to represent
by a single equation the whole light curve, including the saturation region,
and to calculate 70 from the yields observed in high light, using parameters
such as the maximum yield {P"'"-), and the half-saturating light intensity,
i/J.
We have seen in chapters 27 and 28 that theoretical kinetic curves
representing photosynthesis as a function of the supply of "reactants"
(carbon dioxide, reductants, light quanta), are hyperbolae, even when rather
complicated mechanisms are postulated, as long as no "third-order" reac-
tions [such as 2 COo +
A -> A(C02)2, or Chi 2 hv -^ Chi**] are consid- +
ered, and not more than two successive reaction steps are postulated be-
tween the external supply of the reactant and the "rate-determining" step.
(For example, in the case of the carbon dioxide factor, simultaneous con-
sideration of diffusion and carboxylation leads to a hyperbolic carbon dioxide
curve; but if one more supply step is interpolated, the resulting equation
is of the third order.)
The known light curves are much too unreliable to permit a useful in-
quiry into the question whether they actually are hyperbolae {cf. section
7/, chapter 28). If it were possible to demonstrate, by new and more pre-
cise measurements, that the light curves are hyperbolic, then each curve
could be determined completely by three points, i. e., values of P at three
known values of /.
Parameters such as 70 (i- e., the initial slope), .// or P'"''^ could replace
one measurement each. The general equation of a hyperbolic light curve
in terms of 70, i/J and P'^^^- is
P / 270 1/2/ 2_\ P^ ^ jn^

(2*J-5) pmax. _p "I
Wpmax.)2 pm&x.J pmax. _ p pmax J
(This equation obtained from the general equation of a hyperbola by

is
transformation to a set of coordinates with origin in a point on the hyperbola

and the abscissa parallel to the asymptote at a distance —
p™'^''-
from the
latter.)
In treating several particularly simple mechanisms in chapters 27 and
28, we obtained light curves (or carbon dioxide curves) that obeyed an even
simpler relation:
(2i).(i)
p/(^pmu.. _ p) = const. X /
or: P/(Pu.ax. - P) = const. X [CO.]
Comparison shows that this simplification means the disappearance of
the P- term in (29.5), i. e., the validity of the relation:
(29.6a) 70 = P^^^/^/Î
The relationship provides a simple way to check whether the kinetic

mechanism is of corresponding simplicity.
:
Whenever this is the case, and equation (29.6) is obeyed, differentiation

with respect to / shows that the quantum requirement, I/7, is a linear
function of light intensity, with i/p™'"^"- as the slope
(29.7) 1/7 = 1/70 + (I/P^-)
The question arises whether the experimental value of I/7 is a linear

function of the intensity of irradiation and, if so, whether the slope of the
corresponding straight line is equal to the inverse of the saturation value

of photosynthesis in strong light, 1/P"'^^-
Emerson and Lewis (1941)
-
7
Warburg (1948)
02 0.4 06 0.8 1.0
/, einstein/mole Chi x mm.
Fig. 29.8. Quantum requirement of Chiorella as function of light intensity.
Figure 29.8 represents an attempt to apply equation (29.7) to the

quantum yield data of Emerson and Lewis (1943) and of Warburg (1948).
Warburg's values scatter too widely to decide whether they lie on a straight
line or not; but, if this is assumed to be the case, the slope of the straight
line is much higher than in the case of Emerson's measurements (indicating
a much more rapid decline of quantum yield with the intensity of illumina-
:
tion). The abscissae in figure 29.8 are frequencies of absorption of light

ciuanta by each chlorophyll molecule (total number of (juanta absorbed by
the suspension in unit time, divided by the number of chlorophyll molecules
present). The slope of the Warburg curve, equated with 1/P'"'"'", indi-
cates a maximum production of one oxygen molecule per chlorophyll mole-
cule in about 10 minutes; the slope of the Emerson-Lewis curve, the pro-
duction of one oxygen molecule per chlorophyll molecule in about 1 minute.
Table 28. ^' shows that the actual maximum yield in Chlorella in steady
light is of the ortler of one molecule oxj^gen per molecule chlorophyll in 30
seconds. A rapitl decline of the apparent quantum yield with / is consistent
with the assumption that Warburg's values were affected by the inclusion
of the carbon dioxide gush, since the relative importance of this gush must
decrease rapidly with increasing light intensity (as was suggested on
page 1103).
Franck (1949) suggested that the rapid drop of I/70 in Warburg's
experiments with increasing light intensity, is an indication that they re-
flect a gradual repla(;ement of a 4-quanta process (half-way reversion of
respiration), by an 8-quanta process (true photosynthesis).
On page 1 104, we described the more recent experiments of Warburg,
Burk and co-workers, who claimed that the highest quantum yields are
obtainable by intermittent illumination (which prevails in very rapidly
agitated, dense Chlorella suspensions). If this were true (it was men-
tioned on page 1106, that the results of experiments in flashing hght do not
support the contention of Warburg and Burk), then the relation.ship be-
tween quantum yield and the (average) light intensity must be more com-
plicated than was envisaged in the above derivations.
(b) Quantum Yields in Strong Light
It was suggested above that the yields of photosynthesis given for high
light intensities should not contradict the results obtained in quantum
yield measurements in weak light. What we meant can be illustrated by
the following examples taken from the work of Willstatter and Stoll (1918).
According to figure 32.2, green leaves of Sambucus nigra reduce carbon

dioxide, at 6000 lux, at a rate of about 0.23 mg./cm.^ hr. or 1.44 X
10-''
mole/cm. 2 sec. Since 1 lux corresponds roughly to a flux of 5 erg/cm. ^ sec.

in the region 400-700 mix {cf. chapter 25, page 838), and about 80% of this
flux is a])sorbed by a single leaf, we can calculate, for the energy conversion
factor
(1.44 X 10-' X 112 X 10^) cal

0.28
(() X 103 X 5 X 0.24 X 10-' X 0.8) cal
THEORETICAL AND ACTUAL MAXIMUM QUANTUM YIELD 1137
which corresponds to a quantum yield of about 0.13 (assuming 550 m/x as

average wave length) . In j&g. 32.2, the yellow leaves of Samhucus are shown
to reduce, at 3000 mg. COj/cm.^
lux, 0.067 X lO-^" mole/cm.^hr., or 4
sec. These leaves contain less than one tenth the chlorophyll present in
green leaves of the same species. Measurements such as those represented
in figure 22.10 indicate that the aurea leaves absorb, in the region above
500 m/i, not more than 20% of the incident energy. (Blue and violet light
do not contribute much to photosynthesis in artificial light; cf. page 1163.)
Thus, the energy conversion factor of the yellow leaves, can be estimated as:
-
~ (4 X IQ-^" X 112 X 10^) cal
=
* ~ (3 X 103 X 5 X 0.24 X 10"' X 0.4) cal.
o 31
corresponding to a quantum yield of about 0.14.

Similar difiiculties arise in the interpretation of some of the maximum
yields of photosynthesis listed in Table 28.V (0.8 or 0.9 mg. COa/cm.^ hr.).
As can be judged from the known light curves of land plants, it seems
far as
safe to presume that saturation yields can be obtained in light of the order
of 40,000 lux. A yield of 0.9 mg. C02/cm.2 hr. at 40,000 lux means an av-
erage quantum yield of the order of 0.1, obtained in a region of almost com-
plete light saturation!
These estimates contain too many approximations to be used as quanti-
tative arguments against the upper limit 0.10 ± 0.02 for the quantum yield
of photosynthesis; but they show that it would be well to extend future
investigations of the quantum yield to the leaves of higher plants— partic-
ularly those of the aurea varieties— and to cover the entire length of the
light curves.
6. Theoretical and Actual Maximum Quantum Yield
All kinetic theories of photosynthesis agree that the (approximately)

linear lower part of the light curves corresponds to the state in which the
primary photochemical process is so slow that the nonphotochemical reac-
tions—the "preparatory" as well as the "finishing" ones— can supply the
materials and transform the products of this process without delay. It
may thus seem as if the maximum quantum yield, calculated from the
limiting slope of the light ciu-ves, should be equal to the number of quanta
actually needed for photosynthesis (except for the practically negligible
fraction lost by fluorescence). The fact that the experimentally determined
maximum cjuantum yields often are much lower than 0.1 shows that, in
many cases, the photosynthetic apparatus, or parts ofit, are in a noneffi-
cient state, so as to cause the loss of tiu; prodomiiinnt fraction of all the
:
absorbed light quanta. In "aged" cell suspensions, in particular, the

maximum quantum yield can be much smaller than in healthy young cells
(cf. fig. 28.13). The reasons for this inactive state are as yet unknown and
may lie in nutritional or enzymatic deficiencies (we recall, for example,
van Hille's experiments on the revival of photosynthesis in aged Chlorella
cultures by a fresh supply of N2) or in the obstruction of catalytic surfaces
by narcotizing metabolites ("chlorellin"; cf. page 880). Under the action
of external narcotics (cf. fig. 28.9C) the initial slope of the light curves is
clearly depressed, no full quantum yield can be obtained even in ex-

i. e.,
tremely weak light. This must be attributed to the inactivation of a cer-

tain proportion of chlorophyll complexes, probably by adsorption of the
narcotic the light quanta absorbed by these complexes remain unavailable
;
for photosynthesis.
An interesting question—not yet investigated experimentall}^ is —
whether a "substandard" quantum yield can be corrected, at least par-
tially, by an increase in temperature. If the low yield is caused by some
nonphotochemical process that has become so slow as to depress the rate of

photosynthesis even in very weak light, heating should accelerate this
process and thus improve the yield; but, if the inefficiency is caused by
the fact that a certain proportion of the photosensitive chlorophyll com-
plexes are inactive (partially decomposed, or obstructed by adsorption),
heating might have no effect on the yield.
Another interesting question is whether, even in the case when all
photosensitive catalytic complexes are fully efiicient, the experimental
maximum quantum yield must correspond exactly to the number of quanta
actually used in the reduction of carbon dioxide.
This question is raised by consideration of schemes 28.1A,B and 28.11,
all ofwhich envisage a competition between stabilizing "forward" reactions

and primary or secondary back reactions. Examples of primary back reac-
tions are (28.20a', 28.21a' and 28.41a') examples of secondary back reac-
;
tions are (28.20d and 28.21d).

In scheme 28. lA, the "rate-determining" reaction is (28.20b), and the
rate is given by equation (28.23). The quantum yield is:
(29.9) P/Ia = P/k*I Chlo = 7ikr[AC02]/{k' + A-JACOj])
The maximum quantum yield (reached when [ACO2] = Ao) is:
(29.10) 70 = kr\nn/ik' + AvAo)
In scheme 28.11, the rate is described by equation (28.42), and the

maximum quantum yield is given by equation (28.43)
THEORETICAL AND ACTUAL MAXIMUM QUANTUM YIELD 1139
(29.11) 70 = dP/d(k*Ch]oI) = keEln/ik' + A;.E°)
In (29.9), the "forward" reaction of HX-Chl-Z with ACO2 competes

with the primary back reaction (conversion to X- Chi -HZ); in (29.11)
the stabiUzing "forward" reaction of AHC02-Chl- A'HO with the catalyst
Eb competes with the back reaction (conversion to AC02-Chl-A'H20).
In either case, the "theoretical" quantum yield, n, can be closel}^ ap-
proached only if the ratio A-'/AvA) (or k'/keEâ) is much smaller than 1.
Recognition that the maximum observable quantimi yield may be smaller
than the theoretical quantum yield, n, was first reached in the derivations
of Franck and Herzfeld (1941) (c/. their Table 1). The reaction mecha-
nism used by them, although more complicated than the ones considered
here, also was based on competition between stabilizing forward reaction
and back reactions of the immediate reduction products.
The analytical expressions may be more complicated, but the essential
result also remains the same if the effective back reaction is a secondary
one, such as (28.21d). If this reaction competes with the forward trans-
formation of the intermediate reduction product AHCO3 by the catalyst
Eb (or of the intermediate oxidation product A'HO by the catalyst Ec),
the fraction of the products that undergoes back reaction will remain finite
even when the forward reactions have the maximum possible rate con-
stants, i. e., when the full available amounts of the relevant catalysts are
free and can be utilized for transformation (as is the case in weak light).
The Light Factor. II. Maximum Quantum Yield of Photosynthesis
1922 Warburg, 0., and Negelein, E., Z. phijsik. Chem., 102, 235.
1923 Warburg, 0., and Negelein, E., ibid., 106, 191.
Wurmser, R., Cornpt. rend., 181, 644.
1925 Wurmser, R., AnJi. physiol. physicochimie bioL, 1, 47.
1926 Wurmser, R., /. phys. radium, [II] 7, 33.
1929 Briggs, G. E., Proc. Roy. Soc. London, BIOS, 1.
1935 Gabrielsen, E. K., Planta, 23, 474.
1937 French, C. S.,/. (?m.P%s{o/., 20, 711; 21,71.
Sapozhnikov, D. I., Biokhimiya, 2, 18.
1938 Emerson, R., and Lewis, C. M., Carnegie Inst. Yearbook, 37, 216.
Manning, W. M., Stauffer, J. F., Duggar, B. M., and Daniels, F., /. Am.
Chem. Soc., 60, 266.
1140 THE LIGHT FACTOR. TI CHAP. 29
Manning, W. M., Juday, C, and Wolf, M., ibid., 60, 274.

Eymers, J. G., and Wassink, E. C, Enzymologia, 2, 258.
Wassink, E. C, Vermeulen, D., Reman, G. H., and Katz, E., ibid., 5, 100.

Rieke, F. F., J. Chem. Phijs., 7, 238.
Eichhoff, H. J., Biochem. Z., 303, 112.
^
/'^
Petering, H. G., Duggar, B. M., and Daniels, F., J. Ani. Chem. Soc., 61
3525.
Magee, J. L., De\\'itt, T. W'., Smith, E. C, and Daniels, F., ibid., 61,
3529.
Emerson, R., and Lewis, C. M., Carnegie hist. Yearbook, 40, 157.
Franck, J,, Sigma Xi Quart., 29, 80.
Franck, J., and Herzfeld, K. F., J. Phys. Chem., 45, 978.
Franck, J., and Gaffron, IL, Advances in Enzymology, 1, 199.

1942 Wassink, E. C., Katz, E., and Dorrestein, R., Enzymologia, 10, 269.
Emerson, R., and Lewis, C. M., J. Gen. Physiol., 25, 579.
Franck, J., Am. J. Botany, 29, 314.
1943 Emerson, R., and Lewis, C. M., ibid., 30, 165.
1944 Wassink, E. C., and Kersten, J. A. H., Enzymologia, 11, 282.
Tonnelat, Compt. rend., 218, 430.

J.,
1945 French, C. S., and Rabideau, G. S., /. Gen. Physiol, 28, 329.
1946 Tonnelat, J., Thesis, Univ. of Paris, Series A, No. 2092, serial No. 2959.
Wassink, E. C, Enzymologia, 12, 33.
Warburg, and Luettgens, W., Biokhimija, 11, 303.
0.,
1947 Rabinowitch, E., A.A.A.S. Symposium on Photosynthesis, Chicago, Dec.
1947 (unpublished).
Gabrielsen, E. K., Experieniia, 3, 439.
1948 Warburg, 0., Am. J. Botany, 35, 194.
Kok, B., Enzymologia, 13, 1.
1949 Rieke, F. F., in Photosynthesis in Plants, Tlie Iowa State College Press,
Ames, la., 1949, pp. 251-273.
Moore, W. E., and Duggar, B. M., ibid., pp. 238-250.
Emerson, R., and Nishimura, M. S., ibid., pp. 219-239.
Arnold, W., ibid., pp. 273-276.
Franck, J., Arch. Biochem., 23, 297.
Kok, B., Biochim. biophys. Acta., 3, 625.
Van der Veen, R., Physiol, plantaruni, 2, 217.
Burk, D., Hendricks, S., Korzenovsky, M., Schocken, V., and Warbui'g, O.,
Science, 110, 225.

Warburg, 0., Burk, D., Schocken, V., Korzenovsky, M., and Hendricks,
S., Arch. Biochem., 23, 330.
1950 \\'arl)urg, 0., Burk, D., Schocken, V., and Hendricks, S., Biochcm. Bio-
phys. Acta, 4, 335.
^^'al•l)Ul•g, 0. and Burk, D., Arch. Biochem., 25, 410.
Whittingham, C. P., Nishimura, M. S., and Emerson, li., Proc. Soc.
Exptl. Biol, (in press).
Brown, A. H., and co-workers (Columbus meeting of the Soc. Plant
Physiol., October, 1950).
1951 Tanada, T., Am. J. Botan., 38, 276.
Ehrmantraut, H. C. and Rabinowitch, E., Arch. Biochem. (in press).
Warburg, 0. and Burk, D., Naturwiss., 37, 560.
Warburg, 0., and Burk, D., Z. Naturforschung., 6b, 12.
Chapter 30
THE LIGHT FACTOR. HI. PHOTOSYNTHESIS AND LIGHT

QUALITY; ROLE OF ACCESSORY PIGMENTS*
1. Action Spectrum
The dependence of photosynthesis on the spectral quality of Ught was

the subject of much interest, long before the influence of light quantity
was first investigated. As early as 1788, Senebier conducted experiments
on carbon dioxide assimilation in double-walled vessels, filling the space
between the walls with various colored solutions. Since then, the botanical
literature has been replete with observations on the behavior of plants in
light of different color. (For a review of these investigations, see, for ex-
ample, Gabrielsen 1940.) This was to be expected, since everything con-
nected with color has always held, and still holds a captivating interest for
mankind, even though in scientific photochemistry the qualitative cate-
gories of "red," "blue," "yellow" or "green," which were so dear to Goethe
(he thought them to be the "primary phenomena" of optics), have been
reduced to mere quantitative differences between the energy contents of
the light quanta.
About a hundred years ago the effect of color on photosynthesis be-
came a topic of a lively discussion. Its subject was the position of the
maximum of photo synthetic efficiency in the solar spectrum. In 1844, Draper
found that, when the prismatic spectrum of the sun was thrown upon a
plant, the largest amount of oxygen was liberated in the yellow-green re-
gion this result was confirmed by such authorities in plant physiology as
;
Sachs (1864) and Pfeffer (1871). Sachs pointed out that the yellow is also
the region of maximum "luminosity" of light, i. e., of maximum effect on
the human retina. He himself saw in this only a coincidence; but other,
less cautious authors thought that such a correspondence must be sig-
nificant, and attempted to explain it. The belief that photosynthesis pro-
ceeds most actively in green light, which is only weakly absorbed by chloro-
phyll, led to several peculiar hypotheses, such as that light energy is not
used in photosynthesis at all (Pfeffer 1871), or that the role of chlorophyll
in plants is merely to protect the carbon dioxide-reducing system from
injuryby light (Pringsheim 1879, 1881, 1882).
Timiriazev (1869, 1875, 1877, 1885) vigorously fought these miscon-
* Bibliography, page 1 188.
1142
INTRODUCTION 1143
ceptions. He pointed out that "luminosity" is an anthropomorphic no-

tion, without meaning in objective photometry, that utihzation of light
energy is the essence of photosynthesis, that this utilization cannot take
place unless light is absorbed by a sensitizing pigment, and that this pig-
ment cannot be anything but chlorophyll. Timiriazev was the first to
use the concept of sensitization, a phenomenon then recently discovered
by Vogel and Becquerel, in the discussion of photosynthesis.
A similar point of view was taken by several physicists, e. g., Jamin,
Becquerel and, particularly, Lommel (1871, 1872). The latter pointed out
known as Herschel's law ("no
that the basic principle of photochemistry,
photochemical action without light absorption"), requires that the spec-
tral maximum of photosynthetic efficiency coincide with the absorption
maximum of the sensitizing pigment. Timiriazev (1869, 1875), Miiller
(1872), Engelmann (1882) and Reinke (1884) gave experimental proofs of
this coincidence, by showing that the photosynthetic efficiency of green
plants decreases steadily from red through yellow to green, parallel with
the decline in absorbing capacity of chlorophyll. The error of Draper,
Sachs and PfefYer was attributed by Timiriazev to the use of spectrally
impure light. (Timiriazev himself employed light isolated by a mono-
chromator with a narrow slit, and used microanalytical methods to com-
pensate for the weakness of illumination.) Engelmann suggested that the
error may have resulted from the use of thick leaves or thalli, which ab-
sorb light practically completely even in the minima between the absorp-
tion bands of chlorophyll. (He worked with microscopic plant objects,
using motile bacteria for the detection and determination of oxygen.)
Engelmann (1882) noticed that, in addition to the main maximum in
the red, the photosynthetic "action spectrum" of green plants has a second
maximum in the blue or violet, which he associated with the strong ab-
sorption band of chlorophyll in this region. This perfectly natural conclu-
sion became the subject of one of the most vitriohc controversies in the
history of photosynthesis; it was contested even by such enlightened plant
physiologists as Reinke (1884) and Timiriazev (1885). Particularly violent
were the criticisms Pringsheim (1886) directed against Engelmann's method
and his results; and Engelmann (1887) answered these attacks in language
seldom encountered in the pages of scientific journals, even in the quarrel-
some nineteenth century. At the same time, Engelmann also sharply
rebuked Timiriazev for his attempt (1885) to identify the main maximum
of spectroscopic efficiency of photosynthesis with the energy maximum of
the solar spectrum. Timiriazev saw in this alleged coincidence a striking
example of adaptation of organisms to the prevailing conditions, and thus a
triumph of the Darwinian theory. Engelmann answered that a coin-
cidence of the two maxima cannot be postulated without deliberately
1144 THE LIGHT FACTOR. III. COLOR CHAP. 30
twisting experimental evidence : Direct sunlight has an energy maximum

in the yellow and not in the red, as the action spectrum of photosynthesis;
and the energy maximum of diffuse sky light, which is the second com-
ponent of the natural "light field" of land plants, lies even further toward
the blue-violet end of the spectrum. In this controversy, too, Engelmann
was undoubtedly right. Looking backward, one cannot but admire the
unfailing correctness of his conclusions, obtained by means of an experi-
mental method most investigators would hesitate to use even for qualita-
tive, not to speak of quantitative, purposes. l<]ngelmann not only estab-
lished correctly the general parallelism between the action spectrum of
photosynthesis and the absorption spectrum of chlorophyll he also clearly ;
understood the influence on both spectra of the optical density of the

specimen. His conclusions concerning the photosynthetic efficiency of the
carotenoids and phycobilins, long neglected, appear well on the way to
vindication sixty five years later.
Much of the controversy between Engelmann and his opponents can be
attributed to a lack of understanding of what is meant by "action spec-
trum." A primitive definition can be based on the above-mentioned
simple experiment of Draper A spectrum
: is thrown on a plant or cell sus-
pension, and photosynthesis is measured in different spectral bands of
equal width. Such an experiment, performed with a prism in artificial
light, may easily lead to the belief that the action spectrum of photosyn-
thesis has only one maximum, because the energy of most artificial light
sources declines rapidly toward the violet end of the spectrum, while the
dispersion of the prism increases in the same direction. Both factors co-
operate in causing a rapid decline of the yield of photosynthesis (related to a
given spectral band width) as one proceeds from the red to the violet this ;
decline may more than offset any increase caused by the renewed rise in
the absorption capacity of chlorophyll in the blue-violet region.
Obviously, this definition of the "action spectrum" is arbitrary and ir-
relevant. An improved definition can be obtained by using Ught fluxes

of equal intensity at all wave
which can be achieved, e. g., by ap-
lengths,
propriate variation of the width of the monochromator slit, or insertion of
neutral gray filters. By using such methods. Kohl (1897, 1906), von Rich-
ter (1902) and Kniep and Minder (1909) were able to confirm Engelmann's
finding of the existence of a second maximum in the action spectrum of
photosynthesis, at the short-wave end of the visible region.
However, even an action spectrum obtained by the use of spectral
bands of equal energy is not universal, i. e., it cannot claim validity for all
plants, not even for all specimens of a given species (e. g., all Chlorella sus-
pensions). The first cause of variability of "equienergetic" action spectra
is the varying composition of the pigment system (c/. chapter 15); but
—
INTRODUCTION 1145
even for plants with identical contents of all pigments (or suspensions of
identical cells) the action spectrum still depends on two individual factors.
—
The importance of one of them the optical density of the sample was —
recognized by Engelmann In a thick leaf or thallus, or a dense cell sus-
:
pension, the absorption spectrum and the action spectrum both are blurred;
in the limiting case of complete absorption (approximately realized in
Warburg and Negelein's quantum yield experiments; cf. chapter 25, page
844), the action spectrum, too, may lose all structure.
The second factor that affects the action spectrum of an individual plant
or cell suspension (without affecting its absorption spectrum) is the in-
tensity of illumination. If one would use monochromatic light of such high
intensity as to obtain full light saturation at all wave lengths, the rate of
photosynthesis (which, in the hght-saturated state, is determined only by
the velocity of a dark process) will become identical in all parts of the
spectrum. structure of the action spectrum will appear as soon as the
The
intensity of illumination is reduced below the saturating value. Since the
saturating intensity depends on wave length {cf. sect. 4), the shape of the
action spectrum will change ^vith decreasing light intensity, until the latter
will fall within the practically linear range for all wave lengths. The
initial divergence and ultimate convergence of light curves in light of dif-
ferent color is illustrated by figure 30.8B obtained by Gabrielsen (1940)
with green leaves of Sinapis alba.

In the low intensity range, the shape of the action spectrum becomes
constant, and the spectrum thus acquires a definite significance. Here
and only here—can the action spectrum be compared quantitatively with the
absorption spectrum of the specimen, and the question asked whether the
specific photochemical efficiency depends on wave length.
First, however, a "quantum correction" must be applied. According
to Einstein's law of photochemical equivalency, one has reason to expect
equal numbers of absorbed quanta of different wave length to produce the
same photochemical effect, but not equal quantities of absorbed energy.
Thus, action spectra have to be "quantized," i.e. expressed in moles per

einstein, rather than in moles per erg or calorie. In the linear region, it is
legitimate to convert the "equienergetic" action spectrum into the "quan-
tized" spectrum simply by dividing all ordinates by the corresponding
wave lengths. In the saturation range, this is impossible, and the quantized
action spectrum can be obtained only by direct experiment (i. e., by measur-
ing the rate in bands of equal intensity expressed in einsteins per square
centimeter per second), because here the shape of the action spectrum de-
pends on intensity, and spectral bands that have equal intensity if meas-
ured in energy units have different intensities if measured in einsteins.
If the maximum quantum yield of photosynthesis is the same for all
wave lengths, the quantized action spectrum can be expected to parallel
exactly the absorption spectrum; the "equienergetic" action spectrum,

on the other hand, will always be askew, with blue-violet light appearing
ceteris paribus less efficient than red light.
If the quantized action spectrum determined from measurements in
low light, differs markedly from the absorption spectrum, this is a definite
indication that quanta of different wave length have different photochemi-
cal effects in photosynthesis. Recent determinations of the quantum
yield of green and colored algae in monochromatic light, carried out by
Emerson et al., Button and Manning, and Blinks, have established
the existence of such differences, and made speculations as to their origin
legitimate. Similar conclusions have been drawn previously from experi-
ments under badly controlled conditions, in which broad spectral bands
(isolated by means of colored glass filters) and unknown light intensities
were used. Conclusions drawn from experiments of this type, e. g., by
Montfort, have sometimes proved partially correct, but comparison of
Montfort's confused discussions with the concise presentation of Emerson
and Lewis, and Button and Manning gives a most eloquent demonstration
of the progress that can be achieved in plant physiology by the use of better
physicochemical tools.
The explanation wave length on the maximum quantum
of the effect of
yield of photosynthesis can be sought in three phenomena: (a) in the
composite nature of the pigment system and the (qualitatively or quantita-
tively) different photochemical functions of the individual pigments; (h)
in the multiplicity of the excited electronic states of chlorophyll, two (or three)
of which are involved in the light absorption in the visible spectrum {cf.
fig. 21.20) and (c) in the influence that vibrational energy, acquired by the
;
sensitizer together with electronic excitation, may have on its sensitizing

action. The first factor causes the rate of photosynthesis in different spec-
tral regions tobe affected by the apportionment of the absorbed light
energy to the several pigments, while the second and third factors can
cause changes in efficiency with wave length, even in light absorbed by a
single pigment.
Thus, the study of the effect of wave length on photosynthesis should
aim, first, at the qualitative and quantitative determination of the role of
various pigments in sensitization and, second, at the analysis of the rela-

tion between wave length and photochemical efficiency for each pigment.
Needless to say, we are far from having achieved these aims. Even now,
the greater part of newly pubUshed work on photosynthesis in colored
light remains purely descriptive and unsuitable for quantitative interpreta-

tion.
ACTION SPECTRUM OF GREEN PLANTS 1147
In addition to wave length, the quality of light is characterized by its polarization.

Almost universally, no attention has been paid to this characteristic in the study of the
relationship between light and photosynthesis. Dastur and Asana (1932) and Johnson
(1937) found no difference in the rate of photosynthesis in ordinary and linearly polar-
ized light of the same intensity; but Dastur and Gunjikar (1934) observed a marked
deficiency in the absorption by leaves of elliptically polarized light, and later (1935)
also a similar deficiency in the synthesis of carbohydrates. Although we may doubt
the correctness of these results, it must be borne in mind that birefringence of the chlo-
roplasts {cf. Vol. I, p. 362) and the correlated dichroism may conceivably lead to dif-
ferences in the capacity to utilize light of a different state of polarization. However,
only a very weak dichroism has been observed in the chloroplasts in the natural state
(cf. Vol. I, p. 366).
2. Quantum Yield and Wave Length in Green Plants.

Role of Carotenoids
In chapter 29, we maximum quantum yield of photosyn-

discussed the
thesis in green plants, without paying much attention to the quality of
light used for its determination, in the tacit assumption that it is either
altogether immaterial or has only a secondary influence. Quantum yield
measurements -with Chlorella in monochromatic light were first carried out
by Warburg and Negelein (1923). The results are shown in Table 30.1.
The quantum yield in blue light is somewhat higher than in red light, if
referred to the (estimated) absorption by chlorophyll alone, and somewhat
lower, if referred to the total absorption by all pigments.
If we assume that the relative yields of Warburg and Negelein in dif-
ferent spectral regions are significant (even though their absolute values
had been questioned by Emerson et al. chapter 29), the figures in Table
; cf.
30.1 point to a participation of the carotenoids as sensitizers in photosynthe-

sis, but with an efficiency inferior to that of the chlorophylls.
Table 30.1
Quantum Yields (after Warburg and Negelein 1923)
Light X, m/i y e
Red 610-690 0.23 0.59

Yellow 578 0.23 0.54
Green 546 0.21 0.44
Blue 436 0.20" 0.34"
0.28* 0.48''
" Referred to all pigments. ' Referred to chlorophyll alone.
When Warburg repeated his quantum yields measurements 25 years

later (Warburg 1946, 1948), he again found a somewhat lower ;yield (7 =
0.20 and 0.16 in two experiments) in blue light (X = 436 mju) as compared
with yellow light.
1148 THE LIGHT FACTOR. ITT. TOLOR CHAP. 30
Briggs (1929) made yield determinations in light of three colors and

obtained the results listed in Table 30. lA.
Table 30.IA
Photosynthesis Yield of Leaves in Colored Light (after Briggs 1929)
Yield, ml. O2/5OO cal incident light
Species Yellow light Green light Blue light
Phaseolus vulgaris 14-17 9-11 7-8

Uhnus, yellow 8.8 6.5 5.3
green — 20 12
Sambucus nigra 8.7 9.3 8.7
Sambiicus nigra — 19.0 15.0
These results obtained at light intensities 5-10 times stronger than

those used by Warburg and Negelein, show the expected decline in the
energy conversion jaeld with decreasing wave length (the experiment with
Samhucus is an exception). In other words, the "equienergetic" action
spectrum is askew as expected. The decline from yellow to blue is, how-
ever, somewhat stronger than could be explained by the quantum correc-
tion (the ratio of the yields in yellow and blue is 1.8 to 2.0, instead of 1.4),
again indicating a somewhat lower quantum yield in the region of absorption
by carotenoids.
Wurmser (1925) found, for Ulva lactuca, a much higher quantum yield
in the green than in the red, and a comparatively low yield in the blue;
but these results cannot be considered reliable {cf. page 1118). Gabrielsen's
data for Sinapis alba (1935), summarized in Table 30. Ill, (p. 1162) are
more significant. The relation between the quantum yields in the red and
blue is similar to that found by Warburg and Negelein, and thus allows a
similar interpretation. The value in the green —which, in contrast to
Wurmser's result, is lower —
than in the red may perhaps be taken as an
indication that the yellow-green filter used by Gabrielsen transmitted
much light absorbed by the carotenoids.
The only extensive investigation of the quantum yield of photosynthe-
sis ina green plant as a function of wave length was carried out by Emerson
and Lewis (1941, 1943) with Chlorella pijrenoidosa. They used bands
from 5 to 15 m/x wide, obtained by means of a powerful monochromator.
Figure 30.1 shows the results. The scattering of the points is indicative of
limitations to which the biological "standardization" of cell cultures is sub-
ject. Despite this scattering, it appears certain that the yield is approxi-
mately constant between 580 and 685 m/x. (The authors believe that the
shallow minimum at 660 m/x is real for its suggested interpretation, see page
;
1155.) Below 580 mju, the yield declines considerably, reaches a minimum
at 490 mju and then recovers. Roughly, the depression of the quantum yield
ACTION RPECTRTTM OF GREEN TTiANTS 1140
curve in the green, blue and violet covers the regions where carotene, luteol
nnd other carotenoids con1ril)ute markedl}^ to the Hght al)sorption by
ChJorella. An attempt at a quantitative interpretation of the 7 curve meets
with some difficulties. Figure 22.44 shows that, if the absorption by the
carotenoids is calculated on the basis of extract spectra (by shifting all
bands to make their maxima coincide with the absorption peaks of live
cells), significant participation of carotenoids in light absorption cannot be
GIG
009
008
Q
0.07
z
<
a
G.06
G.G5
004
400 440 480 520 560 600 640 680 720
WAVE LENGTH, m/i
Fig. 30.1. Quantum 3-iold of photo.syii thesis as a function of wave length for
Chlorella (afterEmerson and Lewis 1943). Points ol)tained on 19 runs are indi-
cated by distinct symbols. Band half widths used indicated by horizontal lines
of corresponding length.
expected above 540 mju. Thus the decline in y, which first begins at 580
mn, cannot be attributed to the carotenoids, unless one assumes that, in
the living cell, their absorption bands are not merely shifted, but so strongly
broadened as to extend up to 580 mî. The minimum in the quantum
yield curve, at 490 m/x, corresponds satisfactorily to the maximum of c aro-
tenoid absorption, according to fig. 22.44 (more exactly, to the center of
gravity of the two carotenoid peaks); but one notices that in this region
the carotenoids can be expected to account for 60 or 70% of the total al)-
sorption; while the depression of the y curve does not exceed 25%. It
thus again appears that, even though light quanta absorbed by the caro-
tenoids are less efficient than those absorbed by chlorophyll, they are not
entirely inefficient.
—
One may argue that, if the absorption band of the carotenoids in vivo
is strongly broadened (as suggested above), the maximum of this band may
be much lower than in vitro (since the area of the band —which is pro-
portional to the probability of the corresponding electronic transition
is not hkely to be much affected by the state of the pigment) . Consequently
the proportion of light absorbed by carotenoids in the region of the 7 mini-
mum may be smaller than estimated, thus reducing the discrepancy
between this proportion (supposedly, 60%) and the deficiency in 7 (25%).
100
80
O 60
I-
Q-
q:
° 40
m
<
20
42 mm' 40 mm 39 mm''
400 440 480 520 560 600 640 680 720

WAVE LENGTH, m,x
Fig. 30.2. Comparison of total absorption of Chlorella with the absorption ac-
tive in photosynthesis (after Emerson and Lewis, 1943).
It is unlikely, however, that this discrepancy can be completely elimi-

nated by this correction, i.e., that the assumption of a strongly broadened
and flattened absorption band of the carotenoids will permit the explana-
tion of the quantum yield curve on the basis of complete inefficiency of the
carotenoids. The average quantum yield deficiency between 400 and 580
mn according to figure 30.1, is about 15%; the average contribution of the
carotenoids to light absorption in the same region, according to figure
22.44, is close to 30%. Broadening of the absorption band is more
likely to increase than to decrease the latter value. Thus, the most likely
conclusion to be drawn from the results of Emerson and Lewis is that the
sensitizing efficiency of the carotenoids in Chlorella is not zero, but about
one half that of chlorophyll.
It need hardly be stressed that all conclusions of this type are predicated
on the assumption of a uniform distribution of the pigments, and would
have to be revised if it were demonstrated that some pigments form "color
screens" between the external light source and the other pigments.
—
CAROTENOIDS AS SENSITIZERS IN GREEN PLANTS 1151
The sharp dechne in quantum yield, which sets in, according to figure
30.1, above 680 mn, will be discussed in section 5.
Figure 30.1 was obtained with a dense suspension, which absorbed
practically completel}' at all wave lengths. Emerson and Lewis also made
measurements with a thinner suspension, which transmitted about one
half of incident light, to be able to compare directly the "action spectrum"
of photosynthesis in Chlorella with the absorption spectrum of the same
specimen. Figure 30.2 shows the results. The two curves were drawn
to coincide at 660 mû. They show a significant divergence above 690 and
below 570 m/x. The lower position of the action curve in the green, blue
and violet illustrates the relative inefficiency of the carotenoids; but the
comparative narrowness of the gap between the two curves confirms the
conclusion, reached above, that the carotenoids are not entirely inefficient.
The total absorption was measured directly; "active" absorption was calculated
from photosynthesis on the assumption that all light used for photosynthesis gives a
quantum yield of 0.084. This value was chosen to give agreement between the two
curves in the red, where all absorption is assumed to be active. The half widths of the
bands used are shown on the figure. The cells used in the run covering the red part of
the spectrum were from a separate culture.
The low sensitizing efficiency of the carotenoids of green

relatively
plants in photosynthesis, indicated by these experiments, may be either a
uniform property of all pigments of this group, or it may be an average,
e. g., some carotenoids may be as efficient as the chlorophylls, while others
are entirely inactive. On the basis of the absorption analysis in figure 22.44
itseems unlikely that the inactive fraction of the yellow pigments does not
consist of carotenoids at all, but is formed by pigments such as flavones or
anthocyanines.
can be argued that the shape of the quantum yield curve below 570
It
mju could also be explained by assuming that a complete inefficiencj'' of
carotenoids is partly compensated by an enhanced efficiency of chlorophyll.
The possible difference between the photochemical functions of chlorophyll
in the three excited states (corresponding to the blue-violet, orange and
red band systems, respectively) an important problem. The available
is
evidence gives little indication of such a difference. In chapters 21 (page

634) and 23 (page 748) we concluded, from the excitation of the same red
fluorescence band by light of all wave lengths, that chlorophyll molecules,
excited to the electronic states A or B, are rapidlj^ transferred, by a radia-
tionless process, into the lowest electronic excitation state, Y (which is
the upper state of the red fluorescence band). However, on page 752, we
concluded from Livingston's data, that, in the case of the blue-violet ab-
sorption band, the yield of this transformation is far less than 100%
in other words, that the fate of a large proportion of chlorophyll molecules

in state A is different from transfer into state Y {e.g., they may undergo
transition into a metastable state; cf. scheme 23.1). Whether this is true
not only of chlorophyll in solution but also of chlorophyll in the living
cell remains uncertain.
In vivo, direct photochemical dissociation of chlorophyll by blue-violet
light appears even less likely than in vitro (in consideration of the known
photostability of chlorophyll in the living cell) . True, the yield of chloro-
phyll fluorescence in vivo, as a function of wave length of the exciting light
(represented in fig. 24.5A), shows a slight decline at the violet end of the
spectrum; but this decline is most likely due to the presence of carotenoids,
and not to a decreased efficiency of light absorbed by chlorophyll itself.

As a matter of fact, figure 24. 5A was interpreted on page 814 as an indica-
tion that the fluorescence of chlorophyll in Chlorella can be excited — al-
—
though with reduced efficiency also by the light quanta absorbed by the
carotenoids; and this interpretation of the fluoresence curve is in agree-
ment with Emerson and Lewis' quantum yield curve of photosynthesis.
(However, the fluorescence curve in figure 24. 5A shows no minimum at
490 mn, which is so prominent in the quantum yield curve in figure 30.1.)
To sum up, the results of the quantum yield studies of both photosyn-
thesis and fluorescence are best explained by the assumption that a con-
siderable fraction — of the order of one half
—of the quanta absorbed by the carot-
enoids in green plants is passed over to chlorophyll, transferring the latter into
state Y. Therefore, these quanta can be used in the same way as those
absorbed directly by chlorophyll, either for photosynthesis or for fluor-
escence. It is furthermore likely that most or all blue-violet quanta
absorbed directly by chlorophyll are utilized for conversion into the fluore-
scent state Y.
Quantum yield measurements of Noddack and Eichhoff (1939) and
Eichhoff (1939) covered only the range above 515 m/x and therefore
reveal nothing concerning the function of the carotenoids. Their only
interesting feature is the high yield in the infrared; therefore they will be
discussed in the next section.
The action spectrum of the green alga Ulva taeniata, measured polaro-
graphically by Haxo and Blinks (1950), is represented in figure 30. 11 A,
and is in general agreement with the results of Emerson and Lewis.
3. Photosynthesis of Green Plants in Ultraviolet and Infrared
The exten.sion of the photosynthetically active region into the ultra-

^'iolet has not been studied by systematic quantum yield measurements in
monochromatic light, although it seems to be generally assumed that the
—
PHOTOSYNTHESIS OF GREEN PLANTS IN ULTRAVIOLET AND INFRARED 11 53
yield drops rapidly at, or only a little beyond, the violet end of the visible
spectrum (400 m/x)-
In vitro, the chlorophyll abso'rption extends throughout the near and
medium ultraviolet (c/. fig. 21.3). Whether other common components
of the plant cells, which absorb in this region, interfere with the light sup-
ply to chlorophyll (by acting as ''screens"), we do not know (this could
perhaps be elucidated by fluorescence measurements). Ursprung (1917,
1918) found evidence of photosynthesis in Phascolvs ivJgnris down to 330
him; Hoover (1937) and Burns (1942) ol)scr^•cd it in TrUiciun at 305 niju.
Gabrielsen (1940) calculated a yield of about 1 mg. CO2/5O cm.- hr. as the
possible contribution of the ultraviolet part of the solar spectrum to the
photosynthesis of leaves of Sinapis and Conjlus.
Johnson and Levring (1946) found with six marine algae (green and red),
a decline in respiration by about 1 mg. O2 per hr. per g. dry weight (deter-
mined by Winkler's methods) in near-ultraviolet light (366 mn, intensity
5 X 10"^ cal./cm.- min.). This was interpreted as evidence of photo-
synthetic effectiveness of this light. Further in the ultraviolet (260-320
m^, intensity about equal to that of the erytheme-producing radiation in
sunlight), no such effect could be observêd.
Light below 300 m/x is highly injurious to plants. According to Meier
(1932, 1934, 1936) an illumination of 1000 erg/cm.^ sec. kills ChloreUa
cells in 110 sec. at 260 m/i, and in 10,000 sec. at 302 m/x. Preferential in-
hibition of photosynthesis (with the cells still alive and respiring) by the
mercury resonance line 253.6 m^ was described by Arnold (1933) (c/. chap-
ter 13, Vol. I, page 344); it is not associated with a visible destruction of
chlorophyll.
The presence pigments {e. g., flavones, anthocyanines or
of yellow
other hydrophilic compounds, which may be present either in the cell sap
or in the cell walls) may cause a decline or complete cessation of photosyn-
thesis at comparatively long wave lengths. This is the probable reason why
Bums (1933, 1934) found that the photosynthesis of certain conifers e.g.,
spruce and white pine ceases—below 450-465 m/x {cf. page 1164).
In the spectmm of extracted chlorophyll, the absorption declines rap-
idly above 680 m/x; but the absorption spectra of intact cells reveal, in
addition to a shift of the red absorption band from 660 to approximately 680
mn, an extension of absorption to much longer waves. This spreading of
the red band can be recognized, e. g., in figures 22.10-22.24, 22.44,
and 22.48; and it has also been observed in aqueous suspensions of

chloroplastic matter (Smith, fig. 21.28). In the latter case, the infrared
"tail" of the redband disappeared upon clarification of the suspension by
digitonin, and was ascribed by Smith to scattering ("false absorption").
However, in other investigations, e.g., those of Noddack and Eichhoff
(fig. 22.21), the absorption in the far red was found also in measurements
purported to represent "tme" absorption. It may be due either to a

genuine broadening of the red chlorophyll band, or to the presence of other,
infrared-absorbing components (e. g., ferrous salts). The question of the
infrared limit of photosynthesis is closely associated with the interpreta-
The experimental results of Emer-
tion of this infrared absorption "tail."
son and Lewis (1943) and Blinks and Haxo (1950), on the one hand, and
Eichhoff (1939), on the other hand, are in extreme disagreement. As
0.08
246 mm*
493 mm'
986 mm'
)o 0.06
o"
UJ
>-
P 0.04
<
o
0.02
S
J_
680 700 720 740
WAVE LENGTH, m^
Decline in quantum yield in far red (after Emerson and Lewis 1943).
Fig. 30.3.
Points show apparent quantum yield with three different suspension densities,
calculated for equal incident light. Solid curve extrapolated for true yield, (com-
plete absorption), assuming that differences between the curves obtained with
different suspensions is due to incomplete absorption.
shown in figure 30.1, Emerson and Lewis found a sharp drop in quantum
yield above 680 m/x. This part of their curve reproduced in more detail
is
in figure 30.3. To be sure that the decline was not due to incomplete
absorption (we recall that the curve in figure 30.1 was obtained by War-
burg and Negelein's method which presupposes total absorption), Emerson
and Lewis made determinations with several suspensions of increasing den-
sity, and extrapolated the results to infinite density; the result is repre-
sented by the solid curve in figure 30.3. shows that, even with a gener-
It
ous allowance for incomplete absorption, there still remains a drop in 7,
from about 0.08 at 680 mn, to as little as 0.02 at 730 m/x.
— —
PHOTOSYNTHESIS OF GREEN PLANTS IN ULTRAVIOLET AND INFRARED 1155
A above 630 m/n was noted by Blinks and Haxo

similar decline of X
(1950) in the green alga Ulva taniata (fig. 30.11A), by Emerson and Lewis
(1941) with the blue-green alga Chroococcus (fig. 30.10A) and by Tanada
(1950) with the diatom Navicula minima (fig. 30. 9A). Ehrmantraut (1950)
noted the same phenomenon in the Hill reaction in Chlorella, Livingston
(c/. chapter 23, p. 752) noted a drop in the yield of the chlorophyll fluo-
rescence (in ether and acetone) in the same spectral region.

Emerson and Lewis suggested that the decline in quantum yield on the
infrared side of the absorption maximum of chlorophyll a (at 680 m/x) is
repeated on the red side of the absorption peak of chlorophyll h, and that
this is the explanation for the shallow minimum in the y ciu've that figure
30.1 showed near 660 mn. (The absorption maximum of chlorophyll h in
vivo must be situated at about 648 mû; cf. chapter 22, page 702.)
It may be pointed out in passing that the quantum yield curve of Emerson and
Lewis provides a direct argument against Seybold's (1941) hypothesis that chlorophyll b
does not act as a sensitizer in the reduction of carbon dioxide at all, but is a specific sen-
sitizer for the polymerization of sugars to starch.
In sharp contrast to the results of Emerson and Lewis are those of

Noddack and Eichhoff (1939) and Eichhoff (1939), who found (cf. Table
30.11) that the quantum yield of Chiordla remains high even at 832.5 mju.
Table 30.11
Quantum Yields of Chlorella According to Eichhoff

van m/i
832.5 0.164 598 0.238

780 0.204 576.5 0.263
750 0.244 558 0.238
725 0.228 542 0.204
685 0.179 527.5 0.232
650 0.204 515 0.222
622 0.228
Figure 30.4 shows a comparison of the action spectrum with the absorp-
tion spectrum of Chlorella, according to Eichhoff; the two curves remain
closely parallel far above 680 mix. Even if we doubt the correctness of the
absolute values of Eichhoff' s quantum yields (cf. page 1098), i\\ewave length
dependence of these values could be significant e. g., it would not be af-
fected by an on page 1098). One possible

error in calibration (suggested
—
but not very probable explanation of the differences between the two y
curves in the far red is that only Noddack and Eichhoff have measured
true absorption, while others have related the yield to the sum of absorp-
tion and scattering. The elucidation of this point is particularly desirable
because of the theoretical implications of a decline in y with wave length, as
—
obsei-ved by Emerson and Lewis, and Haxo and Blinks. The latter
(1950) observed, in the action spectmm of Ulva (fig. 30.11), a decline

in the far red quite similar to that found by Emerson and Lewis with
Chlorella. Altogether, the weight of experimental evidence seems to be
against the results ofNoddack and Eichhoff This is remarkable, because .
from the theoretical point of view one would rather expect the quantum
yield to remain constant within the red absorption band of chlorophyll.
The general experience in photochemistry is that wave length is not im-
portant for the photochemical effect, as long as one remains within a single
band system, even if this system extends over all colors of the rainbow.
The reason is that, within a single band system, the electronic excitation
energy is constant, and all excess energy absorbed by the molecule serves
14 70
/ \ \ Assimilation
12 60 Dense suspension /,
\
.
1 -^^ Absorption
15 min. V
10 - ^- 50
8 Q. 40
q:
O
6 CD 30
< ^ /••~-,^'^'" suspension \ V'v^
\ / / 30min^ ^ \ \ ^
4 20
2 10
500 600 700 ROO

WAVE LENGTH, mî
I''ig. 30.1. Action spectrum of Chlorella (after Eichhoff 1939). Scale at left
represents assimilation.
merely to increase its vibrational and rotational energy. If the primary

photochemical process is the dissociation of the absorbing molecule, the
only effect of variations in wave length is that the dissociation products
separate with different relative velocities; usually, this excess energy does
not affect the ultimate fate of the dissociation products, because it is lost
by collisions before the next reaction step. If the primary photochemical
process is electronic excitation, the excess vibrational energy acquired by
the absorbing molecules also will usually be lost before the occurrence of
the secondary reaction. However, exceptions to this behavior are known.
In some cases, the electronic excitation energy too small to bring about i.s
dissociation without the assistance of a. certain amount of vibrational

energy (an example is the delayed monomolecular photodissociation of
large molecules, described by Franck and Ilcrzfcld, 1937, and considered
PnOTORYNTHESIS OF GREEN PLANTS IN ULTRAVIOLET AND INFRARED 11 57
in chapter 18. page 484, as a possible consequence of the absorption of

blue-violet light by rhlorophyll). In other cases, the presence of excess
kinetic energy may help the dissociation products, formed in a tightly
packed medium, to escape immediate recombination (Franck and Rabino-
witch 1934). In still others, the vibrational energy of the excited elec-
tronic state may contribute directly to chemical activation; this is par-
ticularly likely in cases when the reaction partner forms a complex with the
absorbing molecule, so that no energy-dissipating collisions intervene be-
tween the primary and the secondary photochemical step.
The be suggested for a decline in the 7 values
latter explanation could
of photosynthesis at long waves, if the experimental 7 curve would indi-
cate that a certain minimum vibrational energy of the excited electronic

state is required for photosynthesis. However, according to Emerson and
Lewis, the decline occurs within the same band (and not upon transition
from one band to another, e. g., from the orange to the red band). This is
difficult to understand, since, within a single band, the absorption leads
everywhere, not only to the same electronic state, but also to the same vi-
brational state (at least, in respect to high-frequency band vibrations).
One could suggest that the red band of chlorophyll is not a single band,
but contains vibrational bands clustering tightly under its wings. How-
ever, vibrational bands situated on the infrared side of the main electronic
band most likely originate in the vibrating states of the ground state, and
lead to the same excited state as the main band; they thus offer no ex-
planation for a diminished quantum yield.
Another possibility is that the red absorption band of chlorophj-ll conceals a band
corresponding to a different electronic transition. In figure 21.20, it was suggested that a
comparatively weak band Xo —> Ao is hidden under the strong A'o —> Yo band; one could
suggest that absorption in the far red, exhibited by live caused by a shift of the
cells, is
Xo — > Ao band, rather than by an extension of the Xo —

Yo band. One could also sug-
>
gest that, in the series of transitions Xq -^ Ao, Xo -^ Ai, Xo —* A2, ., the latter ones,
. .
which lead to the vibrating states Ai, Ai, and give rise to chlorophyll bands in the orange,
yellow and green, can be followed by radiationless transitions into state Y, while the
first one, which leads to the non vibrating state ^0, cannot produce the same result
(because of insufficient energy of the excited state), and is therefore ineffective in bring-
ing about photosynthesis.
Before considering any of these hj^potheses too seriously, one should

ascertain whether the drop in 7 above 680 m^, obsei^ved by Emerson and
Lewis, not due to a more trivial cause, such as scattering (which Emer-
is
son and Lewis considered unlikely); to the presence of ferrous salts (or
other infrared-absorbing inorganic components) or the presence of a photo-
;
synthetically inactive pigment with a band greater than 680 m/i (perhaps
chlorophyll d; see page 1183).
Observations of the low yield of photosynthesis in filtered extreme red
or infrared light (Urspnmg 1918, Gabrielsen 1940, etc.) without the meas-
urement of absorption have verj^ little significance because of the well-

established rapid drop in the absorbing power of leaves in this region.
The only real problem whether the yield of photosynthesis drops 'pro-
is
portionally with absorption, ormore rapidly than the latter.

Hoover (1937) was able to observe the photosynthesis of wheat up to
750 m/i and Burns (1933, 1934), that of different conifers up to 740 rati.
4. Monochromatic Light Curves, and the Action Spectrum of

Photosynthesis in Strong Light
When the intensity of monochromatic light is raised, one soon reaches
the region in which the shape of the action spectrum becomes variable.
The light curves bend earlier or later, and come to saturation more or less
suddenly, depending on the value of the absorption coefficient, and on the
optical density of the sample, respectively. It was postulated above
(page 1145) that, when all curves reach saturation, the rate must become
independent of wave length, and the action spectrum must lose all structure.
The theoretical and experimental foundations of this postulate will be con-
sidered later (pp. 1162, 1165). At present, we will assume it to be valid,
and consider only the effect of wave length on the shape of the transition
from the linearly ascending part of the light curves (the slope of which at a
given wave length is determined by the product of absorption coef-
ficient and maximum quantum yield) to the "saturation plateau," the
height of which we assume to be independent of wave length.
The effect of optical density on light curves was discussed in chapter 28,
and the results were illustrated by the schematic figure 28.20. A change in
wave length is equivalent to a change in optical density; a cell suspension
that is "thin" in green light becomes "dense" in red or violet light. How-
ever, as far as the rate of photosynthesis is concerned, transition from
green to red light is not in all respects equivalent to an increase in cell
concentration, since the saturation level remains unchanged in the first
case,but increases proportionately with the number of cells in the second
case. Thus, the result of a change from strongly absorbed to weakly ab-
sorbed light is likely to be more similar to that of the change from green to
aurea leaves {cf. fig. 32.2), where the maximum rate is approximately the
same for both varieties.
As mentioned before, the comparison of light curves at different wave
lengths should be carried out by plotting the rate against N^p (number of
incident quanta/cm. ^ sec.,) rather than against the energy flux, 7, in erg
(or cal)/cm.^ sec. Otherwise, the light curves for the shorter waves will
remain below those for the longer waves, even if the photochemical ef-
ficiency of both kinds of quanta are identical (fig. 30. 6C). An example of
MONOCHROMATIC LIGHT CURVES 1159
how completely the "quantized" light curves obtained in monochromatic

light of different color coincide in their initial sections is given in figure 30.5.
Although the two uppermost points in this figure fall into the region of
beginning saturation, they still show no difference between the liglit curves
in green and red light. However, a divergence of these two curves in the
7 -
NT
E
O
in
.E 5h
E
O 4 -
_»
O
E 3 -
(O
V)
UJ
X 2 -
I-
z
>-
to
o
I-
o
X
a.
Fig. 30.5.
The same picture (fig. 30.6A) should be obtained also for partially absorb-
ing systems, if P is plotted against the absorbed (rather than against the
incident) intensity. If Nn^ (incident) is used as the independent variable
and the absorption is comparatively weak, the resulting picture must be
that shown in figure 30.6B.
If the monochromatic light curves are plotted against light energy (I,
or A) instead of number of quanta, Nh;,, the relationships become unneces-
sarily obscured {of. fig. 30.6C). (Some investigators, e. g., Montfort, have
JO
52
MONOCITEOMATIC LIGHT CURVES 1161
500 600
INCIDENT INTENSITY, cal/50 cm.'' hr.
Fig. 30.8A. Light curves of Sinapis alba (after Gabrielson 1935). Top, blue-violet
light; center, yellow-green; bottom, orange-red.
0-^-r-»-
7.«—"o X
200 400 600 800 1000 1200

INCIDENT INTENSITY, cal/SOcm'^hr
Fig. 30.8B. Light curves of Sinapis alba in light of different color (after Gabrielsen
1940). The same saturation yield is approached at all wave lengths. Circles, n>d-
orange light; crosses, yellow-green; triangles, blue-violet.
curves, we consider those in figures 30.7 and 30.8. In figure 30.7A, the
relative position of the curves for green and red light is as predicted in
figure 30.6B. In figure 30.7B, the curve for white light, lies, as predicted,
below that for the more strongly absorbed red light, but the difference is
much larger than expected.

It was mentioned in chapter 29 (page 1098) that the ratios between photochemically
equivalent intensities of red and white light, given by Eichhoff, appeared remarkably
small. was reached at an intensity equivalent to <2000 lux
(Satui'ation in red light
of white light!) one arbitrarily multiplies the "monochromatic" intensities by a
If
factor of 2.5 (this would reduce quantum yields from 0.25 to 0.10), fig. 29. 7B would be
changed as indicated by the dotted line, and acquire a much more plausible appearance.
Figure 30.8A and Table 30.III show the results of Gabrielsen (1935).
The difference in the quantum yield in the green and in the red is notable
Table 30.III
Photosynthesis in Colored Light (after Gabrielsen 1935)
Max. quantum Max. P, mg. CO2/ Reached at / =
Light Av. X, ni^ yield cm.^ hr. 11 cal/cm.^ sec.
Red-orange 650 0.100 0.192 1.67

Yellow-green 540 0.083 ca. 0.12 1.85
Blue-violet 430 0.071 ca. 0.05 0.83
(for a discussion of similar results by Emerson and Lewis, cf. page 1148).
The maximum rates in the blue-violet, and particularly in the green, also are
considerably smaller than in the red; however, figure 30.8A shows that
the observed maximum rates may be far from saturation.
still Figure
30. 8B, taken from a later publication by the same author (1940), shows the
coincidence of the maximum rates in the three spectral regions very clearly.
It is obvious from the preceding discussion that the action spectra of
photosynthesis, obtained by illuminating plants with light of partly saturat-
ing intensity, are difficult to interpret —even if precaution has been taken
to use the same incident intensity (or, better still, the same number of
incident quanta) in all spectral regions. For example, in an optically thin
system, the yield per incident quantum should be smaller in the green than
in the red, because of weaker absorption (cf. fig. 30. 6B); while in a dense,
completely absorbing system the relation could be reversed, because of the
better utilization of the more uniformily absorbed green light (cf. fig.
30.6A). Working with systems that are not too dense, and in light that
is not too strong, one may obtain a "quantized" action spectrum resembling
more or less closely the absorption spectrum of the pigments. An example
is given in figure 30.9, which shows the action spectrum of wheat as ob-
served by Hoover (1937) and "quantized" by Burns (1937-38,1942). How-
—
ever, no quantitative agreement between action spectrum and absorption

spectrum can be expected under these conditions, and conclusions drawn
from differences between them, e. g., as to the role of the accessory pig-
ments, are in the nature of more or less plausible guesses.
Because of the coincidence of the absorjjtion bands of the carol enoids in green
plants with the blue-violet bands of the two chlorophylls, correct guessing is in this
case much more difficult than in the case of brown or red algae. Engelmann (1887)
recognized tliis and based his suggestion that the carotenoids of green plants also act as
sensitizers in photosynthesis not on direct experiments with these plants, but on analogy
with the results obtained with colored algae. He quoted, as an additional argument in
favor of this suggestion, the observation that leaves of the aurea varieties have a com-
paratively high yield of photosynthesis, despite their deficiency in chlorophyll. How-
ever, he did not consider this argument as conclusive, at least not without x'enewed study;
and since then Willstatter and Stoll (1918) have shown that aurea leaves possess a high
relative efficiency also in light filtered through a yellow filter. Willstatter and Stoll saw
400 500 600 700 800

WAVE LENGTH, m/i
Fig. 30.9. Action spectrum of photosynthesis of wheat

"quantized" by Burns (1937, 1938) (after Hoover 1937).
in this proof that leaf carotenoids do not contribute to the sensitization of photosynthesis
in aurea leaves. However, this conclusion was not convincing because in their experi-
ments not only the relative, but also the absolute, yields in both green and aurea leaves
were almost unaffected by the interposition of a yellow filter. In other words, the in-
tensity of blue-violet light was negligible; therefore their experiments, while proving
that aurea leaves are liighly efficient in the light absorbed by chlorophyll alone, proved
nothing as to the efficiency or inefficiency of the carotenoids.
Wurmser (1921^) found the rate of photosynthesis of Ulva lactuca in the blue-violet
to be lower than in the green, but higher than in the red (calculated for absorption by
chlorophyll alone), thus indicating a possible active participation of the carotenoids.
Schmticker (1930) found, by bubble-counting experiments with Cabomba and Crypto-
coryne, that the light intensity required to achieve a certain rate of photosynthesis in-
creased from the red to yellow and green inversely proportionately to the wave length
thus indicating a constant quantum yield; in the blue and violet, on the other hand,
the increase was about 15% larger than was required by the quantum correction if all
pigments were assumed to be active, but somewhat less than could be expected if the
carotenoids were entirely inefficient.
After the problem of the role of carotenoids in photosynthesis had been brought
to the foreground by experiments with brown algae (cf. section 5), Montfort (1940)
made a new attempt to determine whether light absorption by the carotenoids of green
plants also contributes to photosynthesis. He compared the rates of photosynthesis of
the green alga Ulva lactuca in red and orange light (\> 550 m/x) with that in blue-green
light (X350-625 ran, maximum at 450-500 m/x) of equal incident intensity. Table
30.IV shows the results. Comparison of the last three figures in the table shows that the
Table 30. IV
Photosynthesis of Ulva lactuca in Colored Light (after Montfort 1940)

P = Rate of Photosynthesis; A = Rate of Absorption
P (blue-green)/P (orange-red) . 89
A (blue-green)/A (orange-red)° 1 . 19
A (blue-green)/A (orange-red)'' 1 .00"
iP/A) (blue-green)/(PM) (orange-red)" 0.75

(P/A) (blue-green)/(P/A) (orange-red)'' 0.89"
Quantum correction: X ( orange-red )/X (blue-green) O.??**
" All pigments.

''
Chlorophyll alone.
" Calculated from data on extracts.
''
For wave lengths 625 and 480 mju.
quantum yield in the blue-green was only slightly lower than in the red, if referred to the
absorption by all pigments, but much higher if referred to chlorophyll alone. This
speaks in favor of an almost equal efficiency of chlorophyll and the carotenoids. Thus,
the earlier observations of Wurmser, Schmlicker and Montfort can all be quoted in
support of the conclusions derived by Emerson and Lewis from the much more con-
vincing measurements in weaker and truly monochromatic light, that the carotenoids of
green plants do contribute actively, but less efficiently than chlorophyll, to the sensitiza-
tion of photosynthesis.
In ChlorcUa, the quantum yield deficiency in blue and violet light is not
likely to be caused by the presence of a yellow pigment other than the carot-
enoids. (The comparison of the absorption spectrum of live cells with
that of the extracted pigments, cf. fig. 22.44, does not indicate the presence
of such a pigment.) In some higher plants, on the other hand, pigments of
the flavone or anthocyanine class often are present in the cell sap or cell
walls and compete with the photosynthetically active pigments for blue-
violet ({uanta, or even serve es "color screens," particularly when they are
located in the* epidermis, or in the cell walls between the chloroplasts and
the external light source. The presence of these pigments should leave the
saturation yield unaffected, but should depress the quantum yield in the
linear range and in the region of partial saturation. Burns (1933, 1942)
noted that the quantum yield of photosynthesis of spruce and pine seedlings
in the blue-violet (390-470 m/x) was only half as large as in the red (630-
720 mn), or red plus orange (560-720 ran). This can be attributed to the
presence, in these conifers, of a nonactive yellow pigment. (It was men-

tioned on page 1153 that photosynthesis in these plants declines to zero
below 450 or 465 m/x.)
The same should be even more pronounced in leaves of the pur-
effect
purea varieties, or other leaves containing large quantities of red antho-
cyanine pigments. Engelmann recognized as early as 1887, in an investi-
gation entitled ''Leaf Hues and Their Importance for the Decomposition
of Carbonic Acid in Light," that the red pigments of land plants do not
actively participate in photosynthesis; and Willstatter and Stoll (1918)
and Kuilman (1930) confirmed that the presence of these pigments has no
influence on the rate of photosynthesis in strong light. Gabrielsen (1940)
concluded, from a review of the older work and new experiments with
Corylus and Prunus leaves in red-orange, yellow-green and blue-violet
light, that for a given amount of incident energy the red varieties have a
minimum yield in yellow-green light, where light absorption by the red
pigment has its maximum. This minimum was most pronounced in weak
light; in strong light, the monochromatic light curves approached the same
saturation level, in conformity with the findings of Willstatter and Stoll.
Gabrielsen estimated that, in red Corylus leaves, the "screen" absorbed
about 37% of incident light in the blue-violet, about 74% in the yellow-
green and about 33% in the red-orange region. In Prunus, the absorption
was somewhat higher, perhaps because in Corylus the red pigment was pres-
ent only in the epidermis cells, while in Prunus it was found also in the
mesophyll. These results cause us to give little credence to speculations
or observations that relate anthocyanines or flavones to photosynthesis.
In 1922 Noack observed a photochemical conversion of flavones into anthocyanines

in vivo and interpreted a chlorophyll-sensitized oxidation-reduction (cf.
this reaction as
chapter 19, page 541). He suggested that flavones and anthocyanines form a reversible
oxidation-reduction system, which may play a catalytic role in photosynthesis. Sen
(1942) asserted that anthocyanine-carrying leaves have a higher photosynlhetic ef-
ficiency than ordinary leaves, despite their lower content of chlorophyll.
We now return to the question, raised on page 1158, concerning the

third part of the light curves, after the linear range and the transitional
region — the saturation plateau. It was })ostulated there that this plateau
should have tlu; same height for all wave lengths. .Vs long as tlu; rate
isdetermined only by the kinetic mechanism of pJKjtosyntliesis, theoretical
arguments in favor of this postulate appear conclusive. Whether satura-
tion brought about by a limited supply of reactants, or by limited avail-
is
ability ofan enzyme, the maximum rate is determined b}^ the velocity of a
dark reaction, and should be independent not only of the quantity, but also
of the quality of illumination.
Light (luality could, however, affect the maxiinuni rote of photosynthe-
sis, if other photochemical processes can interfere with this process. A

selective sensitizaton of oxidative processes in the photosynthetic apparatus
by the light absorbed by the carotenoids, or by chlorophyll in the blue-violet
band, offers one possibility of this type. However, in this case, only light
curves of the time-dependent "optimum" type (which have been observed,
e. g., in some umbrophilic plants; cf. page 994) should be affected, since the
maximum rate in these curves is determined by the (time-dependent) bal-

ance of photos:ynthesis and photoxidation. Light curves with true satura-
tion plateaus should remain unaffected by an earlier onset of "light inhibi-
tion" (except for a shorter extension of the saturation plateau).
The effect of wave length on photoxidation has never been investigated
systematically. Franck and French (1941) found that photoxidation oc-
curs, in carbon dioxide-deprived leaves, in red as well as in blue light,
but this was merely a qualitative observation. A selective effect of blue
light on the respiration of Chlorella, observed by Emerson and Lewis (1943),
was mentioned in chapter 20 (page 568). Another specific function of
—
yellow pigments seems to be well established the sensitization of photo-
tropic movements. As stated on page 681, the changes in the positions of
the chloroplasts in light are caused only by light absorbed by yellow pig-
ments (Voerkel 1933) and the same is true of the phototactic movements
;
of whole cells {of. Castle 1935).

These pigments may be the carotenoids, although Galston attributed
this function to riboflavin (because the action spectrum showed only a
single peak). In purple bacteria, on the other hand, the action spectrum
of phototaxis coincides with that of photosynthesis {cf. p. 1188).
Very extensive studies of the effects of light of different colors in photo-

synthesis and respiration were made by Danilov (1935, 1936), using green,
blue-green, and red algae. He reported very complicated results in which
the yield was found to depend not only on color (in monochromatic light),
but also on the combination of colors (in non-monochromatic light). Fol-
lowing the tendency of what Kostychev proclaimed as a new "physiological"
approach to photosynthesis (cf. chapter 26, p. 872) he discussed these phe-
nomena in vague terms of stimulation and inhibition of different protoplasmic
functions by hght of different wave length. Thus, yellow and green rays
were credited by him with increasing cell sensitivity to red light, and with
making it insensitive to infrared light; blue-violet light was said to assist
in the utilization of infrared light (supposedly for activation of the "dark"

reaction stages in photosynthesis). Blue-green rays were said to counter-
act the stimulation effects of yellow light, and enhance the stimulating
effects of blue rays, and, generally, to create in the cells a "regulator of the
utilization of light energy," and also to determine the reaction of photo-
synthesis to changes in temperature. In another paper, Danilov (1938)

concluded that the effect of various colors of light on photosynthesis depends
on the method of cultivation of the algae. In particular, variations in the
sensitivity to different colors of light were caused by changes in hydration
of the cells (achieved by culturing Scenedesmus in 1% sodium chloride solu-
tion).
Ursprung (1917, 19182), working with detached leaves by means of Timiriazev's

(1890, 1903) "starch spectrum" method (a spectrum is projected on a starved, destarched
leaf, and the "latent starch image" formed by light is "developed" by iodine), found,
in light of uniform spectral intensity, a continuous decrease in the production of starch

from red to violet, without a second maximum, and attributed this result to the closure
of the stomata and consequent quenching of photosynthesis in blue-violet light.
Dastur and Samant (1933), Dastur and Mehta (1935) and Dastur and
Solomon (1937) described observations that purported to show that pure
red light (or pure blue light) is less efficient in photosynthesis than a com-
bination of both. Their experiments were criticized by Montfort (1937),
who found that the addition of blue-violet light to red light has no effect
on the rate of photosynthesis, provided the red light was in itself of saturat-
ing intensity (this agrees with the experiments of Button and Manning
with diatoms, described on page 1172).
Dastur, Kanitkar and Rao (1938) measured the formation of proteins
in leaves in light of different color, and found differences which they related
to the above-mentioned observations on photosynthesis in colored light.
The results of Dastur and co-workers probably are trivial, being caused
by the use of optically dense tissues. As explained above, such tissues
must (and do) utilize moderately intense green or yellow light better than
orange-red (or blue-violet) light of the same intensity, because the latter
is absorbed in too thin a layer and causes saturation effects there. White
light of partly saturating intensity, since it contains green and yellow, will
give, in such tissues, a higher yield of photosj^nthesis than an equally strong
red-orange or blue-violet light.
The hypothesis of Baly (1935) that photosynthesis requires a quantum of red plus
a quantum of blue light, was mentioned before (Vol. I, p. 554) and characterized as
entirely without experimental basis.
In chapter 28 (page 987) we described the different shape of light curves

ofshade-adapted and light-adapted plants. Since these plants differ in the
composition of their pigment systems, they are likely to show differences
also in their response to light of different color. Lubimenko (1923) ob-
served that shade-adapted plants often are relatively more efficient in blue-
violet light (and relatively easily inhibited by red light). This may be as-
1168 THE LIGHT FACTOR. ITT. COLOR CHAP. 30
sociated with their higher content of chlorophyll 6 (which permits a better

utilization of blue light, 450-500 niju), or with a higher content of carot-
enoids (which was indicated by Willstatter and Stoll's figures in Table
15. Ill, but was not confirmed, as a general rule, by Seybold and Egle's
analysis; cf. Vol. I, p. 414).
The composition of the pigment system also depends on the color of
the light under which the plants were grown. As described in chapter 15
(page 130), chlorophyll is more efficiently synthesized by green plants in red
light,and the carotonoids in blue-violet light (although these assertions
have been contested, and may represent over-simplifications). This may
explain why plants have often been found to be most efficient in the light
in which they were grown. Elodea plants cultured in red light produced
more oxygen in the red, while similar plants grown in blue light gave more
oxygen in the blue (Harder, Doring and Simonis 1936, Harder and Simonis
1938, and Simonis 1938). Thus, the physiological chromatic adaptation
of photosynthesis may be in this case a consequence of the chemical adapta-
tion of the pigment system.
5. Quantum Yield and Action Spectrum of Photosynthesis in Brown

Algae
The study of the relation between wave length and photosynthesis in
brown algae and diatoms is of special interest because of the presence in

these algae of the carotenoid fucoxanthol, which is not encountered in
green plants. The distribution of the light absorption by brown algae

and diatoms among the individual pigments was discussed in chapter 22
(page 723) and illustrated by the (very schematic) figures 22.45A, B and
Table 22.IX, all taken from Montfort (1940). For diatoms, we also gave
the much more adequate figure 22.46 of Button and Manning (1941).
The two reasons why even this figure is not too reliable are: first, the un-
certain (and undoubtedly to a certain extent incorrect) assumptions made
regarding the "red shift" of the absorption bands in vivo, and, second, the
neglect of chlorophyll c. According to Tanada's figure 30. 9B, chlorophyll
c adds much to light absorption of pigment extracts from bro^vn algae and
diatoms between 450 and 500 mju. The brown color of these organisms
in VIVO indicates considerable absorption also farther in the green, from
500-550 niM- Part of this absorption may be due to chlorophyll c, but
most is probably due to fucoxanthol (to which it has usually been ascribed,
cj. page 707). Montfort (1940) discussed the experimental action spectra
of photosynthesis in different brown algae and concluded that light ab-
sorbed by fucoxanthol is fully utilized for photosjmthesis; but this conclu-
sion was not very convincing because of the very primitive experimental
approach, which included the use of broad spectral regions, and of light of
ACTION SPECTRUM OF BROWN ALGAE IIG'J
comparatively high intensit3^ Dutton and Manning (1941) arrived at a

similar conclusion by a procedure which was much more satisfactory at —
least, in principle —
namely, the determination of quantum j-ields in weak
and trul}^ monochromatic light. Because the method of Dutton and Man-
ning is so much more adequate than that of Montfort (c/. the criticism of
Emerson 1937) we will discuss their experiments first.
Dutton and Manning used the dropping mercury electrode for the
determination of oxygen (c/. chapter 25, page 850). The diatom {Nitzschia
closierium) was found to be more sensitive than Chlorella to merciuy; how-
ever, its resistance was sufficient to permit measurements of 30 minutes
duration without marked poisoning.
Chromatographic analysis of the pigments of Nitzschia closierium re-
vealed the presence of chlorophyll a, carotene, luteol, fucoxanthol and prob-
ably flavoxanthol, but showed no trace of chlorophyll h. No mention was

made of chlorophyll c, which was subsequently found by Strain and Man-
ning (Vol. I, p. 406) in diatoms and other broMTi algae. Analysis of the
absorption spectmm of the extract (c/. fig. 22.46) indicated that in methanol
solution at least one half the absorption between 400 and 550 m/i was due
to the carotenoids. This is a much larger proportion than in green plants
(c/. page 1150); it indicates that the ratio chloroph.yll a/total carotenoids
was, in the investigated diatoms, considerably lower than 3/1 (which is

the average for brown algae listed in Table 15.III).
Monochromatic light from a high-pressure a. c. mercury arc was used
for illumination (the effect of intermittency being considered unimpor-
tant), as well as bands isolated from the light of an incandescent lamp by
appropriate filters. The density of the suspensions was such as to give
about 50% absorption in the red, and 75% in the blue. Two portions of
the same suspension were placed in two vessels, and a simultaneous deter-
mination of the quantum yield was made in both of them, the one vessel
being illuminated with violet, blue or green light, and the other with red
light. Table 30. V shows the results. This table indicates that the
individual y values varied, for each wave length, within wide limits {e.g.,
in the red, from 0.038 to 0.100); possibly because of various degrees of

mercury poisoning, or other factors affecting the vitality of the cells. The
conclusions therefore depended entirely on the ratios of the yields obtained
in light of different color, in simultaneous experiments with two aliquots of
the same cells. Table 30.V shows that even these ratios varied widely (e. g.,
from 0.90 to 1.43 in the comparison of yields in violet and red light). The
most consistent was the series of measurements at 496 mû, where seven
determinations all gave, for the ratio (quantum yield in blue-green)/ (quan-
tum yield in red) values between 0.07 and 0.08. By averaging the ratios,
1)utton and Manning arrived at the values in the last column of Table 30.V.
Table 30.V
Quantum Yields of Diatoms (after Dutton and Manning, 1941)
% of total
ACTION SPECTRUM OF BROWN ALGAE 1171
the ratios of thequantum yields in the bhie, violet, green and red, but, even
more by the absolute value of the yield at 496 m/x, where, ac-
strikingly,
cording to their estimates, 93% of absorbed light is taken up by the carot-
enoids. They argued that, if all photosynthesis observed in this spec-
tral region were attributed to chlorophyll, the quantum yield would be
0.059/(1 — 0.93) = 0.84, i. e., much larger than the maximum allowed by
thermochemical considerations. However, this estimate was based on the
distribution data in figure 22.46, and therefore is subject to possible grave
errors. True, at 496 mn, the apportionment of energy is not very sensitive
to the postulated specific value of the "red shift"; it would remain almost
the same if a shift of 10 or 30 m/i were postulated, instead of 20 m/z (the
value used by Button and Manning), or if the shift of the carotenoid
— —
bands particularly^ those of fucoxanthol were assumed to be twice or
three times as large as that of the chlorophyll bands. (A difference of this
type is indicated by some data in chapter 22; cf. Table 22. VI and page
706.) It may thus seem as if an extreme and unlikely assumption con-
cerning the enhancement of the absorption of blue-green light by chloro-
phyll in vivo, or the assumption of a spatial distribution of pigments strongly
favoring absorption by chlorophyll would be required to explain the quan-
tum yield observed at 496 m^t without recourse to sensitization by carot-
enoids. This argument, however, ceased to be quite conclusive, since
Strain and Manning (1942) confirmed the presence in blown algae and
diatoms, of a pigment with strong absorption in the blue-green, chlorophyll
c. According to figure 21.5 this component has an absorption peak at 450
m/x in methanol; in the living cell, its absorption maximum must
lie near
470 m/x, if the shift is the same as for chlorophyll

According to figure
a.
30. 9C, at 470 mix, chlorophyll c in a methanol extract from diatoms accounts
for about ten times more absorption than chlorophyll a. The neglect of
chlorophyll c in the calculations of Button and Manning thus may have
shifted the ratio of the absorptions by the chlorophyll pigments and the
carotenoids, from perhaps about 1 to 1, to the extreme value of 9.3 to 0.7.
To sum by Button and Manning sup-
up, the average 7 values found
port the assumption that the carotenoids in diatoms (and fucoxanthol in
particular) contribute directly to the sensitization of photosynthesis; but
the wide scattering of individual results called for reinvestigation with
material and methods giving more consistent results. Furthermore, all
results, and particularly the absolute yields at 496 m/x, were in need of re-
examination in the light of the possible role of chlorophyll c. This re-
examination could have conceivably brought the brown algae in line with
—
green algae organisms in which a distinctly lower quantum yield of
photosynthesis was observed in the regions of the carotenoid absorption,
but a yield not sufficiently low to permit the assumption of complete in-
1172 TITE Lir.TTT FACTOR. III. fOT.OTl CTI.\P. 30
This hypothesis was supported by the action

acth'ity of the carotenoids.
spectrum of the brown alga Coilodesme, determined polarographically by
Haxo and BHnks (1950) and reproduced, together with the absorption
spectrum, in figure 30.1 IB.
Dutton and Manning

also performed experiments in stronger light, in which they
measured photosynthesis in saturating red light, and then added violet light. They
first
— —
found as one would expect no appreciable effect of this additional illumination on
the yield, -.mA interpreted this as a proof that photosynthesis in blue light, although
sensitized by both chloroph3ll atul the carotenoids, is limited by the same dark reaction
as photosynthesis in red light, which is sensitized by chlorophjdl alone.
Wassink and Kersten (1946) studied the diatom Nitzschia dissipata;

the spectroscopic results of this study were presented in Chapter 22 (p. 706).
These investigators made measiu'ements of the rate of photosynthesis in
012
0.10
0.08
0.06
0.04
0.02
synthesis. They found, similarly to Dutton, ISIanning and Duggar

(Chapter 24, p. 814), that chlorophyll fluorescence in diatoms can he
li\'ing
excited also by light absorbed by fucoxanthol; from this they concluded

that the energy absorbed by fucoxanthol is transferred to chlorophyll be-
fore it is used for photosynthesis.
1.0
•\
0.9
0.8
0.7
0.6
OS'*
o
o
L-,
0.4
0.3
0.2
440 520 600 680

WAVE LENGTH, m^
Fig. 30.9B. Absorption spectra of

methanol solutions of pigments ex-
tracted quant ittitively from cells of
\'avic>da minima (after Taiiada,
1951).
.
yield dips by about 20% between 520 and 475 ibm (we recall that a minimum
of efficiency was found in this region also for Chlorella and Chroococcus)
The yield rises somewhat further in the violet, but declines again toward
the ultraviolet, its value at 400 mju being about 30% below the maximum.
This 7 = /(X) curve must be compared with the curve showing the ab-
sorption by the several pigments in extract from these diatoms (fig. 30. 9B),
and with the curve (fig. 30.9D), derived from it, showing the contribution
of each pigment to the total absorption by the cells. The first figure shows
chlorophyll a, chlorophyll c, and fucoxanthol, as the major components.
100
o
80
a:
o
(/)
CD
<
60
u. 40
o
z
UJ
o
cc
UJ
CL
.
(However, the latter value was derived from a small shoulder on the
absorption curve, and is not precise.) The absorption peak of fucoxanthol
in vivo was calculated from the difference between the absorption curves
of the cells before and after extraction with aqueous methanol it indicated ;
a red shift by as —
much as 40 m/x for this pigment from 445 mn in methanolic
solution, to 485 mju in vivo. The blue peak of chlorophyll c was calculated
similarly from the difference between the spectra of cells before and after
extraction with 50% methanol; a red shift of 20 m/x was deduced in this
way.
(The deduced by Tanada for the four blue-violet bands 8 m/x
shifts —
—
and 40 mju for fucoxanthol can be compared with those
for chlorophyll a,
derived in chapter 22, on pages 705-706, from earlier investigations.
The agreement is good for chlorophyll a; for fucoxanthol, the shift found
— —
by Tanada 40 m/x is about twice that estimated previously by Wassink
and Kersten.)
When the blue-violet solution bands of all pigments present in the
diatoms were shifted as indicated, and superimposed, a composite absorp-
tion curve was obtained (see fig. 30.9C). (No attempt was made by
Tanada to analyze the region > 620 mju, where all absorption is due to the
chlorophylls.) The quantitative agreement is not too good, the composite
curve having a higher peak, and a lower valley in the green than the actual
absorption cui-ve of the cells. This may be due, at least in part, to scatter-
ing —
although the cell cur\ê was obtained on Hardy spectrophotometer
equipped with an integrating sphere, and with cells immersed in glycerol to
reduce scattering. Another likely source of discrepancies is the broadening
of the absorption bands in vivo (particular^ of that of fucoxanthol)
The uncertainty implied between the two curves,
in the discrepancy
had to be accepted in estimating the contribution of the several pigments
to total cell absorption at any given wave length. The results of this
estimate are shown by fig. 30.9D. It indicates that in the region 500-
550 ran, fucoxanthol takes up most of the quanta absorbed; and yet, fig.
30. 9A shows no drop in yield in this region —
except below 520 mju, where
absorption by "other carotenoids" sets in. The simplest explanation of the
results is that three pigments — chlorophylls a and c and fucoxanthol — are
fully effective in photosynthesis (70 — 0.11), while the other carotenoids
are either altogether ineffective or have a much smaller efficiency.
Tanada foundfurther that 70 of Navicula minima was the same at 1.5,
10, and 20°C., and that 7 was a smooth function of light intensitj' between
0.9 and 6.9 X 10 ~* einstein/cm.^ min., the curvature becoming noticeable
>2 X 10~* einstein/cm.2 min. Points obtained in red, orange, blue and
green light onto the same slightly curved
all fell line, similar to that found
for ChloreUa by Emerson and Lewis (fig. 30.5).
117G THE LIGHT FACTOR. III. COLOR CHAP. 30
If fucoxanthol and other carotenoids are to a certain extent active as

sensitizers in green plantsand brown algae, the mechanism of their par-
ticipation is be based on a transfer of energy to chlorophyll, rather
likely to
than on a direct interaction with the oxidation-reduction system. This
hypothesis was suggested by Engelmann over fifty years ago; its first
direct confirmation came from the experiments of Button, Manning and
Duggar on the excitation of chlorophyll fluorescence by light absorbed by
the carotenoids, wliich were described in cluipter 24 (page 814). They
were carried out with the same organism {Nitzschia clodcrium) that was
also used for the measurement of the quantum yield of photosynthesis.
The quantitative results of this study also are in need of re-examination for
possible effects of chlorophyll c.
We will now describe in brief the experiments in light of indefinite (and probably
partly .saturating) intensity, which can be aiiduced in support of the hypothesis that
the carotenoids of brown algae actively participate in photosynthesis. Conditions in
these organisms appeared somewhat more favorable for coriect guessing than in green
plants, because of the absence of chlorophyll b, and consequent enhanced importance
of the carotenoids for the absorption in the region between 450 and 500 m/u; liere again
the contribution of chlorophyll c requires consideration.
As early as 1884, Engelmann found that brown algae (Melosira, Navicula, Pinnul-
aria) illuminated by sunlight or gas light, produced the largest amount of oxygen in the
green part of the spectrum, and concluded that the "orange pigment" of these algae must
participate in sensitization.
Fifty years later, Montfort (1934) compared the rate of photosynthesis in white
light of a certain standard intensity with its rate in orange-red light. While greeji algae
{Ulva lactuca) were equally efficient with both kinds of illuminations, brown algae,
Diclyota dichotoma, Alaria and Desmarestia, produced in orange-red light only one half
the oxygen liberated in wliite light. Montfort interpreted this as an indication that
Phaeophyceae have a liigher relative efficiency in blue and violet light, and attributed
this to the presence of fucoxanthol. Later, IMontfort (1936) and his co-worker Schmidt
(1937) found that the removal of blue and violet rays from white light depressed the
rate of photosynthesis inbrown algae {Diclyota and Laminaria) much more strongly
than in the green Ulva. They calculated the ratio P/A (photosynthesis per unit ab-
sorbed energy) for different colors, using incident light of equal intensity in all spectral
regions.
An arbitrary selection from the confusing abundance of their material is shown in
Table 30. VI. Not all the figures in this table may be strictly comparable, but they
show the trend of the results.
In green algae, the decrease in P/I from the red to the green and the renewed in-
crease in the blue reflected more or less clearly the changes in absorption and in the size
of quanta. (These two factors cooperate to make the yield in green light smaller than
in the red; —
but become antagonistic in the blue.) In brown algae with the exception
of Fucus, which Montfort classified as a "xanthophyll alga" (whereas he designated
—
Laminaria and Diclyota as "fucoxanthol algae") the yields were invariably 30 or 40%
higher in the blue than in the red, and would have become twice as high if related
to the absorption by chlorophyll alone. In the green, too, the P/I values were relatively
higher than they should be according to the absorption of chlorophyll and the size of the
quanta. Moatfort and Schmidt concluded from these results that, of all the carotenoids,
only fucoxanthol is able to assist in the sensitization of photosyntlu^sis, whereas the
carotenoids of green algae are inactive (for a criticism of their methods, sec Emerson
1937).
Table 30. VI
Energy Yield of Algae in Light of Different Colors" (after Schmidt 1937)

P /I (green) P/I (blii^
Alga P/I (red) P/I (red)
Green ^ „^
Cladophora 0-49 0.80
Ulva laduca 0.46 0.82
Brown
Laminaria digitata
Strong light (1) 1.29
Medium light (Vs) 0.91 1 .32
Weak light Qi) 1-00 1 .23
Phyllilis fascia — 1-40
Dictyota dichotoma 90.
1 48.
Fucus vesiculosus (brown, but with low fucoxanthol content) 0.38 0.59
" P/I for equal incident intensities.
A certain improvement of methods was attempted by Montfort later (1940). The

pigments were extracted from the algae and their absorption curves determined (in
methanol solution), with results shown in figure 22.45A,B {cf. also Table 22.VIII). These
absorption data were applied to the results of Gabrielsen and Steemann-Nielsen (1938),
who found that, for equal incident light intensity, the rate of oxygen production by di-
atoms is consistently higher in the blue than in the red. (A similar difference was re-
ported earlier by Mothes, Baatz and Sagromsky 1939.) The difference is particularly
strong in low light, as shown by Table 30. VII. The table describes a perfectly under-
Table 30.VII
Ratios of Rates of Photosynthesis by Diatoms in Blue and Red Light
(after Gabrielsen and Steemann-Nielsen 1938)
PjblueJ Incident light.

Incident light,
P (red) cal/cm.2 sec.
^r^.v
P (red)
cal/cm.2 sec.
0.25 1.8 1.7 1.4

0.5 l.fi 2.4 1.3
1.0 1.5 3.7 1.1
standable transition from the conditions at low light intensities (where the yield is pro-
portional to absorption, and may therefore be higher in the blue than in the red, if the
absorption in the blue is so nmch sti'onger as to overbalance the laiger size of the quanta)
to the conditions in strong (saturating) light, where the rate must be (and apparently is)
independent of wave length. Montfort (1940) preferred, however, to average all the
ratios in Table 30.VII and concluded that a ratio of 1.5 between the rates in blue light
and red light indicates an active participation of carotenoids in photosynthesis. Using
the absorption curviis of methanolic extracts he calculated that the energy conversion
rate in blue light is 1.03 times larger than in red light, referred to absorption by all pig-
—
ments, and 2.46 times larger referred to chlorophyll alone; while, according to the size
of the quanta, it should be smaller by a factor of 0.68.
In the case of brown algae {Laminaria digitata) a similar calculation (based on Mont-
fort's own measurements) gave energy yield ratios (related to the combined absorption
by all pigments) of 0.86 and 1.18 (for two different combinations of filters), as compared
with the quantum size ratios of 0.64 and 0.77, respectively, again indicating a higher
quantum yield in the blue-violet than in the red. At their face value, Montfort's figures
appear to indicate that the carotenoids of the diatoms and brown algae are several times
more efficient as sensitizers than chlorophyll!
Mothes, Baatz, and Sagromsky (1939), Baatz (1941) and Sagromsky (1943) have
described observations of the rate of photosynthesis in filtered red and blue light of
equal intensity (in energy units). They found for these two rates, a ratio of 1 1.2 in the
:
diatom Chaetoceras simplicia centrosperma, as against 1 0.7 in two unicellular green algae,
:
and attributed the relatively better utilization of blue light by the brown cells to the
presence of fucoxanthol. These experiments were more satisfactory than those of Mont-
font in that imicellular algae were used rather than thick thalli; but it was equally un-
satisfactory in the use of broad spectral regions, and even less satisfactory in the absence
of any absorption measurements, which would permit approximate allocation of ab-
sorption to the several pigments. Mothes and co-workers pointed out the difficulty
of the latter problem, caused by the difference of the carotenoid spectrum in vivo from
that in vitro. (This difference is clearly indicated by the change of color from brown to
—
green caused by placing brown algae in hot water a treatment which, they assumed,
disrupts the molecular association of carotenoids with chlorophyll and proteins.)
6. Quantum Yield and Action Spectrum of Red and Blue Algae.

Role of Phycobilins
The history of our knowledge of the role of phycobilins in the sensitiza-

tion of photosynthesis in red and blue algae is similar to that of the role of
carotenoids in —
brown algae. Here too we find the we now know, correct
guess by Engelmann, made as early as 1883, that the phycobilins are active
sensitizers of photosynthesis, as well as a series of vague and indecisive
observations and calculations of several authors, mostly tending to confirm
this guess, and finally, quantitative analyses of the quantum yield as a
function of wave length, carried out by Emerson and Lewis (1941, 1942),
and Haxo and Blinks (1950), which brought convincing confirmation of
Engelmann's concept. As in the previous section, we will discuss the
more recent and reliable experiments first.
Table 29. Ill showed that Emerson and Lewis (1941), in comparing
the quantum yield of different plants in yellow sodium light, found no
difference between green plants and the blue-green algae Chroococcus, al-
though in the latter, far more than half the absorption in the yellow re-
gion was due to phycocyanine. This result of a preliminary observation
was in itself a more convincing proof of the activity of phycobilins in photo-
synthesis than could be derived from all the extensive earlier discussions
of this problem. It was confirmed and amplified by the same authors in
ACTION SPECTRUM OF RED AND BLUE ALGAE 1179
1943, by a systematic investigation of the relation between wave length

and quantum yield in this Cyanophycea.
Chroococcus cells are somewhat smaller than Chlorella (2.5 ju in diame-
ter) and are surrounded by a gelatinous sheath. They scatter less light
0.10
008 , .X'
Q
_l
ill
> 006
Observed
3
<
004 J Colculoted
O
0.02
*--..,.
_L
400 440 480 520 560 600 640 680 720
WAVE LENGTH, m^
Fig. 30.10A. Quantum yieldof Chroococcus photosynthesis (after Emerson and Lewis
1942). SoHd drawn through experimental points, values obtained in different
line is
runs being distinguished by different characters. Broken line shows expected dependence
of quantum yield on wave length, on assumption that -yield for light absorbed by chloro-
phyll and phycocyanine is 0.08 at all wave lengths, and light absorbed by carotenoids is
not available for photosynthesis.
100
Direct
From Photosynthesis
10 mm cells/ml 08 mm. ceils/ml.

J L
400 500 600 700
WAVE LENGTH, mp.
Fig. 30.10B. Comparison of action spectrum (broken curve) and ab-

sorption spectrum of Chroococcus (after Emerson and Lewis 1942).
than Chlorella cells (probably because of the absence of chloroplasts cf. ;
chapter 15, page 355). This makes the determination of the absorption
curve easier, and leads to better agreement between the absorption curve
of the intact cells and the curve constructed by the combination of the
(appropriately shifted) absorption curves of the extracted pigments (illus-
1180 THE LIGTTT FAf'TOU. Til. f'OLOU CHAP. 30
trated by fig. 22.485). The main remaining differences between the "cell
spectrum" and the combined extract spectrum is the apparent broadening of
the red chlorophyll band in the intact cells, and a somewhat lower absorp-
tion of the latter at X < 510 mju.
Chroococcus cells were used in carbonate buffer (85% 0.1 Af NaHCOs +
15% 0.1 Af Na2C03). These algae can live without potassium, but not
without sodium. The method of determination of y was the same as in
the work with Chlorclla. (Bands 6-10 mn ^\'ide were used in the rod and
15-20 m/x wide in the blue-violet; photosynthesis and respiration were
measured in alternating 10 minute periods of darkness and light, the value
of P hemg derived from the rate of oxygen production in the second half
of the illumination period.) Both "dense" (fully absorbing) and "thin"
(partially absorbing) suspensions were used.
The quantum yield of photosynthesis in Chroococcus as a function of
wave length is shown in figure 30.10A. As in Chlorella, y is approximately
constant between 570 and 690 nifi (aside from a slight flat maximum at 680
m/x) —despite the fact that in Chlorella all absorption in this region is due to
more than half the total absorption
chlorophyll, whereas, in Chroococcus,
between 560 and 650 mî must be attributed to phycocyanin.
in the region
Judging from figure 22.49, the absorption by phycocyanin at 600 m/u
should be at least six times as large as that by chlorophyll; but the quan-
tum yield is the same as at 660-680 m/x, where chlorophyll accounts for
practically all absorption. Thus, the photosynthetic efficiency of phyco-
cyanin in Chroococcus must equal that of chlorophyll (the maximum
possible difference being of the order of 10-15%). Another way of repre-
senting the same results is shown in figure 30.10B. Here, the absorption
spectrum of a thin suspension of Chroococcus cells is compared with the
quantized action spectrum of photosynthesis. The close parallelism be-
tween the two curves in the region above 570 mju shows the approximately
equal availability for photosynthesis of the light absorbed by both chloro-
phyll and phycocyanin. Particularly convincing are the two separate
maxima shown by both curves near 620 and 670 m^, which must be at-
tributed to phycocyaninand chlorophjdl, respectively. The large dis-
crepancy in the region 420-550 m/x indicates the inefficiency (or relatively
low efficiency) of the light absorbed by the carotenoids. The dotted line in
figure 30.10A shows, however, that assuming complete inefficiency of
the carotenoids leads to an underestimation of the yield between 450 and
550 m/x; the best agreement between measured and calculated yields can
be obtained by assuming a quantum yield y = 0.08 for the light absorbed
by chlorophyll and phycocyanin, and y = 0.016 for the light absorbed by
the carotenoids.
The action spectra of several species of red algae were studied by Haxo
ACTION SPECTRUM OF RED AND ELITE ALGAE llSl
Ulva taeniata
—^ Thollus obsorption
Action spectrum
Delesseria decipiens
80
—^ Thollus absorption
Action spectrum
Aqueous extract
30
c
40
•^. 20
z
o
P
a.
a:
o ""
UIJ
in
m
<
E
and Blinks (1950) with very striking results. The polarographic method
(page 850) was used, with occasional checks by oxygen determination and
manometric measurements. Light intensities used were low enough (about
twice the compensating intensity) for the results to approximate the maxi-
mum quantum yields. In the green alga Ulva taeniata (fig. 30. 11 A) the
action spectrum was found to follow closely the absorption spectrum, with
the exception of a yield deficiency of up to 100% in the far red (>700 m/x),
already noticed by Emerson and Lewis (c/. fig. 30.3), and of a certain
yield deficiencyaround 480 van, also noticed before, and interpreted as evi-
dence of partial inactivity of the carotenoid pigments in green cells. It is,
however, to be noted that the yield deficiency disappears at 415 m/i, al-
though a considerable proportion of light (over 50% in extracts) must be
absorbed in this region by carotenoids.
In the brown Coilodesme (fig. 30.1 IB), the action spectrum also paral-
leled rather closely the absorption spectrum with a moderate deficiency
(of up to 20%) in the region of strong carotenoid absorption. In the red
algae Delesseria decipiens (fig. 30.11C), Porphyra nereocystis (fig. 30.11D),
Porphyra naiadum (fig. 30. HE) (purple, indicating relatively high content
of phycocyanin) and Porphyra perforata (fig. 30.1 IF, G) (a slate-green
vegetative section containing mainly phycocyanin, and a red carposporic
section) the action spectra Avere strikingly different from the absorption
spectra. They showed unmistakable maxima corresponding to the ab-
sorption peaks of phycoerythrin, at 500 and 565 m/x, and also to those of
phycocyanin at 620 mû (fig. 30. HE, F), but only very little of the chloro-
phyll maxima in the red as well as in the violet. The quantum yield was
estimated to be the order of 0.06 in the phycoerythrin bands, and as low as
0.02 in the chlorophyll bands. Similar results were obtained with several
other Bangiales and Florideae. The absorption by carotenoids appeared to
be as little effective as that by chlorophyll. A slight increase in activity
shown by some species at X = 440 mju, coinciding as it did with an increase
in the absorption of the aqueous phycobilin extract, could not be inter-
preted as sign of photosynthetic activity of the chlorophylls or the caro-
tenoids.
These unexpected results indicate that in contrast to all other plants
direct sensitizationby chlorophyll plays only a subordinate role in at least
some of the red algae, and that photosynthesis in them is sensitized pri-
marily by phycobilins. If this is the case, it would seem unlikely that the
energy quanta absorbed by the phycobilins are transmitted to chlorophyll,
since how could indirectly produced excitation of chlorophyll be more
effective than excitation due to energy absorbed directly by chlorophyll?
Rather, these results would seem to indicate that the phycobilins are
sensitizers of photosynthesis in their own right, and that their presence
—
ACTION SPECTRUM OF RED AND BLUE ALGAE 1183
may perhaps even make that of chlorophyll superfluous, although so far no

chlorophyll-free red algae have been found.
We Avill see below, however, that a different — and no less striking
interpretation of these results is possible.

In one species of Iridophycus, which bleaches to almost green color at
high tide, a much greater participation of chlorophyll in photosynthesis
was noted in rough experiments.
Haxo and Blinks said that, in contrast to Emerson and Lewis's results on
Chroococcus, they found only a weak chlorophyll activity also in two blue-
green algae, Anaboena and Oscillatoria, whose action spectra were similar
to those of Porphyra perphoraia (fig. 36.11F). They suggested that culture
conditions may affect the relative activity of different pigments in algae of
the same class or even the same species.
Haxo and Blinks noted that the saturation rate of photosynthesis of
Delesseria is the same in blue light (565 niju) as in red light (672 ran) '•
This
seems to indicate that the enzymatic mechanism of photosynthesis or, —
at least, the rate-limiting enzymatic reaction —
is the same whether the
quanta are absorbed by a phycobilin or by a chlorophyll.

In chapter 24 (p. 815) we desciibed the fluorescence studies of French
et at. and Duysens (1951) that indicated effective transfer of excita-
(1951)
tion energy in red algae from carotenoids and phycobilins to chlorophyll
a, and (in certain of them) from chlorophyll a to d (despite the low con-
centration of the latter). If one assumes that transfer to chlorophyll d

constitutes a "leak" which makes energy unavailable for photosynthesis,
the results of Blinks and Haxo become understandable. The question
remains why energy transferred to chlorophyll a from phycobilins is not
also lost to chlorophyll d but remains available for photosynthesis. It w'as
noted on p. 815 that this energy stays with chlorophyll a long enough to
cause its fluorescence (Avhile most of the energy absorbed by chlorophyll a
itself causes the fluorescence of d). Two suggestions were made there as
to the possible reasons for this difference in the fate of excitation energy;
but further study is needed for a convincing explanation.
Duysens' observations provide the strongest argument at present in

favor of assuming that chlorophyll a is the one pigment directly par-
ticipating in photosynthesis, and that not only the carotenoids, but also
the phycobilins, sensitize photosynthesis by transferring their excitation
energy to chlorophyll a. Another argument supporting this view is the
observation of French and co-workers (1951) that the yield of fluorescence
of the phycobilins in algae shows none of the induction effects and of the
peculiar changes with hght intensity which were discussed at length in
chapters 24 and 28 (part B) and are indicative of an intimate relationship
between chlorophyll and the chemical processes of photosynthesis.
1184 THE LIGHT FACTOR. III. OOLOli CHAP. 30
Beside the measurements of Emerson and Lewis and of Blinks and Haxo,
all earlier observations on the role of phycobilins in photosynthesis have
only the weight of corroborative evidence. Most of this evidence pertains
to red algae, and has been gathered in connection with Engelmann's theory
of "complementary chromatic adaptation" of these algae to the blue-green
light that prevails under the sea. (Obviously, this color adaptation can
only be useful to the algae if the light absorbed by the red pigments can be
used for photosynthesis.) Because of the absorption of red and blue-
A'iolet light by water, a full utilization of the central part of the visible
—
spectrum which is only insufficiently absorbed by chlorophyll is of —
vital importance for the plants living deep under the sea. This considera-
tion was the basis of the theory of chromatic adaptation, developed by
Engelmann in 1884. This subject was almost lost sight of in the first quar-
ter of the new century, while new methods of quantitative study of photo-
synthesis by green leaves were being developed by Blackman and co-
workers and by Willstatter and Stoll. Later, Warburg made the green
Chlorella cells the favorite subject of photosynthetic studies. In the
work on green pigments was con-
plants, the presence of accessory yellow
sidered to be scarcely more than a nuisance. These pigments were not
—
prominent enough both in concentration and in the part they took in
light absorption —
to make them a desirable subject of independent study;
but their presence interfered with the quantitative study of chlorophyll-
sensitized photosynthesis in the short-wave region of the visible spectrum.
The fact that blue and red algae offer a much more promising field for the
study of the part played by the "accessory" pigments in photosynthesis
was almost forgotten. Engelmann had noticed, however, as early as
1883 (using motile bacteria for the oxygen determination) that the maxi-
mum of the photosynthetic efficienc}^ of red algae (CaUithamnion and Cer-
amium) lay in the green part of the spectrum, and that of blue algae (Oscil-
latoria and Nostoc), in the yellow. As in the case of green plants, the posi-
tion of the maximum of photosynthesis coincided roughly with that of the
maximum of light absorption. A year later (1884), Engelmann described
a "microspectrophotometer," which he was able to show that
by means of
the parallelism between the absorption spectra and the "photosynthetic
action spectra" of the colored algae is quantitative. He concluded that
all pigments that contribute to light absorption by the algae also con-
tribute to photosynthesis, and expressed this result by the equation
-E'abs. = E'assim. (£" Standing for energy), w^hich was a direct challenge to the
concept of the exclusive sensitizing role of chlorophyll in photosynthesis.
—
One of the developments of Engelmann's theory ^the concept of "chro-
matic adaptation" as a factor determining the composition of the pigment
—
system in plants has been discussed in chapter 15. Here, we are con-
ACTION SPECTRUM OF RED AND BLUE ALGAE 11S5
cemed with the other aspect of the same phenomenon —

chromatic adapta-
tion as a factor enabhng plants to make better use of available light energy.
It was mentioned in chapter 15 that Oltmanns (1893) and others (most
recently, von Richter 1912, and Sargent 1934) objected to Engelmann's
theory (as well as to its extension by Gaidukov to color changes induced
artificially in blue-green algae) and insisted that algae responded only to
changes in light intensity, and not color.
\m\ Richter (1912) raised a further objection and asserted that "chro-
matic adaptation" could not achieve the purpose that was suggested by
Engelraann, because phycobilins do not act as sensitizers in jjliotnsynthe-
sis. Although, in comparing the ratio of the photosynthetic productions
of the green alga Ulva lactuca in red and green light with the corresponding
ratios for Plucaniiiim, CallUhamnion, Delesseria and other red algae, von
Richter could not help confirming the results of Engelmann and finding
that red algae are two or three times as eflicient in green light as the green
algae, he nevertheless denied that this proved the photosynthetic activity
of the red pigments. He pointed out that similar differences are obtained
also by changing the intensity of light, and suggested that the red algae
utilize green light better not because of its wave length but because it is
only weakly absorbed by chlorophyll — and red algae are adapted to weak
light.
However, the views von Richter have not been confirmed by later
of
investigators. Wurmser and Ducleaux (1921) compared the photosyn-
thetic eflSciencies of red and green fronds of Rhodymenia palniafa and
Chondrus crispus and found that the red varieties give yields two or three
times greater than the green ones. Wurmser (1921) compared the photo-
synthesis of the green alga Ulva lactuca with that of the red alga Rhodymenia
palmaia in red, green and violet light. He found that, if the rate in red is
taken as imitj^ that in green is 0.24 in Ulva and 0.49 in Rhodymenia, and
that in violet 0.81 and 0.16, respective^ (for equal intensity of incident
light). Thus, the red algae are more eflScient in the green, but le::s ef-
ficient in the violet than the green ones. Wurmser pointed out that, even
if von Richter's intensity not mean that color ef-
effects are real, this does
consequences of changes in absorption intensity.
fects are only indirect
by Harder (1923), who concluded that
Similar results were obtained
both intensity adaptation and color adaptation are real phenomena. He,
as well as Ehrke (1932), interpreted the result of the rate measurements
with red algae as indicating active participation of the phycobilins in photo-
synthesis, and the same conclusions were reached by Montfort (1936) and
Schmidt (1937), who found the spectral maximum of the efficiency of phyco-
cyanin algae in yellow light, and that of phycoer>'thrin algae in green
light.
Levring (1947) determined "action spectra" of photosynthesis of a

number of marine algae in filtered sunlight. (Only qualitative results can
be expected from such measurements, because of the relatively high light
intensityand the relatively strong absorption of the thalli). He found
evidence of particularly strong photosynthetic efficiency (high ratio yield/
absorption) in green light in ten species of red algae, and concluded that
the red phycobilin pigment is (if not more active) than the green
as active
chlorophyll. Combining these with those of his measurements of
results
spectral distribution of light in different depths, he concluded that because
of the presence of the red pigment, the Rhodophyceae utilize the blue-green
light deep under the sea better than the Chlorophyceae, as postulated by the
Engelmann-Gaidukov theory. He agreed, however, that adaptation to
low light intensity is an alternative way of adjustment to life in great
depths; an important element of it is low respiration.

Thus, even more uniformly than in the case of brown algae, the crude
observations on the relative efiiciency of photosynthesis of red algae in light
of different color support the assumption that the accessory pigments of
these organisms are active sensitizers in photosynthesis, and that Engel-
mann's theory of chromatic adaptation was fundamentally correct. And
one may ask oneself, how could it have been otherwise? Would it not be
strange if the appearance of orange or red pigments in deep-water algae
would be only a coincidence, and these pigments were helpless in performing
the task so obviously set to the plants by the character of the "light field"
in which they live —to catch and utilize for their maintenance and propaga-
tion radiations in the middle of the visible spectnim, which are the only
ones to reach them in some intensity?
It may be argued that not all deep-water algae are red, some green
algae being encountered in great depth. In other words, algae can sur-
vive without phycobilins in the greatest depths where life occurs. How-
not a convincing argument against Engelmann's theory.
ever, this in itself is
Algae could adapt themselves to great depths in two ways: by reducing

and weakly
respiration to a level permitting growth even in extremely Aveak,
absorbed light; and by adjusting pigment systems to enhance light
their
absorption. The fact that the first adjustment has been sufficient for
some green species does not invalidate the hypothesis that red algae have
also used chromatic adaptation for the same purpose.
Another objection to Engelmann's theory is that many red algae live
on or near the surface and that phycobilins are found in blue-green algae,
which are surface organisms. It is known, however, that red algae often
tend to lose their phycobilin and become green when exposed to sunlight
(c/. Vol. I, Chap. 15); and even if many of them (as well as the blue-green
algae) apparently find their phycobihn content useful, or at least not harm-
—
ACTION SPECTRUM OF PURPLE BACTERIA 1187

I
even on the surface, this does not prove that phycobiHns are not pig-
ful,
ments primarily intended to permit photosynthesis deep under the sea.

—
One may speculate particularly in the light of Blinks' experiments
whether photosynthesis with phycobilins may not be an older process than
photosynthesis with chlorophyll; perhaps, the development of the green
pigment and its substitution for the phycobilins have been the product of a
later development, in which plant life, originating in the depths of the
ocean, migrated to the surface and finally spread overland.
7. Action Spectrum of Purple Bacteria
Engelmann found, in his fundamental work on photosynthetically ac-

tive bacteria (1888) that, if a spectrum was thrown on their cultures, they
developed only in the absorption bands of the green pigment (which we
now call bacteriochlorophyll). Purple bacteria also contain numerous
carotenoids, with absorption bands clearly separated from those of bac-
teriochlorophyll (c/. fig. French (1937) found that the action spec-
22.21).
trum determined by the rate of consumption of
of Streptococcus varians (as
hydrogen) sho\\Ti in figure 30.12 closely parallels its absorption spectrum
in the yellow and red part of the spectrum but does not show maxima in the
green or blue that correspond to the absorption bands of the carotenoids
(c/. Table 30. VIII). French concluded that the red carotenoid pig-
ments of purple bacteria are photosynthetically inactive. It must be
noticed that, from the spectroscopic point of view, the conditions in purple
o
<
1/5
if)
<
O
LJ
<
<r
400 600 800

WAVE LENGTH, m^
Fig. 30.12. Rate of CO2 assimilation of a very dilute suspension
of Streptococcus varians as a function of wave length of incident light
French 1937).
(after Rate scale represents molecules CO2 X 100 per
incident quantum.
' ;
bacteria are particularly favorable for an investigation of the part played

by the carotenoids in photosynthesis, since the absorption peaks of the
bacterial carotenoids are not concealed behind the absorption bands of
chlorophyll, as is the case not only in green plants, but also in "fucoxanthol
algae."
Table 30.VIII
Position of Wave Length Maxima (m/x) in Spectrum of Streptococcus varians
(after French 1937)
Methanol solution Cells Action spectrum

~~
410 420 —
— 490" —
— 510" —
— 550" —
605 590 590
770 880 900
° Carotcnoid bauds.
Vermeulen, Wassink and Reman (1937), too, found that the develop-
ment of Chromatiuni takes place mainly in light belonging to the absorp-
tion bands of bacteriochlorophyll (infrared, red and a narrow band at 590
mju; cf. figs. 22.19 and 22.21), but not in those of the red carotenoid pig-
ments.
More recently, however, Manten and Thomas found the situation to be
more complex. First, Manten (1946), in studying the action spectrum of
phototaxis of Rhodospirillum ruhrum, found peaks corresponding to the
absorption peaks of bacteriochlorophyll (590 m/x) as well as to those of some
of the carotenoids (but not the main carotenoid of these cells, spirilloxan-
thol). He suggested that the mechanism of phototaxis involves in Rhodo-
spirillutN, primarily, a stimulation of photosynthesis, and that the action
spectrum of phototaxis therefore indicates that some (but not all) caro-
tenoids are photosynthetically active also in purple bacteria. Thomas
(1950) investigated the action spectrum of photosynthesis of the same cells
manometrically by measuring carbon dioxide consumption in the presence
of 0.015 M sodium butyrate as hydrogen donor, (cf. chapter 5, p. 106). Be-
cause of the sigmoid shape of the light curve, the action spectrum was
determined by measuring the ratio of ACO2 in light of a given wave length
(bands isolated by Christiansen filters) to ACU-j in light of a standard wave
length (yellow sodium line) Between 460 and 650 mii the action spectrum
.
had a peak at 590 m^ belonging to bacteriochlorophyll (see fig. 22.27)

and three peaks attributable to carotenoids. However, no maximum of
photosynthesis is noticeable at 550 mû, whore a peak is present in the
absorption curve of the cells (compare fig. 22.27 and table SO.VHI above)
this peak has been attributed to the most abundant bacterial carotenoid,
BTBLIOGRArHY TO CHAPTER 30 1180
spinlloxanthol, which is thus shown to be inactive, both in photosynthesis

and in phototaxis. The
resuh, shoAvs close similarity of the two action spec-
tra,and thus supports Manten's hypothesis that phototaxis is a consequence
of enhanced photosynthesis.
The observations by Duysens (1951) of carotenoid-sensitized fluores-
cence of bacteriochlorophyll in Chromatium and Phodo spirillum rubrum,
described in chapter 24 (p. 810) indicates that the capacity of the caroten-
oids in [)urplc bacteria (o sensitize phototaxis and photorodudioii of COo
may be based on transfer of their excitation energy to bacteriochlorophyll.
The Light Factor. III. Photosynthesis and Light QuaUty; Role of Accessory Pigments
1788 Senebier, J., Experiences sur Vadion da la lumiere solaire dans la vegetation.
Barde-I\langet, Geneva.
1844 Draper, J. W., ^4nn. chim. phys. [3] 11, 214.
1864 Sachs, J., Botan. Z., 22, 353, 361, 369.
1869 Timiriazev, C, ibid., 27, 169.
1871 W., Arch, botan. Inst. Wurzburg, 1871 [1] 1.

Pfeifer,
Lommel, E., Ann. Phys. Chem. (Poggendorf) 143, 568. ,
1872 Lommel, E., ibid., 145, 772.

Miiller, N. J. C, Botanische Untersuchwigen, vol. I, Heidelberg, 1872, p. 3.
1875 Timiriazev, C, Investigations on the Assimilation of Light by Plants. St.

Petersburg, 1875.
1877 Timiriazev, C, Ann. chim. phys., [5] 12, 355.
1879 Pringsheim, N., Monatsber. preuss. Akad. Wiss., Berlin, 1879, 532, 860.
1881 Pringsheim, N., ibid., 1881, 117, 504.
Pringsheim, N., Jahrb. iviss. Botan. 12, 288.
1882 Pringsheim, N., ibid., 13, 377.
Engelmanii, T. W., Botan. Z. 40, 419.

1883 Engelmann, T. W., ibid., 41, 1, 17.
1884 Reiiike, J., ibid., 42, 1.
Engelmann, T. W., ibid., 42, 81.
1885 Timiriazev, C, Aiin. sci. nat., botan. et biol. vegetale [7] 2, 99.
1886 Pringsheim, N., Ber. dent, botan. Ges., 4, XC.
Pringsheim, N., Jahrb. iviss. Botan., 17, 162.
1887 Engelmann, T. \\., Botan. Z., 45, 100.
Engelmann, T. W., ibid., 45, 393, 409, 425, 441, 457.
1888 Engelmann, T. \\., ibid., 46, 661, 677, 693, 709.
1890 Timiriazev, C, Compt. rend., 110, 1346.
1893 Oltmanns, F., Jahrb. wiss. Botan., 23, 349.
1897 Kohl, F. G., Ber. deut. botan. Ges., 15, 111, 361.
1902 von Richter, A., Rev. gen. botan., 14, 151, 211.
1903 Timiriazev, C., Proc. Roy. Soc. London, B72, 424.
1906 Kohl, F. G., Ber. deut. botan. Ges., 24, 222.
1909 Kniep, H., and Minder, F., Z. Botan., 1, 619.

1912 von Richter, A., Ber. dent, botan. Ges., 30, 280.
1917 Ursprung, A., ibid., 35, 44.
1918 Ursprung, A., ibid., 36, 73.
Ursprung, A., ibid., 36, 87, 111.
Willstatter, R., and StoU, A., Untersuchungen iiber die Assimilation der
Kohlensdure. Springer, Berlin, 1918, pp. 114, 141.
1921 Wurmser, R., Compt. reJid., 171, 820.
Wurmser, R., Arch. phys. bioL, 1, 33.
Wurmser, R., and Ducleaux, J., Compt. rend., 171, 1231.
Wurmser, R., and Ducleau.x, J., Recherches sur V assimilation chlorophyllienne,
Paris.
1922 Noack, K., Z. Botan., 14, 1.
1923 Warburg, 0., and Negelein, E., Z. phijsik. Chem., 106, 191.
Lubimenko, V., Compt. rend., 177, 606.
Harder, R., Z. Botan., 15, 305.
1925 Wurmser, R., Ann. physiol. physicocfmn. bioL, 1, 47.
1929 Briggs, G. E., Proc. Roy. Soc. London, BIOS, 1.
1930 Kuilman, L. W., Rec. trav. botan. neerland., 27, 287.

Schmiicker, T., Jahrb. xoiss. Botan., 73, 824.
1932 Ehrke, G., Planta, 17, 650.
Dastur, R. H., and Asana, R. D., Ann. Botany, 46, 879.
Meier, F. E., Smithsonian Inst. Pub. Misc. Collections, 87, No. 10.
1933 Dastur, R. H., and Samant, K. M., Ann. Botany, 47, 295.
Arnold, W., /. Gen. Physiol., 17, 135, 145.
Burns, G. R., Playit Physiol. 8, 247.
Burns, G. R., Science, 78, 130.
Voerkel, S. H., Planta, 21, 156.
1934 Burns, G. R., Plant Physiol, 9, 645.
Franck, J,, and Rabinowitch, E., Trans. Faraday Soc, 30, 120.
Montfort, C, Jahrb. wiss. Botan., 79, 493.
Sargent, M. C., Proc. Natl. Acad. Sci., U. S., 20, 251.
Dastur, R. H., and Gunjikar, L. K., Ann. Botany, 48, 1003.
1935 Dastur, R. H., and Gunjikar, L. K., ibid., 49, 273.
Castle, E. S., Cold Spring Harbor Symposia Quant. Biology, 3, 224.
Gabrielsen, E. K., Planta, 23, 474.
Baly, E. C. C., Proc. Roy. Soc. London. B117, 218.
Dastur, R. H., and Mehta, R. J., Ann. Botany, 49, 809.
Danilov, A. N., Sovet. Botan., No. 4, p. 3.
1936 Danilov, A. N., Arkh. Biol. Naiik, 43, 365; Eksp. Botanika, 2, 5.
Harder, R., Doring, B., and Simonis, W., Nachr. Ges. Wiss. Gottingen,
Math.-natur. Kl, VI, 2, 129.
Montfort, C., Jahrb. tviss. Botan., 83, 725.

1937 Hoover, W. H., Smithsonian Inst. Pub. Misc. Collections, 95, No. 21.

Vermeulen, D., Wassink, E. C, and Reman, G. H., Enzymologia, 4, 254,

Dastur, R. H., and Solomon, S., Ann. Botany, 1, 147.
Johnston, E. S., Smithsonian Inst. Pub. Misc. Collections, 96, No. 3.
Montfort, C, Ber. deut. botan. Ges., 55, 142.

Schmidt, G., Jahrb. wiss. Botan., 85, 554.
Emerson, R., Ann. Rev. Biochem., 6, 535.
Burns, G. R., Am. J. Botany, 24, 257.

1938 Burns, G. R., ibid., 25, 166.
Gabrielsen, E., and Steeraann-Nielsen, E., Conseil permanent intern, ex-

ploration de la mer; rapp. proc. verb., 108.
Harder, R., and Simonis, W., Nachr. Ges. Wiss. Gottingen, Math.-natur. KL,
VI, 3, 129.
Simonis, W., Planta, 29, 129.
Dastur, R. H., Kanitkar, U. K., and Rao, M. S., Ann. Botany, N.S. 2, 943.
Danilov, A. N., Eksper. Botanika, 3, 1.

1939 Eichhoff, H. J., Biochem. Z, 303, 112.
Mothes, K., Baatz, I., and Sagromsky, H., Planta, 30, 289.
1940 Montfort, C., Z. physik. Chem., A186, 57, 253.
Gabrielsen, E. K., Dansk. Botan. Arkiv., 10, 1.
1941 Seybold, A., Botan. Arch., 42, 254.

Baatz, I., Planta, 31, 726.
Emerson, R., and Lewis, C. M., Carnegie Inst. Yearbook, 40, 157.
Franck, J., and French, C. S., /. Gen. Physiol, 25, 309.
1942 Burns, G. R., A7ner. J. Botany, 29, 381.
Emerson, R., and Lewis, C. JM., J. Gen. Physiol, 25, 579.
Sen, P., /. Indian Botan. Soc, 19, 147.
Strain,H. H., and Manning, W. M., /. Biol Chem., 144, 625.
1943 Sagromsky, H., Planta, 33, 299.
Emerson, R., and Lewis, C., Am. J. Botany, 30, 165.
1946 Warburg, 0., and Llittgens, W., Biokhimija, 11, 303.
Wassink, E. C., and Kersten, J. A. H., Enzymologia, 12, 3.
Johnson, H. G., and Levring. T., Goleborg K. Vetensk. Vitt-Samh. Handl
B5, No. 3.
1947 Levring, T., Goleborg K. Vetensk. Vitt-Samh. Handl, B5, No. 6.
Manten, A., Anionic van Leeuwenhoek, 14, 65.
1948 Warburg, O., Am. J. Botany, 35, 194.
1950 Thomas, J. B., Biochim. Biophys. Ada., 5, 186.
Haxo, F. T., and BHnks, L. R., /. Gen. Physiol, 33, 389.
Ehrmantraut, H., Thesis, Univ. of Illinois.
1951 Tanada, T., Am. J. Botany, 38, 276.
Duysens, L. N. M., Nature (in press).
French, C. S., and Koski, V. M., Proc. Soc. Exptl Biol (in press).
AUTHOR INDEX OF THE MAIN INVESTIGATIONS
DESCRIBED IN VOLUME II, PART 1*
Albers, V. M., and Knurr, 11. V.: Absorption spectra of single chloroplasts, 692, GOS,
699, 701,
Alkiro,. G. I. See Van Rysselberghe, P.
Augelslein, U. : Carbonate ions enhance photosynthesis, 887.
Arens, K.: Passage of carbonate ions throiigli leaves, 887-888.
Arnold, A. : Decline of photosj'nthesis of aquatic plants with time under constant con-
ditions, 877.
Arnold, W.: Calorimetrie measurement of photosynthesis, 854; calorimetric quantum
yield determinations, 1 122-1 12;>; inhil)ition of photosynthesis by ultraviolet,
1153. See also Emerson, II.
and Oppenheimer, R. J.: Energy transfer between pigments, 758-759; enhance-
ment of phycobilin fluorescence by cell destruction, 801, 816; fluorescence yield of
Chroococcus, 813.
Aronoff, F.,and Calvin, M.: Spectra of hexaphenjd porphins, 623-625.
Asana, R. I. See Dastur, R. H.
Aufdemgarten, H.: Thermal conductivity measurements of CO2 exchange in photo-
sj'nthesis, 853.
Auerbacher, F. See Hagenbach, A.
B
Baatz, I. See Mothes, K.
Baehiach, h]., and Dh^re, C: Fluorescence of diatoms, 806.
Baldwin, W. C. G. See Mecke, R.
Ballai-d, L. A. T.: Inhibition of photosynthesis by excess CO2, 903.
Barker, H. A.: Light curves of diatoms, 969; CO2 curves of purple bacteria, 893, 904.
Bazyrina, K., and Chesnokov, V.: CO2 fertilization, 902. See aho Chesnokov, \'.:
Kost>Thev, S. P.
Beadle, B. W. See Zscheile, F. P.
Berthold, G.: No
chromatic adaptation?, 995.
Biermacher, O.: Absorption spectrum of chlorophyll a and b in different solvents, 637-
639. See also Dhere, C.
Blackman, F. F.: Limiting factors, 859-861; light curves and theory of limiting factors,
965.
(with Matthaei, G. L. G., and Smith, A. M.): Light curves of photosynthesis, 964.
and Smith, A. M.: CO2 concentration as limiting factor, 891 CO2 curves of leaves,
;
892, 894, 904.

Blagoveshchenskij, A. V.: High rate of photosynthesis of mountain plants, 997, 1001.
Blinks, L. R. See Haxo, F. T.
* Complete author index will be found at the end of Volume II, Part 2.
1193
1194 AUTHOR INDEX
Bohning, R. H.: Midday depression and CO2 content in air, 875, 876; constant photo-
synthesis of land plant under constant conditions, 879; decline of photosynthesis
of shade plants in strong light, 989.
Bose,J. C: Energy conversion by plants under natural conditions, 1003-1004.
Boysen-Jensen, P.: Light curves of Fagus and Sinapis, 966; light curves of heliophilic
and umbrophilic plants, 987; maximum rates of photosynthesis, 991; photo-
synthesis of leaves under natural conditions, 998.
and Miiller, D.: Constant photosynthesis under field conditions, 876; light curves
of ferns and compensation points of trees, mosses, and lichens, 982;
lichens, 966;
light curves of umbrophilic and heliophilic plants, 987, 988, 989; photosynthesis
under natural conditions, 997-1001.
Brackett, F. S.: Empirical equation for light curves, 1046. See also Hoover, W. H.
Brewster, D.: Chlorophyll fluorescence, 740.
Briggs, G. E.: Yield measurement with leaves, 1118; yield in blue-violet lower than in
yellow and green, 1148.
Brilliant, V. A.: Adaptation phenomena, 873.
Brown, A. H.: Tracer experiments on respiration in light, 1 117.
Brown, H. T., and Escombe, F. Rate of CO2 uptake by leaves from air, 912; theory of
:
CO2 diffusion through stomata, 913; rate of photosynthesis under natural condi-
tions, 1000; energy conversion under natural conditions, 1003-1004.
Brown, W. H., and Heise, G. W.: Criticism of Blackman's law of limiting factors, 861,
965.
Buder, J.: Fluorescence of bacterioviridin, 811.
Buhr, H. See Guttenberg, H. von.
Burk, D., and Lineweaver, H.: Calculation of carboxylation equilibrium from CO2
curves of photosynthesis, 935; kinetic analysis of light curves, 1046. .See also
Warburg, O.
Burns, G. R. Absorption of light by conifer needles, 684 no photosynthesis
: ; in conifers
below 465 m/x, 1153; photosynthesis up to 740 niyu, 1158, 1164-1165.
Burr, G. O. See Miller, E. S.
Calvin, M. See Aronoff, F.; Lewis, G. N.

and Dorough, G.: Long-lived infrared fluorescence of chlorophyll, 748, 795.
Carr. See Loomis, W.
Chesnokov, V., and Bazyrina, K.: CO2 concentration threshold of photosynthesis, 907;
CO2 exhaustion effects in land plants, 908; gas exchange balance at low CO2
concentrations, 985. See also Bazyrina, K.; Kostychev, S. P.
Coblentz, W. W. See Stair, R.
Comar, C. L. See Zscheile, F. P.
Daniels, F. See Magee, J. L.; Manning, W. M.; Petering, H. G.

Danilov, A. N.: Cooperative and antagonistic effects of different colors on photo-
synthesis, 1166-1167.
Dastur, R. H. (with Asana, R. D., and Gunjikar, L. K.): Has polarization an effect on
photosynthesis?, 1147.
(with Samant, K. M., Mehta, R. Y., Solomon, S., Kanitkar, U. K., and Rao. M. S):
Cooperative and specific effects of different colors in photosynthesis, 1167
AUTHOR INDEX 1195
Decker, J. P. See Kramer, P. J.
De Witt, T. W. See Magee, J. L.
Dezelic,M. See Stern, A.

Dh6re, C. (with Fontaine, M., Raffy, A., and Biermacher, O.): Fluorescence spectrum of
chlorophylls a and h, 740-746, 748; of chlorophyll c, 747; of protochlorophyll,
748; of phycobilins, 799, 801; of algae, 806, 807, 808, 809, 811. See also Bach-
rach, E.
Dorcas, M. J. See Osterhout, W. J. V.
Doring, B. See Harder, R.
Dorough, G. See Calvin, M.
Dorrestein, R. C; Katz, E.
See Wassink, E.
Drautz, R.:- "Shade" and "sun" regions in a leaf, 873; interpretation of midday de-
pression, 874.
Ducleaux, J. See Wurmser, R.
Duggar, B. M. See Dutton, H. J.; Manning, W. M.; Moore, W. E.; Petermg, H. G.
Duntley, S. C.: Absorption and scattering in inhomogeneous systems, 712-713.
Dutton, H. J., and Manning, W. M.: Action spectrum of diatoms; sensitizing activity
of fucoxanthol, 1169-1172; distribution of light energy among pigments in di-
atoms, 725-726; quantum yields in Nitzschia dosterium, 1096.
, Manning, W. M., and Duggar, B. M.: Chlorophyll fluorescence sensitized by
carotenoids in diatoms, 814-815.
Duysens, L. N. M.: Excitation transfer from chlorophyll 6 to a in solution, 790; spectro-
photometry of fluorescence of red algae and purple bacteria, 806, 807; fluores-
cence of chlorophyll d (?) in Porphyra excited by energy transfer from a, 811-812,
815-816; chlorophj'll a fluorescence excited by energy transfer from 6 in Chlorella,
809; bacteriochlorophyll fluorescence in Chromatium and Rhodospirillum sensi-
tized by carotenoids, 810; two kinds 815-
of chlorophyll complexes in red algae?,
816; chlorophyll a fluorescence excited energy transfer from phycobilins, 815-
bj^
816; inactivity of chlorophyll a in red algae due to energy transfer to chlorophyll
d?, 1183.
E
Egle, K.: Absorption spectrum of chlorophyll in different solvents, 637-639; absorption
and reflection of leaves in infrared, 692. See also Seybold, A.
Ehrke, G.: Compensation points of green, brown and red algae, 983; photosynthesis of
red algae in colored light, 1185.
Ehrmantraut, H. C, and Rabinowitch, E.: Quantum yield of Hill reaction in Chlorella
and chloroplasts, 1130-1131.
Eichhoff, H. J.: Light curves of Chlorella suspensions of different density, 1008-1009;
quantum yield in Chlorella high in near infrared?, 1097-1098.
Emerson, R. Light curves of normal and chlorotic Chlorella, 967. See also Whitting-
:
ham, C. P.
and Arnold, W.: CO2 curves of Chlorella, 899.
and Green, L.: Photosynthesis in Chlorella insensitive to pH changes, 835; C0»
saturation in Chlorella depends on concentration of CO2 molecules only, 891 (^02 ;
curves of Chlorella, 893, 896; of Gigarlina, 893, 904, 906, 908; light curves of
Gigarlina, 967; maximum rate of GUjartina, 991, 992.
and Lewis, C. M.: Transmission spectra of Chlorella and Croococcus, 691, 692, 699,
705; carotenoid bands in algae, 706; phycobilin bands in Chroococcus, 707-708;
relative intensities of absorption bands in Chlorella and Chroococcus, 708; en-
hanced absorption in far red in vivo, 715; distribution of energy among pigments
1196 AUTHOR INDEX
in Chlorella, 722-723; in Chroococcus, 728-729; linear range of liglit curves of

Chlorella, 980, 981; CO2 "burst" in quantum yield measurements, 1086, 1087-
1088, 1091-1094; quantum jdelds of photosj'nthesis in different species, 1094-
1095, in the blue-green alga Chroococcus, 1096, in monochromatic light, 1148-1152;
carotenoids in Chlorella sensitize photosj'nthesis with lower efficiency than
chlorophj'll, 1149-1150, 1151-1152; decline of photosj'nthesis in Chlorella and
Chroococcus above 680 mju, 1154; monochromatic light curves of Chlorella, 1159;
action specti'um of Chroococcus, full activity of phycobilins, 1178-1180.
and Nishimura, M. S. : Criticism of Warburg's quantum yield determinations,
1101-1104.
Engelmann, T. W.: Chromatic adaptation, 994-995; a second spectral maximum of
photosynthesis, 1143-1144; red pigments of leaves inactive in photosynthesis,
1165; evidence of photosynthetic efficienc}'^ of fucoxanthol in brown algae, 1176;
of phycobilins in red algae, 1178, 1183-1184.
Escombe, F. See Brovn\, H. T.
Evstigneev, V. B., Gavrilova, V. A., and Krasnovsk}', A. A.: Effect of oxygon, alcohol
and watci' on absorption spectra of chloroph}!! and pheophytin in nonpolar
solvents, 648; effect of solvents on chlorophyll fluorescence, 771-772; activation
of fluorescence of chlorophyll, pheophytin and phthalocyanin by oxygen and
water, 788.
Eymers, J. G., and Wassink, E. C: Effect of thiosulfate concentration on CO- reduction
b}' Chromaliutn, 946; linear range in purple bacteria, 980, 981; quantum yield of
CO2 reduction by Thiorhodaceae, 1127.
Ewart, A. J. : Inhibition of photosynthesis by excess light, 964.
Filzer, P. : Periodicity of photosynthesis in detached leaves, 874. See also Harder, R.

Fong, J. See Pratt, R.
Fontaine, M. See Dhere, C.
Forster, T.: Transfer of electronic energybetween molecules, 758-759; as mechanism of
mechanism of quenching by admixtures, 785.
self-quenching, 759, 774; as
Franck, J.: "Narcotization" of chlorophyll by metabolites as cause of enhanced fluores-
cence, 824-826; effect of CO2 on fluorescence as evidence of CO2 association with
chlorophyll, 941 effect of CO2 and reductants on fluorescence in plants and bacteria
;
caused by internal narcotization, 942-943, 950; narcotization as kinetic factor in

photosjai thesis, 1033, 1034, 1041-1043; interpretation of light curves of fluores-
cence, 1070, 1071, 1076, 1077, 1078; mechanism of the CO2 burst, 1086-1087;
interpretation of Warburg's and Kok's measurements as indicating partway re-
versal of respiration by light, 1117. See also Shiau, Y. G.; Weller, S.
and French, C. Photoxidation in CO2 deprived leaves, 1166.
S.:
, French, C. S., and Puck, T. T.: Fluorescence of leaves and algae, 806; its relation
(() photosynthesis, 819, 824; effect of CO2 concentration on fluorescence, 940;
liuorescence-light curves of Hydrangea, 1048-1049; effect of CO2 on these curves,
1051; of temperature 1055-1056; of cyanide 1057-1058.
and Herzfeld, K. F.: Nondissociable acid as first product of CO2 fixation in photo-
synthesis, 917, 918, 919, 927-930; kinetic model of photosynthesis, 1018, 1021-
1022, 1032, 1035, 1036, 1038, 1040; energy requirements and quantum yield of
])liot(isynthesis, 1089-1090; theoretical vs. maximum observable quantum yield,
1139.
and Levi, H.: Quenching of chlorophyll fluorescence by oxygen, 778-779; by
benzidine and KI, 780.
and Livingston, R.: Mechanism of self-quenching, 755-760, 774.
AUTHOR INDEX 1197
(with Pringsheim, P., Pollack, M., and Terwood-Lad, D.): Quenching of phos-
phorescence a sensitive way of measuring O; production in photosynthesis, 851.
Freeland, O. R.: CO2 penetration through cuticle, 911.
French, C. Absorption spectrum of bacteriochlorophyll, 616-617; spectra of purple
S.:
bacteria, 692, 693, 702; carotenoid bands in purple bacteria, 707; effect of H2
pressure on photoreduction of CO2 in bacteria, 944-945; sigmoid shape of light
curves in bacteria, 948, 964; quantum yield of purple bacteria, 1124-1125, 1126-
1127, 1332; action spectrum of purple bacteria, inactivity of carotenoids?, 1186-
1188. See also Franck, J.; Rabideau, G. S.
and Koski, V. M.: Absorption spectra of phycobilins, 664-665; absorption band

of protochlorophj'll in leaves, 705; spectrophotometric study of fluorescence of
phycobilins, 799-801; protochlorophyll fluorescence in partially green leaf, 811;
light curves of phycobilin fluorescence in red algae linear when those of chlorophyll
curved, 1051.
and Rabideau, G. S. : Quantum yield of Hill reaction in chloroplasts and Chorella
cells, 1094, 1128-1130.
(with Van Norman, R. W., Macdowall, F. D., and Koski, V. M.): Spectrophoto-
metric study of fluorescence of blue-green and red algae, 806, 807, 809, 811-812;
phycoerythrin-sensitized fluorescence of chlorophyll in red algae, 815.
Fuller, H. J. : CO2 concentration near ground, 902-903.
Gabrielsen, E. K.: CO2 compensation point, 899; re-utilization of respiratory CO2, 900-
901; light curves of Sinapis, 966; of sun and shade leaves of Fraxtnus, 967; of
Triticum, 967; high saturating intensity of sun leaves, 987; rate of photos3-n-
thesis of Sinapis under natural conditions, 998; quantum yield measurements on
leaves, 1188-1189; equal saturation rates in light of different colors, 1145; con-
tribution of ultraviolet to photosj^nthesis in sunlight, 1153; monochromatic light
curves, 1161-1162; screening effect of red leaf pigments, 1165.
and Steemann-Nielsen, E.: Photosynthesis of diatoms in blue and red light, 1177.
Galanin, M. D. See Vavilov, S. I.
Gavrilova, V. A. See Evstigneev, V. B.
Geiger, M.: Closure of stomata as cause of midday depression, 875.
Gessner, F.: Absence of midday depression in aquatic plants, 876; photosynthesis of
aquatic plants constant if medium effectively renewed, 878, 879; leaf shape and
CO2 exhaustion effects, 905; light curves of higher aquatic plants, 967; of shade
and sun plants, 988; maximum rates of aquatic plants, 991, 992, 1002; no light
inhibition of shade-grown Elodea, 994.
Giltay, E.: Rate of photosynthesis of tropical plants, 1001.
Green, L. See Emerson, R.
Greenfield, S. S.: Light curves of Cu + + and Ni + + inhibited Chlorella, 975.
Gunjikar, L. K. See Dastur, R. H.
Guttenberg, H. von, and Buhr, H.: Carbohydrate accumulation as cause of midday
depression, 875.
H
Hagenbach, A. : Red bands in leaves, 697.
shift of chlorophyll
, Auerbacher, F., and Wiedemann, E. Visible and ultraviolet absorption spectra
: of
chlorins and porphins, 606, 629.
Harder, R.: Modified law of limiting factoi-s, 862; adaptation to light intensity and
temperature, 873; time course of photosynthesis under constant conditions, 877-
1198 AUTHOR INDEX
878, 994; CO2 curves of aquatic plants, 892, 895, 897-898, 904; light curves of
Fontinalis, 967, 971 ; compensation points of algae, 983, 984.
and coworkers Interpretation of midday depression, 874.
:
Doring, B., and Simonis, W. Photosynthesis of Elodea most efficient in light of the
, :
color in which it grew, 1168.

Filzer, P., and Lorenz, A.: Photosynthesis of desert plants, 997, 1001.
,
Hartel, O.: CO2 supply through roots and midday depression, 910.
Harris, D. G., and Zscheile, F. P.: Absorption spectrum of chlorophyll in different
solvents, 637-639, 643-645. See also Zschiele, F. P.
Haxo, F. T., and Blinks, L. R.: Action spectra of red algae, 1180-1182; of Ulva (green
alga), 1152, 1181, 1182; oi Coilodes me (hrown alga), 1172, 1181, 1182; of blue-
green algae, 1183; drop of quantum yield of Ulva in far red, 1153, 1156.
Heise, G. W. See Brown, W. H.
Hendricks, R. H. See Thomas, M. D.
Hendricks, See Warburg, O.
S. B.
Henrici, M. Photosynthesis of mountain plants, 997.
:
Herzfeld, K. F. See Franck, J.

Hill, G. R. See Thomas, M. D.
Holt, A. S. See Rabideau, G. S.
Honert, T. H. van der: CO2 curves of Hormidium, 893, 895, 904; assimilation numbers
of Hormidium, 991.
Hoover, W. H.: Photosynthesis in near ultraviolet, 1153; in far red, 1158; action
spectrum of photosynthesis in average light, 1162-1163.
Johnston, E. S., and Brackett, F. S. Constant photosynthesis of land plants under
, :
constant conditions, 879; CO2 curves of wheat, 892, 896, 904; light curves of
wheat, 966, 970; linear range, 980.
Hubert, B. Absorption spectrum of different chlorophyll colloids, 649.
:
Jaag, O. See Jaccard, P.

Jaccard, P., and Jaag, O. Variations of photosynthesis under constant conditions, 877.
:
James, W. O., Enhancement of photosynthesis by bicarbonate a buffering effect?, 887;

CO2 curves of aquatic plants, 892, 903.
Johnson, H. G., and Levring, T.: Photosynthesis of algae in near ultraviolet, 1153.
Johnston, E. S.: Photosynthesis in polarized light, 1147. See also Hoover, W. H.
Juday, C. See Manning, W. M.
Kaempfert, W. See Schanderl, H.

Kanitkar, U. K. See Dastur, R. H.
Karrer, P., and Solmssen, V.: Absorption spectra of carotenoids from purple bacteria,
656, 658.
and Wi'irgler, E.: Absorption spectra of fucoxanthol and other carotenoids, 656,
657, 661.
Kasha, M. See Lewis, G. N.
Katunsky, V. M.: CO2 fertilization, 902.
Katsurai, T. See Svedberg, T.
Katz, E.: Interpretation of light curves of fluorescence, 1077. See also Wassink, E. C.
and Wassink, E. C. Absorption spectra of chlorophyll in different solvents, 637-
:
642; of colloidal chlorophyll-protein extracts, 653-656; of colloidal bacterio-

AUTHOR INDEX 1199
chlorophj^ll-protein extracts, 654-656; of blue-green algae, 692; of green sulfur

bacteria, 694, 704; of Chlorella, 699, 700.
, Wassink, E. C, and Dorrestein, R.: Inhomogeneity of light absorption in cell
suspensions, 864-866; light curves of purple bacteria, 969, 972; of suspensions of

different density, 1009-1011; fluorescence-light curves of C/irowa/nm(, 1049-50.
Kautsky, H., and coworkers: Quenching of chlorophyll fluorescence by oxygen, 778;
nonquenching by allylthiourea and isoamylamine, 788-789; phosphorescence
quenching by oxygen, 793-794; time course of fluorescence in leaves and algae,
806; its relation to photosynthesis, 819, 820, 822; effect of temperature on
fluorescence of leaves, 1055; of cyanide, 1057; of narcotics, 1063: of O2, 1063.
Kersten, J. A. H. See Wassink, E. C.
Kniep, H.: Maximum rates of Ulva and Porphyra, 991, 992.
and Minder, I.: Bubble counting study, 847; action spectrum of photosynthesis
has two peaks, 1144.
Knorr, H. V. See Albers, V. M.
Kohl, F. G. Double-peaked action spectrum of photosynthesis,
: 1 144.
Kok, B. : Chlorella light curves have two linear ranges?, 981 ;
quantum yield of Chlorella
doubled below compensation?, 1113-1116, 1132; a photosynthesis mechanism
involving high energy phosphates, 1115-1116.
Komor, J. See Noddack, W.
Kopp, C. See Noddack, W.
Korzenovsky, M. See Warburg, O.
Koski, V. M. See French, C. S.
and Smith, J. H. C: Absorption spectrum of protochlorophyll, 618-619.
Kostychev, S. P.: "Physiological concept" of photosynthesis, 872-873; variations
caused by internal factors make short-time rate measurements meaningless?, 876.
and coworkers: Midday depression and accumulation of carbohydrates, 875; no
effect of stomatal opening on photosynthesis?, 915; rate of photosynthesis under
various climatic conditions, 874, 875, 996-1001.
Bazyrina, K., and Vasiliev, G.: CO2 exhaustion effects in land plants, 908.
,
and Soldatenkov, S. V. Midday depression in algae, 876.

:
Kramer, P. J., and Decker, J. P.: Deciduous plants umbrophilic, conifers heliophilic,
989.
Krasnoselskaja-Maximova, T. A. See Maximov, N. A.
Krasnovsky, A. A. See Evstigneev, V. B.
Kundt, A. Effect of solvents on chlorophyll spectrum, 635-637.
:
Kuilman, L. W.: Inactivity of red leaf pigments in photosynthesis, 1165.

Kursanov, A. L.: Carbohydrate accumulation and midday depression, 875; midday
depression in algae, 876; diurnal course of photosynthesis, 874; rate of photo-
synthesis of subtropical plants, 999.
Lai, R. N. See Singh, B. N.

Levi, H. See Franck, J.
Levring, T.: Underwater light fields, 735; action spectra of algae, 1188. See also
Johnson, H. G.
Lewis, C. M. See Emerson, R.
Lewie, G. N. (with Kasha, M., McClure, D. S., and Calvin, M.): Long-lived triplet
states, 790-795.
Lemberg, R.: Absorption spectra of phycobilins and their proteids, 664-667.
1200 AUTHOR INDEX
Liebig, J. : "Law of the minimum," 858-859.

Lineweaver, H. See Bm-k, D.
Lipmann, F., and Tuttle, L. C: Reductive carboxylation, 937.
Livingston, R.: Absorption spectrum of allomerized chlorophyll, 613-614; search for
infrared chlorophyll fluorescence, 748, 795-796; drop of fluorescence yield of
chlorophyll in far red, 1153. See also Franck, J.
and Ke, C.-L.: Concentration quenching of chlorophyll fluorescence, 774-775;
quenching of chlorophyll fluorescence in solution by admixtures, 781-788.
, Watson, W. F., and McArdle, G.: Effect of alcohols and amines on absorption
spectrum of chlorophyll, 646-649; solvent effect on chlorophyll fluorescence, 747;
yield of chlorophyll fluorescence in solution as function of wave length of exciting
752-753; fluorescence jdeld of allomerized chlorophyll, 754; nonfluores-
light,
cence of chlorophyll in nonpolar solvents and its activation by water, alcohols
and amines, 766-771, 777.
Loewy, A. See Scarth, J. W.
Lloyd, F. E.: Chloroplast fluf)rescence under the microscope, 806.
Loomis, W., Carr, and Randall, H. M.: Transmission, scattering and reflection of light
by leaves, 676, 677, 683; leaf spectra, 688, 691, 699; absorption and reflection of
infrared by leaves, 692, 694, 695; spectra of autumnal leaves, 696, 697; relative
intensities of absorption bands in leaves, 708. See also Verduin, J.
Lorenz, A. See Harder, R.
Lubimenko, V. N.: Chlorophyll bands in leaves of different species, 699, 700-702; no
carotenoid bands in leaves?, 705; light threshold of photosynthesis?, 964; light
curves of umhrophilic and heliophilic plants, 987, 989; shade plants more effective
than sun plants in blue light, 1167-1168.
Lundegardh, H. Modification of the law of limiting factors, 862; COo exhaustion effects
:
in land plants, 908; light curves of Oxalis and Stellar ia, 966; of umbrophilic and
heliophilic plants, 987; rate of photosynthesis under natural conditions, 999.
M
Macdowall, F. D. See French, C. S.
McArdle, G. See Livingston, R.
McAlister, E. D., and Myers, J.: Time course of fluorescence in plants, 806; its relation
to photosynthesis, 819, 824; infrared photometry as method for measuring
photosynthesis, 852-853; effect of CO2 concentration on fluorescence in leaves,
940; fluorescence-light curves of wheat, 1048; effect of CO2, 1051; effect of
oxygen, 1066-1067.
McClure, D. S. See Lewis, G. N.
McGee, J. M. See Van Rysselberghe, P.
McLean, F. T. Oxygen release during midday
: depression, 874; photosynthesis of
tropical plants, 1001.
Mackinney, G.: Spectroscopically pure chlorophyll, 603-604.
Magee, J. L., De Witt, T. W., Smith, E. C, and Daniels, F.: Calorimetric measurement
of photosynthesis, 854; calorimetric quantum yield determinations, 1123.
Manning, W. M., Juday, C, and Wolf, M.: Chemical quantum yield measurements with
Chlorella at different depths, 1120. See also Button, H. J.; Strain, H. H.
,Stauffer, J. E., Duggar, B. M., and Daniels, F.: Quantum yield measurements
with Chlorella by gas analytical methods, 1119-1120.
and Strain, H. H.: Absorption spectrum of chlorophyll d and derivatives, 616;
light absorption by chlorophyll d in red algae, 720-721; fluorescence spectrum of
chlorophyll d, 748.
AUTHOR INDEX 1201
Man ten, A. : Action spectrum of phototaxis of purple bacteria shows activity of bacterio-
chlorophyll and of carotene ids except spirilloxanthol, 1188.
Maskell, E. J.: "Mutual interaction of factors" and the law of limiting factors, 863;
periodicity of photosj-n thesis in detached leaves, 874-875; role of stomata in
midday depression, 875; stomata as a limiting factor, 915.
Matthaei, G. L. G. See Blackman, F. F.
Maximov, N. A., and Krasnoselskaja-Maximova, T. A.: Fluctuations of photosynthesis
in leaves, 876.
Mecke, R., and Baldwin, W. C. G.: Reflection of infrared light by leaves, 094, 696.
xMehta, R. Y. See Dastur, R. H.
Meier, F. E.: lulling of Chlorella by ultraviolet light, 1153.
Menke, W.: Carotenoid bands in chloroplasts, 706; fucoxanthol band in brown algae,
706, 707.
Metzner, P. : Fluorescence burst in heated chloroplasts, 817.
Meyer, K. P.: Absorption spectra of different chlorophyll colloids, 649-650; leaf spec-
tra, 687, 691, 699, 705; carotenoid bands in leaves, 706; relative intensities of
bands in leaves, 708; scattering and absorption in leaves, 710; "light sieve"
effect in seedlings, 715.
Miller, E. S.: Absorption spectra of carotenoids, 656, 660.
and Burr, G. O. : CO2 compen.sation point, 898-899; compensation at low CO2, 985.
Minder, I. See Kniep, H.
Mitchell, J. W.: Constant photosynthesis under constant field conditions, 876, 877.
Molvig, H. See Stern, A.
Monch, I.: Carbohydrate accumulation and midday depression, 875; rate of photo-
sjTithesis of alpine plants, 997, 1001.
Montfort, C: Distribution of light energy among pigments in Ulva (green), 719-720,
725; in Fucus and Laminaria (brown), 724-725; rigid and adaptable umbrophiles
and heliophiles, 989; light inhibition of deep sea algae, 995-996; photosynthesis
of Ulva in colored light indicates efficiency of carotenoids, 1164; that of brown
algae shows activity of fucoxanthol, 1168-1169.
and Neydel, K.: Midday depression in stomata-free plants, 876.
and Schmidt, G.: Efficiency of brown algae in light of different color, 1176-1177;
of blue-green and red algae, probable activity of phycobilins, 1185.
Moore, W. E., and Duggar, B. M.: Quantum yield of Chlorella in blue, red and mixed
light measured by polarographic method, 1121-1122, 1132.
Mothes, K., Baatz, I., and Sagromsky, H.: Photosynthesis of green and brown algae in
red and blue light, 1178.
Miiller, D.: Light curves of arctic plants, 966; rate of photosynthesis of normal and N-
deficient leaves, 998; yield of photosynthesis of arctic plants under natural condi-
tions, 998. See also Boy sen- Jensen, P.
Mulliken, R. S.: Theory of polyene spectra, 663-664.
Myers, J. : Photosynthetic characteristics of Chlorella cultures, 834. See also McAlister,
E. D.
N
Nathansohn, A. : CO2 molecules —the only substrate of photosynthesis?, 887.
Negelein, E. See Warburg, O.
Neuwohner, W.: Interpretation of midday depression, 874, 875; midday depression in
algae, 876.
Neydel, K. See Montfort, C.
Nishimura, M. S. See Emerson, R.; 'VMiittingham, C. P.
1202 AUTHOR INDEX
Noack, K.; Fluorescence of colloidal leaf extracts, 776-777; participation of flavones in
photosynthesis?, 1165. See also Sierp, H.
Noddack, W., and Eichhoff, H. J.: Light scattering by Chlorella suspension, 676-677;
absorption spectrum of Chlorella, 690, 692, 699, 700; absorption ratio in red
and green in Chlorella, 708; enhanced absorption in far red in vivo, 715-716;
light scattering measured by "ellipsoid photometer," 843; constant photosyn-
thesis in Chlorella, 877; light curves of Chlorella in red, yellow and white light,
969,1160,1162; linear range, 980; compensation point, 983; quantum yield of
Chlorella in monochromatic light, 1155-1156.
and Komor, J.: Yields of organic matter in grass plots, 1004-1006.
and Kopp, C: Light curves of Chlorella at different temperatures, 969, 973; linear
range, 980; assimilation numbers of Chlorella cultures, 991, 993.
Ochoa, S.: Reductive carboxylation, 937, 939.

Oltmanns, F.: No chromatic adaptation?, 995.
Oneto, J. F. See Pratt, R.
Oppenheimer, R. J. See Arnold, W.
Osterhout, W. J. V., and Dorcas, M. J.: Penetration of carbonic acid into Valonia, 887.
Osterlind, S. Can algae use bicarbonate ions more effectively than CO2 molecules?, 890-
:
891; CO2 compensation point lowered by bicarbonate, 895; inhibition by excess

CO2 in algae, 903.
Overkott, O.: CO2 supply through roots, 910.
Paauw, P. van der: CO2 curves of Hormidium, 893, 904; adaptation of Hormidium to
weak light, 989, 994.
Paetzold, I. See Stocker, O.
Pantanelli, E.: Photosynthesis as function of light intensity, 964.
Pauling, L. Theory of polyene spectra, 663.
:
Pekerman, F. M. See Vavilov, S. L

Petering, H. G., Duggar, B. M., and Daniels, F.: Polarography applied to measurement
of photosynthesis, 850-851; quantum yield of Chlorella determined by polaro-
graphic method, 1120-1121.
Plaetzer, H. : Compensation points of aquatic plants, 982; of algae, 983.
Pokrovski, G. L Reflection, absorption and transmission of infrared light by
: leaves, 696,
Pollack, M. See Franck, J.
Pratt, J. See Pratt, R.
Pratt, R.: Inhibition of photosynthesis by a metabolite of Chlorella, 833; effect of and K
Na bicarbonate on time course of photosynthesis in Chlorella, 835-836, 877.
(with Fong, J., Oneto, J. F., and Pratt, J.): A growth-nihibiting substance ("chlo-
rellin") formed in Chlorella suspensions, 880; its extraction and inhibiting effect on
photosynthesis, 880-881; decline of photosynthesis with age of Chlorella suspen-
sions, 881-882.
Pringsheim, P. See Franck, J.
Prins, J. A.: Life-time of excited chlorophyll, 633-634; yield of chlorophyll fluorescence
in solution, 752, 754.
Pruckner, F.: Solvent effect on spectra of porphin, chlorophyll and bacteriochlorophyll,
642. See also Stern, A.
Puck, T. T. See Franck, J.
AUTHOR INDEX 1203
Purevich, K.: Energy conversion by plants under natural conditions, 1003-1004.

Putter, A. Energy conversion by plants over a whole season, 1005-1006.
:
Rabideau, G. S., French, C. S., and Holt, A. S.: Absorption spectra of chloroplast dis-
persions, 654-655; of leaves and chloroplast suspension, 688-689, 691. See also
French, C. S.
Rabinowitch, E.: Interpretation of spectra of bacteriochlorophyll, 617-618; of porphin,
621; of hexaphenyl porphin, 623; of chlorophyll and bacteriochlorophyll, 630-633;
absorption and reflection in plane-parallel vessels, 672-674; interaction of scatter-
ing and absorption, 711-712; interpretation of fluorescence spectra of chlorophyll
and bacteriochlorophyll, 750-752, 753-754; mechanisms of quenching, 755-758;
yields of photosynthesis and fluorescence, 820, 821-822, 823-824; three types of
saturation curves of photosynthesis, 866-872, 1012-1017; fluorescence-light
intensity curves of plants in strong light, 941 effect of reductants on fluorescence
;
in bacteria, 950-951; light curves of thin and dense suspension, 1007-1008. See
also Ehrmantraut, H. C.
Rabinowitch, E., and Epstein, L. F.: Fluorescence quenching by dimer formation, 761-
762, 765.
(with Jacobs, E. E.): Kinetic analysis of the CO2 factor in photosjTithesis, 916-939;
calculation of carboxylation equilibrium constant, 935-936; analysis of light
curves for different kinetic models, 1017-1047; interpretation of light curves of
fluorescence, 1067-1078; extrapolation of maximum quantum yield, 1133-1135;
theoretical and experimental maximum quantum yield, 1137-1139.
Raffy, A. See Dh6r6, C.
Randall, H. M. See Loomis, W.
Rao, M. S. See Dastur, R. H.
Rehm, S. See Stacker, 0.
Reinau, E.: CO2 fertilization, 902.
Reinke, Discovery of light saturation, 964.
J.:
Reman, G. H. See Wassink, E. C.
Richter, A. von: Action spectrum of photosynthesis has two peaks, 1144; phycobilins
inactive in photosynthesis?, 1184-1185.
Riecke, F. F.: Quantum yield of photosynthesis —
repetition of Warburg and Negelein's
experiments, 1090-1091; new measurements, on Scenedesmus and Chlorella, 1095;
quantum yield of CO2 reduction by anaerobically adapted Scenedesmus, 1 128.
Riley, G. A.: Yield of photosjTithesis in sea, 1006.
Roach, J. R. See Zscheile, F. P.
Rommel, L. G. : Reaction kinetics and law of limiting factors, 863-864; phase boundary
air-cell wall as diffusion barrier, 916.
Rudolph, H. : Absorption spectrum of protochlorophyll, 618-619.
Ruttner, F.: Photosynthesis of aquatic plants at high pH, 890, 906; CO2 compensation
point lowered by bicarbonate, 899.
Sachs, J.: Bubble counting as method of measuring photosynthesis, 846; cardinal

points, 858; maximum photosynthesis in yellow-green light?, 1142.
Samant, K. M. See Dastur, R. J.
Sandoval, A. See Zechmeister, L.
;
1204 AUTHOR INDEX
Schanderl, H., and Kaempfert, W.: Changes in light transmission caused by chloroplast
movements, 680-681; "light sieve effect" in leaves, 715; inhomogeneity of light
absorption in a leaf, 864-865.
Scarth, J. W., Loewy, A., and Shaw, H. H.: Fluctations of photosynthesis in detached
leaves, 877.
Schmidt, G. See Montfort, C.
Schocken, V. See Warburg, O.
Schoder, A.: Interpretation of midday depression, 874; stomata do not limit photo-
synthesis, 915.
Schroeder, H. Diffusion resistance of air channels as limiting factor, 916.
:
Senn, G.: Chloroplast movements in algae, 679.

Seybold, A.: Absorption bands of protochlorophylls a and b, 619; absorption of lightby
leaves, 677, 678, 683; by algae, 683; by single chloroplasts, 683; by nonplastid
pigments, 684; leaf spectra, 687, 691, 699; spectra of aquatic plants and algae,
689, 690, 691, 699, 706; relative intensities of absorption bands in leaves, 708;
light absorption by carotenoids in Phaseolus leaves, 721-722;
"natural light
fields," 731-735.
See also Sierp, H.
and Egle, K.: Nonfluorescence of adsorbed chlorophyll, 775, 776, 777; fluorescence
changes in leaves caused by boiling, drying and changes in humidity, 817-818;
chlorophyll present in leaves in two forms?, 818-819.
and Weissweiler, A.: Absorption spectrum of adsorbed chlorophylls a and b, 651-
652; absorption by leaves with different chlorophyll content, 678; by single
chloroplast layers, 683; by aurea and purpurea leaves, 685; leaf spectra, 686, 691
spectra of algae, 690, 691; spectra of boiled and ether-filled leaves, 698; of
Chlorella, 700; ratios of absorptions in red, green and blue in various leaves, 710;
assimilation numbers of aurea leaves, 993.
Shaw, H. H. See Scarth, J. W.
Shiau, Y. G., and Franck, J.: Fluorescence changes in algae, 806; their relation to
photosynthesis, 819-821; effect of quinone on fluorescence of Chlorella, 1067;
fluorescence-light curves of Chlorella and Scenedesmus, 1049; effect of oxygen,
1067.
Shull, C. A. : Reflection spectra of leaves of different age, 696, 697, 699.
Sierp, H.,Noack, K., and Seybold, A.: Diffusion through septa, 913.
Simonis, W. See Harder, R.
Singh, B. N., and Kumar, K.: Sigmoid light curves of leaves?, 964; light curves of
Raphanus sativum, 966, 987.
, Lai, R. N., and Kumar, K.: CO^ curves of higher plants, 892, 903, 904, 908.
Smith, A. M. See Blackman, F. F.
Smith, E. C. See Magee, J. L.
Smith, E. L.: Absorption spectrum of colloidal leaf extracts, 652-653; CO2 curves
of Cabomba, 892, 897, 903, 904; are CO2 curves nonhyperbolic?, 937-938; light
curves of Cabomba, 967, 968, 971, 987; higher order equations for light curves,
1044, 1046-1048.
Smith, J. H. C. See Koski, V. M.
Soldatenkov, S. V. See Kostychev, S.
Solmssen, V. See Karrer, P.
Solomon, S. See Dastur, R. H.
Spoehr, H. A. (and coworkers): Separation of an antibiotic from Chlorella, 882-883;
organic matter production by field crops and trees, 1006.
AUTHOR INDEX 1205
Stair, R., and Coblentz, W. W. : Infrared absorption spectrum of chlorophyll and phytol,
610-612; of carotenoids, 656.
Stalfelt, M. G.: Closure of stomata as cause of midday depression, 875; CO2 diffusion
through cuticle, 911; effect of partial closure of stomata on photosynthesis, 915-
916; light curves of mosses, 966; of lichens, 967; compensation points, 982.
Stauffer, J. E. See Manning, W. M.
Steemann-Nielsen, E.: Utilization of bicarbonate by Myriophyllum, 888-890; CO2
curves of Myriophyllum and Fontinalis, 893, 903; light curves of green and brown
algae, 969; induction losses following reduction of light intensity, 1040. See also
Gabrielsen, E. K.
Stefan, M. J.: Theory of from point source, 912-913.
diffusion
Stern, A.: Typical porphin and chlorin spectra, 620.
and Molvig, H.: Absorption spectrum of porphin, 620-622.
, Molvig, H., and Dezelic, M.: Fluorescence of porphyrins and chlorins, 749-750.
, Wenderlein, A., Molvig, H., and Pruckner, F.: Effect of substitutions on porphin
spectrum, 621-629.
Stern, K.: Fluorescence of chlorophyll colloids, 775-776.
Stocker, O.: Rate of photosynthesis of tropical plants, 1001.
, Rehm, S., and Paetzold, I.: Fluctuation m photosynthesis and in CO2 content of
air, 876-877; maximum rates of photosynthesis of leaves, 991.
Stokes, G. G.: Composite nature of chlorophyll deduced from fluorescence spectrum,
740; discovery of leaf fluorescence, 805; fluorescence of pigments from red algae,
759.
Stoll, A., and Wiedemann, E.: Fluorescence of colloidal leaf extracts, 777. See also
Willstatter, R.
Strain, H. H.: Absorption spectra of leaf carotenoids, 656, 658, 660. See also Manning,
W. M.
and Manning, W. M. Absorption spectrum of chlorophyll c (chlorofucin), 614-615;
:
light absorption by chlorophyll c in diatoms, 720-721.

,Manning, W. M., and Hardin, G.: Absorption spectra of carotenoids of brown
algae, diatoms and dinofiagellates, 656-657, 659, 661.
Svedberg, T., and Katsurai, T.: Absorption spectra of phycobilins, 664-665.
Tanada, T.: Absorption spectrum of diatoms, 699; relative band intensities in diatoms,
708, 710; distribution of light energy between pigments in diatoms, 726; quan-
tum yield in Navicula minima, 1097; action spectrum of N. minima, full activity
of fucoxanthol and chlorophyll c, 1173-1175.
Terwood-Lad, D. See Franck, J.
Thoday, D.: Midday depression of photosynthesis, 874.
Thomas, J. B.: Action spectrum of photosynthesis of purple bacteria shows peaks be-
longing to bacteriochlorophyll and carotenoids, other than spirilloxanthol. 1188.
Thomas, M. D., Hendricks, R. H., and Hill, G. R.: CO2 compensation point, 899.
and Hill, G. R.: CO2 fertilization, 902.
Timiriazev, K.: Maximum of photosynthesis coincides with maximum of chlorophyll
absorption, 1142-1143; coincides with maximum of solar spectrum?, 1143.
Tonnelat, J.: Calorimetric measurement of photosynthesis, 854; calorimetric quantum
yield determinations with Chlorella, 1123-1124.
1206 AUTHOR INDEX
Tswett, M. Fluorescence of chloroplasts under the microscope,

: 806.
U
Ursprung, A.: Photosynthesis in near ultraviolet, 1153.
Van der Honert, T. H. See Honert, T. H. van der.

Van der Paauw, P. See Paauw, P. van der.
Van der Veen, R. See Veen, R. van der.
Van Norman, R. W., French, C. S., and Macdowall, F. D. H.: Absorption spectra of red
algae, 692, 699; fluorescence spectra of extracts from red algae, 799. See alto
French, C. S.
Van Rysselberghe, P., Alkire, G. I., and McGee, J. M.: Electrolytic reduction of COj,
939.
Vasiliev, G. See Kostychev, S. P.
Vavilov, S. Galanin, M. D., and Pekerman, F. M.: Transfer of electronic energy be-
I.,
tween molecules, 758-759; fluorescence quenching through energy transfer to

nonfluorescent pigments, 778.
Veen, R. van der: Thermal conductivity as method of measuring photosynthesis, 853;
kink in light curve near compensation point, 1116; a mechanism of photosynthesis
involving formation of high energy phosphates by back reactions, 1116-1117.
Verduin, J.: Mutual interference of pores in diffusion through septum, 913-914.
and Loomis, W. E.: CO2 concentration near ground, 902.
Vermeulen, D., Wassink, E. C., and Reman, G. H.: Absorption spectra of purple bac-
teria, 692, 693; spectrophotometric study of bacteriochlorophyll fluorescence,
747, 748; fluorescence spectrum of Chlorella and Chromatium, 806, 807, 809, 810;
quantum yield of fluorescence in Chlorella and Chromatium, 813; its dependence
on wave length, 813-814; action spectrum of growth of purple bacteria, inactivity
of carotenoids, 1188. See also Wassink, E. C.
Voerkel, S. H.: Chloroplast movements in Funaria, 679-682.
Volkov, A.: Discovery of proportionality between photosynthesis and light intensity,
964.
von Guttenberg, M. See Guttenberg, M. von.
von Richter, A. See Richter, A. von.
W
Wakkie, J. G.: Absorption spectrum of chlorophyll in different solvents, 637, 641; at
high concentration, 651; fluorescence of chlorophyll emulsions, 776.
Warburg, O.: Use of unicellular algae for the study of photosynthesis, 833-834; mano-
metric methods of measuring photosynthesis, 847-850; CO2 curve of Chlorella,
893, 906, 908; effect of O2 pressure on photosynthesis, 960- light curves of
Chlorella, 967; second series of quantum yield measurements, 1098-1107.
and Burk, D. (also Schocken, V., and Hendricks, S.) Third series of quantum yield
:
determinations, by the two-vessel method, 1104-1109, 1110, 1132.

, Burk, D., Schocken, V., Korzenovsky, M., and Hendricks, S. B.: No light effect
on respiration in Chlorella when CO2 is effectively removed, 901.
and Negelein, E.: Early curvature of light curves in dense suspension of Chlorella,
980; determination of quantum yield of photosynthesis, 1085-1086, 1088;
first
quantum yields in colored light, 1 147.

AUTHOR INDEX 1207
and Schocken, V.: Chlorophyllide-sensitized autoxidation of thiourea in pyridine
as an actinometric reaction, 839-841.

Wassink, E. C: CO2 exhaustion effects in leaf discs in buffer, 905; light curves of
horticultural plants, 967, 970; linear range, 980, 981; maximum photosynthesis
of horticultural plants, 991 quantum yields of leaf discs, 1096.
;
(with Katz, E., Dorrestein, R., and Kersten, J. A. H.): Fluorescence-time course
in algae, 806; quantum yield of fluorescence in diatoms and purple bacteria, 812;
relation of fluorescence to photosynthesis, 819, 820, 824, 825. See also Eymers,
J. G.; Katz, E.; and Vermeulen, D.

and Katz, E.: Light curves of Chlorella cultures of different age, 978 cyanide
stimulation of fluorescence in Chlorella, 1057, 1060, 1061.
, Katz, E., and Dorrestein, R.: Absorption spectra of purple bacteria, 692, 702-704;
fluorescence of colloidal extracts from purple bacteria, 777; CO2 curves of purple
bacteria, 893, 903-904; effect of concentration of CO-, and of reductants on
fluo-
rescence, 941, 942, 949-950; effect of concentration of thiosulfate, H2S and H2

on
rate of C0> reduction, 945-947; CO2 reduction with mixed reductants, 947-948;
^gmoid light curves in bacteria, 948, 964; effectson photoreduction and fluores-
cence of bacteria, of pH, 952-954; of cyanide, 954-955; of hydroxylamine, 955-
at
957; of azide, 958-959; of urethans, 959-960; light curves of purple bacteria
different temperatures, 969, 974; in presence of KCN, NH2OH, NaN, and ure-
than, 977; at different pH's, 978; linear range, 980; fluorescence-light curves
of Chromatium, 1049-1050; effect of CO2 on them, 1052; of reductants, 1052-
1055; of temperature, 1057, 1059; of cyanide, 1061-1062; of hydroxylamine and

azide, 1062-1064; of pH, 1063, 1065; of narcotics, 1063, 1066; interpretation
of
light curves of fluorescence, 1077; quantum yield determinations with purple

bacteria, 1125-1126, 1127-1128, 1132.
and Kersten, J. A. H.: Absorption spectra of carotenoids from diatoms, 657, 659;
of diatoms, 692, 699; fucoxanthol bands in diatoms, 706; relative intensities of

absorption bands in diatoms, 708; effect of CO2 concentration on fluorescence of
diatoms, 940-941 ; effect on photosynthesis and fluorescence of diatoms of cya-
nide, 945-955; of urethan, 959; light curves of diatoms, 969, 972; in presence
of
cyanide and urethan, 976; fluorescence-light curves of diatoms, 1050-1051;

effect ofCO2, 1051; of temperature, 1056-1057, 1059; of cyanide, 1062; quantum
yield in Nitzschia disupata, 1097; action spectrum of N. dissipata; efficiency of
fucoxanthol, 1172.
, Vermeulen, D., Reman, G. H., and Katz, E.: Effect of urethan concentration on
photosynthesis in CMoreifc, 959; of O2 pressure, 960 photosynthesis-light curves
;
of Chlorella at different temperature, 967, 973; in presence of

KCN, 974; in
presence of urethan, 975; in O, and N2, 976; fluorescence-light curves of Chlorella,
presence of cyanide, 1057, 1060; of
1048; at different temperatures, 1055; in
narcotics, 1063, 1065; of O2, 1063.

Watson, W. F. See Livingston, R.
Waugh, V. G.: Fluctuations of photosynthesis in leaves, 876.
Weil-Malherbe, M. -See Weiss, J.
Weis, F.: Sun and shade plants, 987.

Weiss, J. : Mechanism of fluorescence quenching, 780.
and Weil-Malherbe, H.: Concentration quenching of chlorophyll fluorescence, 772-
774, 789-790; quenching by oxygen, 779.
975.
Weller, S., and Franck, J.: Light curves of hydroxylamine-inhibited Chlorella,
Wenderlein, A. See Stern, A.
White. J W. See Zscheile, F. P.
1208 AUTHOR INDEX
Whittingham, C. P.: Time course of photosynthesis of Chlorella in carbonate buffers,

908; calculation of carboxylation equilibrium, 935.
, Nishimura, M. S., and Emerson, R.: Criticism of Warburg and Burk quantum
yield determinations, 1109-1113.
Wiedemann, E. See Hagenbach, A.; Stoll, A.
Willstatter, R., and Stoll, A.: Constant photosynthesis of detached leaves, 877; light
curves of excised leaves, 966; maximum rates of photosynthesis in strong light,
assimilation numbers, 990, 992; yield of photosynthesis in strong light in green
and yellow leaves, 1136-1137; inactivity of earotenoids?, 1163; of red leaf pig-
ments, 1165.
WUschke, A.: Fluorescence bands of brown and green algae, 806, 807.
Wolf, M. See Manning, W. M.
Wiirgler, E. See Karrer, P.
Wurmser, R.: Absorption and scattering in inhomogeneous systems, 713-714; absorp-
tion ratio red blue enhanced in vivo, 716; quantum yield measurements with Ulva
:
by Winkler's method, 1118, 1148.

and Ducleaux, J.: Relative efficiency of red and green algae in colored light, 1185.
Zechmeister, L., and Sandoval, A.: Fluorescent carotenoid derivatives, 798-799.

Zscheile, F. P. See Harris, D. G.
and Comar, C. L.: Absorption spectrum of chlorophylls a and b, 605.
, Comar, C. L., and Harris, D. G. Preparation of spectroscopically pure chlorophj-ll,
:
603-604.
and Harris, D. G.: Absorption spectra of chlorophylls a and b in ultraviolet, 609-
610; spectrophotometric study of the fluorescence of chlorophyll, 741-746, 748;
effect of solvent on fluorescence yield of chlorophyll, 763-764, 769.
, White, J. W., Beadle, B. W., and Roach, J. R.: Absorption spectra of earotenoids,
656, 659, 660.

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mammnaM-i iBHJumii

By EUGENE I. RABINO WITCH

INTERSCIENCE PUBLISHERS, INC., NEW YORK

INTERSCIENCE PUBLISHERS, Inc., 250 Fifth Avenue, New York 1, N. Y.

For Great Britain and Northern Ireland:

PRINTED IN THE UNITED STATES OF AMERICA

The manuscript of ^'oUlme II of this monograph was ready in draft

material, research in the field of photos^Tithesis picked up after the war-

of pigments (in solution and in chloroplasts) and with studies of the ,

chemistry of carbon dioxide reduction by means of radioactive carbon.

chemical composition of the intennediates and the nature of the enzymes

* As in Volume I, an index of the most important investigations described is pro-

and biochemists have disentangled its chemical processes as thoroughly

to thank him for his unstinted assistance.

PART THREE— SPECTROSCOPY AND FLUORESCENCE

Chap. 22. Light Absorption by Pigments in the Living Cell 672

Spectra of Living Plants 697

Chapter 22, contd. PaGE

PART FOUR—KINETICS OF PHOTOSYNTHESIS

Methods of Kinetic Measurements 833

5. Measurements of Carbohydrate Production and Energy Con-

External and Internal Factors in Photosynthesis

1. The "Cardinal Points" and the "Luniting Factors" 858

(a) Sources of Saturation in Photosynthesis

5. Rate of Photosynthesis under Constant

Role of the Stomata 910

Interpretation of Carbon Dioxide Curves 916

(g) Calculation of Carboxylation Constant

Chapter S7, contd. PaGE

Chap. 28. The Light Factor. I. Intensity 964

A. Light Curves of Photosynthesis 964

1. Quantum Yield Measurements by the Manometric Method. . . . 1085

Chapter 29, Contd. PaQE

ABSORPTION SPECTRA OF PIGMENTS IN VITRO

A. Absorption Spectra of Chlorophyll

The spectra of chlorophylls a and b have been studied in detail because

Zscheile,Comar and Mackinney (1942) studied samples of chlorophyll prepared by

extinction coefficients by means of two different photoelectric spectrophotometers.

A spectroscopically important impurity likely to be present in many

10% of chlorophyll a. can easily be recognized by increased

He recommended extraction with hexane (which dissolves chlorophyll a

residue no longer shows the chlorophyll b band.

The absorption curves given by Mackinney (1938, 1940, 1941) and

Table 21.1. Chlorophyll Solutions in Ethyl Ether

A. Absorption Maxima. Main bands in italics.

CHLOROPHYLLS a AND 6 607

Table 21.1 Continued

Chlorophyll b in ethj'l ether

Observers (643 m/i) (453 m>i) (510 m^)

Sprecher von Bernegg et al.

(1935) 4.7 9.4 0.28 2.0 17

As pointed out once before, particularly wide discrepancies can be

band, and sometimes as a separate peak, almost as prominent as the main

We recall that the three structures of chlorophyll, A, B and C, described

Chlorophyll Extinction Measurements in Various Solvents

Methanol, Rabinovvitch and Weiss (1937) (ethyl chlorophyl-

The absorption spectra of the two chlorophylls in the ultraviolet are

The absorption spectra of chlorophyll, ethyl chlorophyllide and phytol

260 280 300 320 340 360 380 400 420