Banana Polyphenoloxidase.

Preparation and Properties 1, 2

James K. Palmer
Central Research Laboratories, United Fruit Company, Norwood, Massachusetts

Griffiths ( 13) has presented evidence in(licating 7.0: 10- 5 EDTA; 4.2 X 10- ) i% ascorbic acid;
that the browning reactions of banana fruit result 5 X 10-4 31 dopamline and enzyme in a final volumie
from the enzymic oxi(lation of (lopamiiine (3.4 (lih-- of 3 ml. The reaction rate wN'as proportional to

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droxyphenylethylaminei) hy pIolyphenoloxi(lase. Al- eiiz) iie concentrationi xNhen the decrease in optical
thoughi (lopamine does occur in various fruits andl lensitv was between 0.02 and 0.2 per minute. All
vegetables (23), it has not previously b)een imiplicat- extracts assayed by this method were free of inter-
ed as an important substrate in browning reactions. fering ascorbic acid oxidase.
The present investigation was undertaken to develop PPO was also assayed manometrically by stand-
metho(ds for preparation of banana polvphenoloxidcase ardl \Varburg tecli(lue at 30°. Reaction mi.xtur-es
(PPO) 3 an(l to determinie solile of its properties in are (lescribed for the individual experiments.
comparison with phenol oxi(lases frolim othier sources. One unit of PPO activity is expressed wherever
lOxsible as that amilount of enzy-me which ill catalyze
Materials and Methods the transformation of 1 Mmilole of substrate peri min-
ute un(ler the con(litions of the assay'. Michaelis
Materials. The chemiiicals use( an 1 their sup- conistanits were obtai ne(l froml Lineweaver- Burk ploti
pliers were as follows: Dopamiline HCI: D.l,-ar- (16 ).
terenol HCI; L(- ) arterenol bitartrate hydrate; The protein conteIlt of the (letergenit extracts \\.as
chlorogeniic acid-, tyramine HCI: California Cor- estimlate(l frolm nlitrogenl determinations (1 1)ill tile
poration for Biochemi cal Researchi. 3.4-dihydroxv- extraCIct after a 4 h1our d(ialsis agailnst 400 volumes
phenylalanine (DOPA ): Nutritionial Bliochemicals of 0.02 M\ potassiumii phosphate, pH 7.0 in a stirrinig
Corp. L (-) tyrosine: Pfalistielil Laboratories. Inc. dialyzer. Removal of nonproteiin nitrogen wx as es-
Catechlol; o-cresol: Fishier Scientific Co. p-Cresol senitially coliiplete undler these con(litions. but little
(redistilledl); 8-hydroxyquinoline: L( + )'cysteine or n1o I'PO (or (letergent) wvas lost. Protein in the
HCI: Matheson, Colemialn, and Bell. DE,AE-cellu- fractions frolim DEAE cellulose column1i1s w\as estilimt-
lose (Type 20, 0.82 mie(qjg ): Schileicher- & Schuell ed by the miethio(d of Wkaddell (25). Egg albumileni
Co. MWNNIT = 40.000) an(l gamma globuliin (Bovine Frac-
Assayv Procediures. The standard assay for ba- tion II, MWNV 150,000 ) gave i(lentical standilard
nana PPOC was adaptedl fromil tile tyrosine assav of Fox curves. This teclniqlue could not be usedl onl tile
and Burnett (10). The increase in optical (lensity at (letergent extracts as the detergent interfered.
470 mia after mixing enzyme and substrate wvas fol- The spectromiietric technique of Mason (17 ) as
lowed at 25° in a DK-2 sl)ectrophotomieter eqluippe(l use(l to studly the reactioni mechanism. A series ot
with a time (Irive. The reactioin mlixture, in a final 1 yiumole saniples of (lopanmine in 10 ml of 0.05 Mr po-
volume of 3 ml, contained 0.033 \)potassium phos- tassium phosphate. pH 6.0 wvere oxidizecl by 50 nlg
phate, pH 7.0; 5 X 10-: -\ (lopaiiine and sufficient of Ag.,O ith shaking for periods vary ing bet een
enzyme to cause an increase in optical density of 0.02 1.5 ancd 20 mlinutes. The solutionls w-ere filtere(d
to 0.1 per minute. Rates were calculate(d from the quickly, and the resulting spectra were compared
initial slope of the curve alid were proportional (+ with those of the enzyimic oxidationl products. D,
10 %) to enzyme concentration. L-DOPA was oxidlize(l un(ler simililar- conldlitionis to
Some extracts vere assayed indirectly (8) by chieck the procedlure.
following spectrophotonmetricallv at 265 nm/L the rate Dopamiline f8-oxidase activity was assayed by the
of disappearance of ascorbic acid at 25° in a reaction techni(lue of Smlithi andl Kirshner (21). A one nil
mixture containing 0.05 pl otassiunl phosl)lpate, pH ali(luot of diluted (1: 50) detergent extract was ill-
cubated with 1 Aimole of dopamine at 370 for 1 hour.
1 Revised manuscript received Jan. 28, 1963. The reaction was stopped with 1 nil of 10 9 tri-
2 A preliminary report of this work lhas appeared in
Planit Physiol. 36, Suppl. xxxviii, 1961. chloroacetic acidl an(l the nmixture centrifuge(d at
3 Terminology used in this paper: Pheniol oxidase, low speed to sedimilent protein. A 1.2 mln, ali(quot was
generic term to include all enzymes which catalyze the extracted 3 times with ether and thein assaye(h fluori-
oxidation of phenols; tyrosinase, enzyme which catalyzes mietrically (9) to (letermine arterenol (Beckmain DK-
the oxidation of both mono- and diphenols; polyphenol- 2 spectrophotometer. No. 22850 fluorescence at-
oxidase, enzyme which catalyzes the oxidation of ortho
dihydric phenols only. tachlimlent, 515 Illn).
508
 PALMER-BANANA POLYPHENOLOXIDASE 509
Results Table I
Polyphenoloxidase Activity of Banana Pulp Extracts.
Extraction of Soluble PPO. Ripe banana fruits
(Musa acuminata, var. Hort. Gros Michel) were the Q°2* (IAI 02 per mg dry wt per hr)
Experiment
source of the enzyme. All operations were conduct-
ed at 0 to 50 unless otherwise specified. number Homogenate Detergent
extract
Initial experiments were carried out with buffer-
ed homogenates (1-2 g of tissue in 20 ml of 0.1 M 1 105 154
potassium phosphate, pH 6 or 7) of banana pulp or 2 168 403
peel tissue. These homogenates catalyzed the * Assay conditions: Experiment No. 1, 0.067 M dop-
aerobic oxidation of dopamine and other diphenols, amine; 0.067 M potassium phosphate, pH 6.0; 0.5 ml
resulting in the production of brown pigments. All enzyme (undiluted detergent extract). Experiment

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activity was lost on boiling these homogenates and No. 2, 0.017 M dopamine; 0.067 M potassium phos-
phate, pH 6.0; 0.2 ml enzyme. Final volume (includ-
the browning reactions were inhibited by ascorbic ing 0.2 ml 5 % KOH in center well) was 3.0 ml in
acid, sodium hydrosulfite, cysteine, diethyldithiocar- each case.
bamate, and potassium ethyl xanthate. Pulp tissue
was chosen for further study as the pulp homogenates
were essentially colorless whereas peel homogenates the properties and purification of banana PPO, un-
darkened rapidly during the preparative procedure. less otherwise noted.
The browning reactions in pulp homogenates in- Dopamine can also be oxidized directly or in-
variably took place on the surface of the tissue frag- directly by laccase, monoamine oxidase, cytochrome
ments. When the homogenates were centrifuged at oxidase, or peroxidase. The banana fruit undoubt-
500 X g for 10 minutes, 80 to 100 % of the PPO ac- edly contains some or all of these enzymes. How-
tivity was sedimented with these fragments. Any ever, the detergent extracts were free of significant
remaining activity was in the supernatant fraction amounts of these enzymes as determined by appropri-
after further centrifugation at 20,000 X g for 15 ate assay (12, 20) and by failure of the extracts to
minutes. These results suggested that the PPO was oxidize monophenols, /3-phenylethylamine, p-phenyl-
adsorbed on or structurally associated with the cell enediamine or ascorbic acid (3, 6, 22).
wall. Releasing the enzyme into solution was ac- Piurification of Banana PPO. The enzyme was
complished by extraction of the pulp with buffered freed of excess detergent by precipitation from the
detergent solution. Pulp (2 g) was homogenized undiluted detergent extract at -8° with 1.6 volumes
in a 50 ml Duall Tissue Grinder (Kontes Glass Com- of acetone. Significant amounts of detergent were
pany) in 18 ml of a 1 % detergent solution buffered not precipitated from these extracts until 1.7 volumes
at pH 7.0 with 0.02 M or 0.1 3& potassium phos- of acetone had been added. The precipitated mate-
phate. The non-ionic, polyoxyethylated detergents rial was collected by centrifugation (3500 X g, 20
known as Cutscum (Fisher Scientific Company) and minutes, -8°) and then stirred up in a volume of
Igepal CO-630 (General Aniline and Film Company) 0.02 Ai potassium phosphate, pH 7.0, equal to one-
were equally effective in rendering the enzyme solu- half the original volume of detergent extract. After
ble. The PPO activity was in the supernatant solu- standing overnight the PPO had redissolved and the
tion after centrifugation at 20,000 X g for ca. 15 min- mixture was centrifuged at 20,000 X g for 10 min-
utes. These detergent extracts were appropriately utes to remove the inactive residue. The superna-
diluted (usually 1: 50) with 0.02 I potassium phos- tant fluid was added to a 1.0 cm X 10 cm column of
phate, pH 7.0 for use in the assay procedures. DEAE cellulose which had been previously washed
This procedure not only solubilized banana PPO, and equilibrated with 0.04 M phosphate, pH 8.0 by
but also increased the activity by a factor of 1.5 to the procedure of Keller et al. (15). The flow rate
2.5 (table I). Cotzias et al. (5) reported similar of the column (1 ml/min; 1 lb pressure) was usually
results in their studies on monoamine oxidase. De- reduced as the sample entered the column and addi-
tergent at 1 % approached the minimum concentra- tion was stopped when pressures of 8 lbs/sq in were
tion required for efficient extraction of banana poly- required to maintain a flow rate of about 8 ml/hr.
phenoloxidase from pulp tissue. Little or no addi- The column was then washed with 100 ml of 0.04 M
tional activity was extracted at 5 % while extracts phosphate, pH 8.0 at 8 ml/hr, followed by 0.08 M
with 0.1 % detergent contained about one-half of the phosphate, pH 8.0 at the same flow rate, according
activity at 1 %. Extracts of comparable pulp sam- to the procedure of Frieden and Ottesen (11). Some
ples with 1 % detergent solution had essentially preparations did not reduce the flow rate appreciably
identical activity. However, extraction of the and these were eluted at 15 ml/hr under about 1 lb
sedimented debris with fresh aliquots of detergent pressure. Fractions of 2.5 to 3.5 ml were collected
yielded about 15 % additional activity. and assayed for protein content and PPO activity
The detergent extracts were clear to slightly (standard assay).
cloudy, faint yellow-brown in color and free of sig- Figure 1 shows the results of a typical chromato-
nificant endogenous activity. These readily prepar- graphic separation. A 10 to 12 fold purification of
ed extracts were used for all subsequent studies on the PPO from detergent extracts was obtained in
510 PLANT PHYSIOLOGY

PHOSPHATE , pH 8.0
1,800 I 0.040M O.o-o-
I

1,600

,4001-
i-
I.-

0 1,200 J
4(
I--2
-
200
CN a

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z
0 t
z

o a -i
-J
z
1 00
600 60 -

a.
2
0 a-
IL
400 o PPO ACTIVITY 40 z

x PROTEIN
0 0
w
200 20 0 10 20 30 40
.,LI DOPAMINE CONCENTRATION-mM
0.
- I
0
-

10 20 30
X-X
40 50
u0
60
FRACTION NUMBER
FIG. 1. Chromatography of banana polyphenoloxidase on DEAE cellulose. See text for details.
FIG. 2. Effect of dopamine concentration on activity of banana polyphenoloxidase. Standard assay.

the fractions of highest specific activity from the than that determined from the oxygen uptake (table
DEAE column (table II). These pooled fractions, III), presumably because the spectrometric assay
hereafter referred to as purified extract, were used specifically measures the initial rate of formation of
in some of the substrate studies described below. the first pigmented product, while the 09 uptake
Suibstrate Studies. The substrate specificity of arises from a series of oxidations involved in melano-
detergent extracts and purified extracts was identical. genesis. Similar differences in the Km of chloro-
A variety of o-dihydric phenols were oxidized, genic acid as a substrate for the polyphenol oxidase
dopamine having the lowest Km value among the of Solidago zvirgaurea leaves have been reported by
substrates tested (table III). Monophenols such as Bjorkman and Holmgren (2).
tyrosine, tyramine, o-cresol or p-cresol were not The enzyme was saturated by dopamine concen-
oxidized. No reaction was observed for up to 1 trations higher than 3 mar, but inhibition set in
hour after mixing the enzyme with the monophenols above 12 mbL (fig 2).
at 0.002 -i to 0.02 M. Addition of catechol or dopa- pH Optinmumiii. Banana PPO had a pH optimum
mine in an attempt to eliminate any induction period of 7.0 when catalyzing the oxidation of dopamine
had no effect on the oxidation of tyrosine. (fig 3). Negligible autoxidation of this substrate
The Km for dopamine determined with the stand- occurred in the standard assay at pH 7 or below.
ard spectrometric assay was considerably smaller The values at pH 7.5 and 8.0 in figure 3 were cor-

Table II
Purification of Polyphenoloxidase from Bantana Fruit
Specific
Volume Activity* Total Protein activity Recovery
Fraction ml units/ml activity mg/ml units/mg
units protein
40 4.7 188 0.48 9.8 100
Detergent extract
Acetone ppt 19 8.9 169 90
DEAE eluate 9.5** 2.3 21.8 0.02 115 12***
* Standard assay at pH 7.0.
** Pooled fractions of highest specific activity from DEA E separation.
*** Actual recovery in this experiment, where only 10.5 ml of the acetone ppt was separated on DEAE column.
Normally the final yield of high specific activity enzyme will be nearer 20 %.
 PALMER-BANANA POLYPHENOLOXIDASE 511

100
0
CH2 FAST CH
2 FAST C-HZ
I 75 HO
H A I
oH
2
-PPO2
.CH2 +Io 0
I

CHt
H2 LH

DOPAMINE DOPAMINE 2,3-DIHYDROINOOLE

QUINONE -5,6 QUINONE (RED)

MAX.. 300mju,LOG f .3.95

4 70 yLOG f -3.40

I-F 50
Jsiow

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HO
C-) MELANIN J FAST

II
< 25 (GENERAL
ABSORPTION)
N a °t HO
H H
0N __ _ INDOLE-5,6 OUI NONE 5,6-DIHYDROXYINDOLE
(PURPLE)
MAX 540m

0
3 4 5 6 7 8
pH
FIG. 3. Effect of pH on activity of banana polyphenoloxidase. Standard assay, dopamine substrate. pH 6 to 8,
phosphate buffer; pH 3.8 to 6, citrate buffer.
FIG. 4. Proposed reaction mechanism for the oxidation of dopamine by banana polyphenoloxidase. Spectrochem-
ical evidence was obtained for the presence of the bracketed compounds only. The remaining intermediates are as-
sumed by analogy with DOPA oxidation (18), and not all the probable intermediates are shown.

rected for 9 and 20 % autoxidation, respectively. least 70 % for a month at 2 to 50. The diluted de-
Variation in the final phosphate buffer concen- tergent extracts and the purified extracts were some-
tration from 0.01 M to 0.067 M or substitution of what less stable, sometimes losing 30 % of their
citrate buffer for phosphate buffer at pH 6 had no activity in 1 week at 2 to 50. Detergent extracts
effect oni the activity. could usually be frozen and thawed without effect,
Stability of Enzyme. The undiluted detergent but occasional preparations lost significant activity
extracts usually retained at least 90 % of their orig- after such treatment. At room temperature the en-
inal dopamine oxidase activity for 2 weeks and at zyme in detergent extracts was stable for at least
24 hours. In one case, it retained 60 % of its activity
after 1 week at room temperature.
Table III Reaction Mechanismn. The first spectroscopically
Mlichaelis Constant of Substrates for observable product of the PPO catalyzed oxidation
Banana Polyphenotozidase of dopamine was a red pigment with absorbancy
peaks at 300 mn and 470 m/. If the oxidation was
Km continued beyond about 15 minutes, a purple pigment
Substrate M Assay (Xmax = 540 mu) was produced and finally a grey
to black precipitate appeared. Oxidation of dopa-
Dopamine 6.3 x 10-4 Standard mine for increasing periods of time with silver oxide,
D,L-arterenol 2.4 x 10-3 p

L-arterenol 3.6 X 10-3 " as described by Mason (17), yielded the same pig-
D,L-DOPA 4.4 X 10-2 ments. Log e for the red pigment was 3.95 at 300
L-DOPA 6.6 X 10-2 m/' and 3.40 at 470 m. Log e for the purple pig-
D-DOPA 3.0 x 10-2 ment could not be determined because of its transi-
Catechol 2.6 x 10-8 Indirect
Dopamine 4 x 10-3 Manometric** tory nature. Essentially identical spectral data were
Chlorogenic acid 3 X 10-2 " also obtained for the enzymic and chemical oxida-
* Average of at least 2 determinations with either de- tion products from DOPA, in agreement with Mason
tergent extracts or purified extracts. No significant (17).
differences were noted for the 2 different prepa- Based on this spectrochemical evidence, the re-
rations. action mechanism shown in figure 4 is proposed for
** Assay conditions: 0.025 M potassium phosphate, pH the enzymic oxidation of dopamine by banana PPO.
7.0; 0.2 ml enzyme (undiluted detergent extract); It is analogous to that proposed by Mason (18) for
0.2 ml 5 % KOH (center well) ; substrate and water
to the final volume of 2 ml. DOPA oxidation.
 512 PLANT PHYSIOLOGY

One other possible mlechanisnm for the production It is significant that dopamine is the most reac-
of melanins in banana extracts was considered. tive substrate for the banana enzyme since this com-
Snlith and Kirshner (21) reported the presence of pound occurs at exceptionally high levels (1-2 mgng
an enzyme (termed dopamine /3-oxidase by Senoh fr wt) in the peel of the banana fruit (E. H. Buckley,
et al. (19)) in bananas which converts dopamine to unpublished) and has been shown to be the only
arterenol. Since arterenol is a substrate for banana significant substrate in the browning reactions of
PPO (table III), this compound could be the actual this fruit (13). To the author's knowledge, this is
substrate for melanin formation in banana extracts. the clearest link between the substrate specificity of
Furthermore, the initial oxidation products of arte- a phenol oxidase from a particular plant material and
renol would have spectra essentially identical to the denmonstrated substrate for browning reactions in
those derived from dopamine. that material. Further study of this unique relation-
The dilute(l detergent extracts contained sut- ship could help to answer some of the problems con-

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ficient dopamine 3-oxidase to convert up to 20 % of cerning the synthesis, localization, andI role of phenol
the original dlopanmine to arterenol in 1 hour, when oxidases in plant tissue.
assayed by the technique of Smith and Kirshner Dopamine is also the primary substrate for PPO
(21). However, because of the markedl difference in the roots and rhizome of the banana plant (M. E.
in the Km of the 2 compounds, it is unlikely that Mace, unpublished). Although it occurs in a variety
arterenol plays any significant role in the in vitro of other plant tissues (23). it has seldom been tested
ssystemii. For example. if w e nmake the unlikely as- as a substrate for phenol oxidase. Dopamine has re-
sumption that 10 % of the dopamine in the standlardI cently been shown to be the most reactive substrate
polyphenoloxidase assay -as instantaneously con- (Kill = 2 X 10-4 At. spectral assay at 465 mL) for
vertedl to arterenol by the dopamnine P-oxidase pres- the purifiecl PPO from blowfly larvae, where it ap-
ent, the concentration of arterenol would be only 5) pears to be a key intermedliate in sclerotization (14).
X 10-4 .r. No significant pigment formation re- This comlpound could prove to be important in the
sultedl when this concentrationi of artelrenol was in- brownvling reactions of many plant and animal tissues.
cubate(l w ith banana PPO. These observation. (0lo There are 2 other noteworthy points regarding
not preclude the possibility that both mechanisms may the substrate specificity of banana PPO. First, al-
occur in living tissue. though (lopamiiine and L-DOPA (liffer chemically
only by the presence of a carboxyl group on the
Discussion .idlechain, the Km values for these 2 compounds dif-
fer by a factor of 100. Second, in contrast to other
Phenol oxidlases are copper proteins of wvide oc- phenol oxidases, D-DOI'A appears to be a more
currence in nature wlhich catalyze the aerobic oxidla- reactive substrate than L-DOPA. Additional study
tion of certain phenolic substrates to quinones wshich will be requiredl to assess the significance of these
are autoxilizeed to dark brown pignments generally observations.
know-n as melanins. For discussion purposes. these
enzymes are assume(l to be single enzymes with Summary
broad specificity, although there is sonme evidlence
for the presence of more than one phenol oxi(lase in Polyphenoloxidase has been shown to occur in
certain tissues (4). the pulp and peel of the banan<a fruit. It can be
The phenol oxidase extracted w-ith dletergent froml readily extracted from the pulp an(d rendered soluble
the pulp of the ripe banana fruit is by substrate spe- in buffered detergent solution. These preparations
cificitv a polyphenoloxidase an(l is sinmilar inl this were essentially colorless, and had little or no en-
respect to the enzynmes of sweet potato (7, 24), (logenous activity. The enzyme was further purifie(d
tobacco (4), tea leaf (24), and(I Solidago virgaufrca by acetone precipitation and chromatography on
leaf (2). In contrast, the enzyNmes fromimost other DEAE-cellulose.
.sources such as potato, mushrooml, anid several mlam- The soluble enzy-me catalyze(d the oxidation of a
malian tissues are tvrosinases (or true tvrosinases) variety of diphenolic substrates. Km values for a
w-hiclh catalyze the oxidationl of both imiono- anid niumiiber of these were (letermine(l and thev indicate
(liphlenols (24). that (lopaminine is miiost readily oxidized. Banana
Yasunobu (24) has recently reviewe(d the sub- lpolyphenoloxidase in detergent extracts was relative-
strate specificity of a number of phenol oxidlases lv stable and hadl optimum activity at pH 7 with
from various sources, both plant and animal. He (lopamiiine as the substrate. The miiechanismii of (lop-
concluded that these enzymes catalyze the oxidation amiiine oxidation appears to be analogous to that for
of a wride variety of substrates, but that each indi- the oxidation of DOPA by tyrosinase.
viclual enzy-me tends to catalyze the oxidlation of one
particular phenol (or a particular type of phenolic Acknowledgments
compound) more readily than others. The results The author is indebted to Dr. G. R. Mandels for con-
reported here show that banana PPO follows this tinuing advice and encouragement and to Mrs. Anne
general pattern, with (lopamine being the most rea(lilv Desmond and Miss Leslie Short for technical assistance.
Miss Short was a participant in the Summer Science
oxidizedl substrate. Program of Thayer Academy, Braintree, Massachusetts.
 PALMXrER-BANANA POLYPHENOLOXIDASE 513
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