DENATURATION CHANGES IN EGG ALBUMIN W I T H UREA,

RADIATION, AND H E A T
BY JANET H, CLARK
(From the Division of Biological Sciences, The University of Rochester,
Rochester, New York)
(Received for publication, August 11, 1943)

The change produced in native proteins by various agents which results in

loss of their characteristic properties and changes in solubility is called denatura-
tion. The term, however, may be applied to structural and physical changes
in the protein molecule which differ widely with the particular agent used.
Urea denaturation differs in many respects from both heat and radiation
denaturation. Urea has a strong dispersivO action on proteins so that when a
high concentration of urea is added to a protein the solution remains clear until
the solution is diluted or dialyzed when a certain amount of insoluble protein is
formed. Hopkins (1) found that the rate of denaturation of egg albumin by
urea varied inversely with the temperature, but Diebold (2) found a positive
temperature coefficient for the denaturation of fibrinogen by urea. Owing to
its dispersive action high concentrations of urea will dissolve proteins coagulated
by the action of heat or radiation, or other denaturing agents. Laporta (3)
reports a certain amount of reversal of denaturation as a result of this disper-
sion by urea. Steinhardt (4) has shown that the functional properties of
hemoglobin and pepsin are retained in urea solution so that the loss of solubility
after treatment with urea may not be a true denaturation.
M a n y observers (2, 5, 6) have found that sulfhydryl groups appear after
denaturation which are not detectable in native proteins. In the case of urea
denaturation the number of SH groups is independent of the protein concentra-
tion and depends on the concentration of urea.
In the presence of urea most proteins split into smaller molecules. Burk and
Greenberg (7) found that in 40 per cent urea hemoglobin had half of its normal
molecular weight but that egg albumin was unchanged. Recently Williams
and Watson (8) found some dissociation of egg albumin into smaller molecules
in 50 per cent urea.
This investigation was undertaken with the idea of comparing the effect bf
different denaturation agents on molecular structure and shape and of studying
the interrelations between the effects of different denaturation agents. In the
course of the study certain new observations on urea denaturation were made.
1The term "dispersion" is used here to denote the opposite of "aggregation" as
in the articles by Hopkins (1).
101

The Journal of General Physiology

102 DENATURATION CHANGES IN EGG ALBITMIN

Methods
The material used was isoelectric egg albumin (0.9 per cent except where otherwise
stated) prepared by the method given in previous publications (9, 10).
Ultraviolet radiation was given by the General Electric Portable A.C. Uviarc.
Measurements of the opalescence of the solution were made by means of the Tyndall
meter and Macbeth illuminometer described in a previous publication (9) and in obser-
vations of the depolarization of the Tyndall beam a polarizing eye piece was used with
the Macbeth illuminometer and readings taken with the nicol in two positions. The
ratio of the dark to the bright component multiplied by 100 gave the per cent de-
polarization.
Optical rotation measurements were made with a Hilger polarimeter giving readings
to 0.01 °. The D line of sodium was used throughout and the experiments were
carried out at room temperature.
Hydrogen ion determinations were made colorimetrically.

3 .3.5~ UREA

2 /
/ .30 % UREA
!

i
O* 10" 20" 30" 40"G.
FIG. 1. Effect of temperature on rate of urea denaturation of egg albumin. Ordi-
nates = opalescence of Tyndall beam in apparent foot-candles. Abscissae = tem-
perature.

RESULTS

Urea Denaturation.--When urea is a d d e d to isoelectric egg a l b u m i n solutions

in concentrations of 10 to 50 per cent there is a shift of p H to a b o u t p H 5.2-5.8
depending on the concentration of urea. Even if these solutions are b r o u g h t to
p H 4.8 t h e y remain clear as long as the urea is present.
If the urea is dialyzed out and the solutions a d j u s t e d to the isoelectric point
there is a precipitate formed and the degree of opalescence was taken as a
measure of the denaturation. C o n t r a r y to the observations of H o p k i n s (1)
the rate of d e n a t u r a t i o n was found to have a positive t e m p e r a t u r e coefficient,
the rate increasing r a p i d l y with t e m p e r a t u r e above 20 ° C. T h e results are
given in Fig. 1 and Table I for solutions standing 2 hours a t t e m p e r a t u r e s
from 4-40 ° C.
The rate of d e n a t u r a t i o n a t 40 ° C. for different concentrations of urea is shown
in Fig. 2. The urea was a d d e d to isoelectric albumin bringing the p H to 5.4-5.6
 JANET H. CLARK 103

and subsequently dialyzed and a d j u s t e d to p H 4.8. W i t h concentrations of

urea less t h a n 20 per cent the rate of d e n a t u r a t i o n was too slow to be a ~
preciable.

TABLE I
Temperature Coe.~cient of Denaturation of 0.9 Per Cent Egg Albumin by Urea
Temperature coefficient
Temperature
30 per cent u r e a 35 per cent urea

10-20 1.33
20-30 2.0 1.66
3O--40 2.5 2.0

DEN AT. AT ,40°C

3 ~ p 05 ~, UREA

t / as i:¢
/ / /

TIME 1 2 HRS.

FIG. 2. Rate of urea denaturation of egg albumin at 40° C. Ordinates opales- =

cence of Tyndall beam in apparent foot-candles. Abscissae -- time of immersion in

water bath at 40 ° C.
TABLE II
Opalescence
pH Urea concentration
6 days 21° 7 days 4 °

ft.-candles ft.-candles
4.8 25 2.35 0.41
30 3.70 1.38
40 5.30 2.00

Although urea d e n a t u r a t i o n is v e r y slow a t 0 ° after 9 to 10 d a y s in the ice

box the following results are obtained (see T a b l e I I ) .
One m a y conclude, therefore, t h a t 20 per cent urea produces practically no
d e n a t u r a t i o n in a 0.9 per cent egg albumin solution. Urea denatures slowly a t
a concentration of 25 per cent. 35 per cent is r a p i d l y effective a t room temper-
a t u r e a n d 30 per cent is rapidly effective a t a somewhat higher temperature.
104 DENATURATION CHANGES IN E G G A L B U M I N

Conflicting results on urea denaturation are undoubtedly due to the fact that
the concentration necessary to produce denaturation varies with the protein
used and its concentration as well as with the concentration of urea and the
temperature of the solution.
Effect of Urea on Heat Denaturation.--It has been stated that urea, because of
its dispersive action prevents the heat coagulation of proteins. I t was found
that in the presence of 50 per cent urea, egg albumin solutions brought to boiling
will not precipitate until the urea is dialyzed out and the p H adjusted to 4.8.
Also it was found that heat-coagulated albumin will redissolve in 50 per cent
urea. However, the protein is dispersed without reversal of denaturation.
Concentrations of 20 to 40 per cent urea did not prevent the heat denaturation
of albumin and indeed seemed to accelerate the denaturation. If tubes con-

TABLE III
Effea of Urea on Heat Denaturation
pH Urea Temperature Opalescence
per cent Jr.-candles
4.8 58 ° C. ½ ram.
4.8 25 58 ° C. ½ ram. 5.2
4.8 30 58 ° C. ½ ram. 6.2
4.8 4O 58 ° C. ½ ram. 6.2
4.8 50 58 ° C. ½ mm.
4.8 58-60 ° C. 10 rain. 1.8
4.8 25 58-60 ° C. 10 min. 4.7
4.8 62 ° C. 3 mm. 1.9"8
4.8 25 62 ° C. 3 mm. 4.4

taining egg albumin only and egg albumin plus 25, 30, and 40 per cent urea,
readjusted to p H 4.8, were heated simultaneously, opalescence appeared first in
the tubes containing 30 and 40 per cent urea. These show some opalescence as
the temperature of the water in which the tubes are immersed reaches 54 ° C.
If the water is brought to 58 ° C. and the tubes then removed the tube containing
egg albumin is still clear and those containing urea show a dense precipitate, the
precipitate being heavier in the tubes containing 30 and 40 per cent than in the
tube containing 25 per cent.
Some results are summarized in Table I I I .
This would seem to indicate that urea in concentrations too low to split the
molecule and disperse the heat-denatured protein lowers the heat coagulation
temperature of egg albumin and accelerates the rate of heat coagulation. Actu-
ally a different interpretation was found to be more consistent with the facts
as it is probable that heat below the temperature of heat coagulation breaks the
urea-protein complex and allows the precipitation of urea-denatured protein.
 JANET H. CLARK 105

Effect of Urea on Radiation Denaturation.--In a previous publication (9) it

was stated that egg albumin exposed to ultraviolet radiation at 4 ° C. remained
clear until heated for a short time to a moderate temperature (400 C.). With
the stronger arc used in these experiments, and consequently less perfect tem-
perature control, there was slight opalescence after radiation on ice, although
this opalescence was prevented by adding 25 to 30 per cent urea to the solutions.
The zone in which opalescence occurs is also narrowed in the presence of urea.
If the solutions are radiated on ice and then put in a water bath at 40° C. the
opalescence that normally occurs is diminished by 25 per cent urea and com-
pletely prevented by 35 per cent (see Fig. 3).

~ NO UREA

3
?'/
~_~25=,; UREA
2 M
tl
, Inl

.4 ,5 6 FH ~UREA 25 30 35

FIG. 3 FIG. 4
FIG. 3• Effect of urea on denaturation by radiation followed by heating to 40° C.
Ordinates = opalescence of Tyndall beam in apparent foot-candles. Abscissae = pH.
FIG. 4. Concentration of urea necessary to prevent aggregation in egg albumin after
radiation and heat (40° C.) at the isoelectric point. Ordinates = concentration of
albumin in per cent. Abscissae = concentration of urea in per cent.

That radiation changes have taken place is evidenced by color and smell and
by the fact that the solutions precipitate when the urea is dialyzed out. As 30
to 35 per cent urea will denature fairly rapidly at 40 ° there will be urea denatura-
tion as well as radiation denaturation, but as the results are the same if the urea
is dialyzed out before heating to 40 ° C. it is probable that the radiation changes
take place in the presence of urea and urea only prevents the flocculation of the
denatured protein• If the concentration of egg albumin is reduced the opales-
cence is abolished by a lower concentration of urea. A 0.45 per cent albumin
solution remained clear if radiated and heated in 30 per cent urea. The con-
centration of urea needed to prevent flocculation after radiation and moderate
heat is proportional to the concentration of the protein (see Fig. 4).
If albumin solutions are radiated at pH 7.4 or 3.8 and heated to 400 C. for an
hour they remain clear but precipitate on being brought to pH 4.8. This pre-
106 D E N A T U R A T I O N C H A N G E S IN E G G ALBLrM-IN

cipitation is prevented if 25 per cent urea is present but the solutions precipitate
if the urea is dialyzed out.
Chick and Martin (11) found heat denaturation to be a reaction between
protein and water with an extraordinarily high temperature coefficient. Heat
coagulation involves two processes, the denaturation of the protein and the
separation of the denatured protein in focculated form.
The coagulation of proteins after exposure to ultraviolet radiation has been
shown (9) to take place in three steps. Step 1 is a change produced by radiation
shorter than ~, 310 m# which takes place at any temperature and any pH. Step
2 is a heat change taking place in the light-denatured molecule with a high
temperature coefficient which takes place at any pH and is effective at a tem-
perature which does not produce any appreciable heat denaturation in the
unradiated protein. Step 3 is the aggregation of the light- and heat-denatured
molecules which occurs only near the isoelectric point.
One may summarize these changes as follows:

Steps in process of denaturation

Denaturingagent
(a) (b) (c)

Heat -- Heat denaturation Separationof denatured

protein in flocculated
form
(a) (b) (c)

Ultraviolet radiation Light dena- Heat change in light-de- Separation in floccu-

followed by moder- turation natured molecule lated form
ate heat

Urea in concentrations below 50 per cent (the concentration at which it splits

the egg albumin molecule) appears to accelerate change (b) in heat denaturation
although the precipitate formed is probably due to urea-denatured not to heat-
denatured molecules. Above 50 per cent it prevents change (c) as the result of
splitting the molecule.
In the case of radiation denaturation, steps (a) and (b) take place when egg
albumin solutions are radiated and subsequently heated in the presence of urea.
The third step, (c), of flocculation is prevented by 35 per cent urea ff the solu-
tions are radiated and heated at pH 4.8 and by 25 per cent urea if they are radi-
ated outside the zone of opalescence. This is a concentration of urea which does
not prevent the flocculation accompanying heat denaturation. As the concen-
tration of urea needed to prevent step (c) in radiation denaturation is propor-
tional to the concentration of protein, there is obviously a urea-protein complex
formed which reacts to radiation and to heat in a way that differs from the
protein molecule alone. The changes may be diagrammed as in Fig. 5. A1-
 jm~mT m CLAR~ 107

though f o r diagrammatic purposes the complex is shown as one molecule of

protein plus one molecule of urea the relative concentration would suggest that
the protein molecule may be enveloped in a shell of urea molecules. According
to this interpretation the urea-protein complex will not aggregate producing
flocculation, hence preventifig step (c) in radiation denaturation, as the tempera-
ture of 40 ° C. used in radiation denaturation is not high enough to break down
the complex. In heat denaturation, however, a temperature of 54° and over
breaks down the urea-protein complex before a heat change per se takes place in
the molecule and there is consequent flocculation of the urea-denatured mole-
cule. When the molecules have been split by urea in concentrations of 50 per

R~
.o ! .oc
UREA
HEAT'+ FLOC.

UREA []

50~ ~ -4' I'~,~JGI~ [ ] E NO FLOC.

UREA

HEAT FLOC.

[P'INATIVE ~ DENAT. C. FLOC.

~ SPLIT [ ] UREA

FIG. 5. Diagram showing interaction of heat, radiation, and urea on egg albumin
molecule at different temperatures and concentrations of urea.

cent or over they are apparently no longer capable of flocculation until the urea
is removed by dialysis.
Tyndall Beam Resutts.--An attempt was made to investigate the effect of
ultraviolet radiation, heat, and urea on the shape of the albumin molecule by
the depolarization of the Tyndall beam. The Tyndall beam from a solution
in which the molecules are roughly spherical and small compared with the wave
length of light is completely polarized. Depolarization is caused by increase
in asymmetry or increase in size of the particles. The results were not satisfac-
tory as the increase in size when opalescence developed at the isoelectric point
completely masked the effect of change in shape. Opalescence less than 1.5
foot-candles was found to be determined more by asymmetry than by change
in size. The results from the clear and slightly opalescent solutions are of
doubtful significance, but are given in Table IV and indicate the following
results.
1. The egg albumin molecule after radiation only has a fair degree of sym-
108 DENATURATION CHANGES IN E G G ALBUMIN

m e t r y except a t the isoelectric p o i n t and the same is true after radiation a n d

heating to 40 ° C.
2. A s y m m e t r y develops with aggregation which is prevented in the presence
of urea.
3. E v e n when there is some degree of aggregation as evidenced b y opales-
cence the presence of urea p r e v e n t e d a n y great degree of depolarization so t h a t
the protein-urea complex tends to remain a fairly symmetrical molecule.

TABLE IV
Depolarization of Tyndall Beam
pH TT.~o [ Total ] Bright Dark Depolariza-
Treatment ~*~ !opalescence compom nt component tion

per cent [ per cent

i
Native albumin 4.8 -- 0.07 I 0.13 0.05 37

Urea-denatured 4.8 35 0.2 0.4 0.10 25

4.8 35 1.3 1.9 0.5 26

Radiation only 4.4 -- 0.85 1.33 0.29 22

4.8 -- 0.14 0.24! 0.1 40
5.4 -- 0.5 0.85 0.18 21

Radiation + urea 4.8 30 0.14 0.36 0.09 25

Radiation + heat 40o + 4.6 25 0.35 0.6 0.12 20

urea 5.0 25 1.0 1.7 0.34 2O
5.2 25 0.57 1.05 0.2 20

Heat 100° at pH 3.8 or 7.4 4.4 m 0.82 1.35 0.28 20

brought to different pH 4.8 1.75 2.5 1.0 40
after heating 5.8 0.92 1.6 0.34 21
5.0 25 2.2 3.4 1.28 37
5.6 25 0.55 0.95 0.17 18

4. H e a t d e n a t u r a t i o n a t a p H outside the zone of opalescence leaves mole-

cules fairly symmetrical until they are brought near the isoelectric point.
5. Urea does n o t p r e v e n t this a s y m m e t r y in h e a t - d e n a t u r e d molecules near
the isoelectric p o i n t in 25 per cent concentration, which is p r o b a b l y due to the
fact t h a t h e a t breaks down the protein-urea complex.
These results tend to confirm the picture of the reaction of the protein mole-
cule and of the protein-urea complex to radiation and h e a t t h a t is given in
Fig. 5.
Optical Rotation.-~The change in optical rotation with h e a t d e n a t u r a t i o n
previously observed (12) was confirmed a n d additional results obtained with
 JANET H. CLARK 109

ultraviolet radiation and urea denaturation. Observations on undenatured

albumin were m a d e over a range of concentration and from p H 3.4 to 10.5. T h e
results are given in T a b l e V.

TABLE V
Optical Rotation of Egg Albumin. Effect of Denaturation and Ckanges in pFl and Concentration

Condition Concentra- pH (Angle of Denatured

tion rotation)
~er cent deg./dm. per Cent
Fresh 1.2 4.8 --0.35 ° --29.0
0.6 4.8 --0.340 --28.0
0.3 4.8 --0.19 ° --32.0
0.15 4.8 --0.10 ° --33.0

Fresh 1.2 3.4 --0.34 ° --28.0

4.8 --0.34 ° --28.0
6.0 --0.340 --28.0
6.8 --0.33 ° --27.5
7.4 --0.32 ° --27.0
9.0 --0.33 ° --27.5
10.5 --0.340 --28.0

Boiled 5 min. 1.2 3.4 --0.60 ° --56.0

6.4 -0.74 ° --61.7 100
7.2 --0.61 ° --50.8
0.6 7.0 --0.60 ° --50.0

Ultraviolet radiation
20 min. (4° C.) 1.2 6.4 --0,39 ° --32.5 35
1 hr. (4° C.) 1.2 6.4 --0.40 ° --40.0 50
20 min. (4° C.) 0.6 7.0 --0.38 ° --32.0
40 min. (4° C.) 0.6 ' 7.0 --0.50 ° --42.0

Urea 25 per cent 0.6 4.8 --0.33 ° --27.5

45 per cent 0.6 4.8 --0.34 ° --28.0
60 per cent 0.6 4.8 --0.43 ° --36.0

As has been previously observed (13) it was found t h a t fresh u n d en at u r ed

albumin shows no v a r i a t io n in optical rotation between p H 3.4 and 10.5. T h e r e
was evidence of a slight increase in optical rotation in a v e r y dilute solution
(0.15 per cent) b u t observations with such dilute solution are subject to large
errors and the increase observed m a y n o t be significant. Boiling for 5 m i n u t es
a t p H 3.4 or 6.4 --~ 7.2 approximately doubled the optical rotation, the increase
being greater, as pointed out b y Barker (12) the closer the p H is to the isoelec-
tric point.
W h e n albumin is exposed to ultraviolet radiation at a t e m p e r a t u r e of 4 ° C. or
110 DENATURATION CHANGES IN EGG ALBUMIN

less, the first step in the denaturation process occurs, the coagulation which
results from radiation being the result of a three-step process (9). The result of
the first step is to increase the optical rotation and the increase is roughly
proportional to the amount denatured as determined gravimetrically by subse-
quent coagulation of the light-denatured protein. Indeed a measure of the
optical rotation of the radiated protein would serve as an excellent and very
simple method for estimating the amount of denaturation following radiation
for this particular protein, and shows a similarity here between radiation and
heat denaturation.
Urea concentrations up to 45 per cent had no effect on optical rotation but
there was a marked increase at 60 per cent urea. At higher concentrations it
was impossible to obtain readings.
Where changes in optical rotation can be checked with ultracentrifuge
determinations of molecular weight it is evident that association of molecules
into larger complexes is accompanied by a decrease in optical rotation and dis-
sociation by an increase. The stable values of optical rotation in all proteins
in the pH range where the molecular size of proteins remains constant and the
increase in optical rotation outside this range is one example of this. Associa-
tion and dissociation, however, are not the only possible causes of change in the
optical rotation values of a protein molecule.
Egg albumin denatured with 40 per cent urea has the normal molecular weight
of 35,000 to 40,000 but it has been reported (8) that there is some dissociation
of the molecule with concentrations of urea above 50 per cent. The change in
optical rotation with urea above 50 per cent is probably accounted for by disso-
ciation of the molecule.
The increase in optical rotation with heat and radiation denaturation, how-
ever, must be due to structural changes and not to change in size. No ultra-
centrifuge results for the molecular weight of egg albumin denatured outside the
pH range where aggregation occurs are available. However, osmotic pressure
measurements show no evidence of dissociation to account for the observed
optical rotation changes.
One may conclude, therefore, that the egg albumin molecule shows an in-
crease in optical rotation as the result of structural changes when denatured by
radiation or heat. Urea denaturation does not affect the optical rotation of
egg albumin~ but splitting of the molecule by high concentrations of urea
increases the optical rotation.

CONCLUSIONS

The extent of urea denaturation depends on the concentration of protein and

urea and also on the temperature of the solution. Egg albumin solutions (0.9
per cent) are not denatured by 20 per cent urea, denature slowly with 25 per
 JA~T ~. CLAm< 111

cent urea, and denature rapidly with 35 per cent urea at room temperature. At
a higher temperature 30 per cent urea is rapidly effective.
Denaturation of the egg albumin molecule by radiation or by heat is accom-
panied by structural changes as evidenced by optical rotation values, but is not
accompanied by association or dissociation of the molecule in the pH range out-
side the zone in which aggregation follows denaturation.
Denaturation of the egg albumin molecule by urea produces no change in
optical rotation until the concentration of urea is high enough to dissociate the
molecule.
In the presence of urea a urea-protein complex is formed in which the protein
is denatured but cannot flocculate because of the dispersive action of the urea.
This prevents flocculation of proteins exposed to radiation and subsequent
heating to 40 ° C. as the urea-protein complex is not broken down at a tempera-
ture of 40 ° C. The presence of urea therefore prevents the flocculation of
proteins denatured by radiation.
The urea-protein complex is broken down by heating to 55-58 ° C. so that the
molecules aggregate at a temperature below the temperature of rapid heat
denaturation. This appears to be an acceleration of heat denaturation or a
lowering of the heat denaturation temperature, but in reality is an effect of heat
on the urea-protein complex which frees the urea-denatured protein and permits
its aggregation.
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1. Hopkins, F. G., Nature, 1930, 126, 328, 383.
2 Diebold, W., and Juhling, L., Biochem. g., Berlin, 1938, 296, 389.
3. Laporta, M., Kolloicl-Z., 1932, 61, 376.
4. Steinhardt, J., Y. Biol. Chem., 1938, 123, 543.
5. Mirsky, A. E., and Anson, M. L., J. Gen. Physiol., 1936, 19, 427, 439.
6. Greenstein, J. P., J. Biol. Chem., 1938, 125, 501; 1939, 128, 233.
7. Burk, N. F., and Greenberg, D. M., J. Biol. Chem., 1930, 87, 197.
8. Williams, J. W., and Watson, C. C., Nature, 1937, 139, 506.
9. Clark, J. H., J. Gen. Physiol., 1935, 19, 199.
10. Clark, J. H., Am..Tour. Physiol., 1925, 73, 649.
11. Chick, H., and Martin, C. J., J. Physiol., 1910, 40, 404; 1911, 4.l, 1.
12. Barker, H. A., J. Biol. Chem., 1933, 103, 1.
13. Almquist, H. J., and Greenberg, D. M., J. Biol. Chem., 1934, 105, 519.