JOHN HOLME
782
I n most instances the difference between calculated and experimental results is within a few hundredths of a unit and in no instance do they differ by more than 0.04. The substitution of n log a, for the BM term in eq. 4 led to a poorer fit of the experimental data. The addition of an n log a, term did not result in a significantly better fit. This is unlike the situation for uncharged bases.lS The log QBH values obtained for formic and acetic acids in LiCl are different (see Tables I and 11). These results indicate that, if an acidity function, H-20 (19) D . Rosenthal and J. S. Dwyer, Can. J . Chem , 41, 80 (1963); K. N. Bascoinbe and R. P. Bell, Discussaons Faraday Soc., 24, 158 (1957).
Vol. 67 f€I+fB-
PKBH - log- [BH 1 = -log [H+]-[B- 1 fBH -log [H+l - log QBH (9) is defined in terms of a particular indicator or series of indicators for LiCl solutions, the degree of protonation cannot be calculated directly from H - measurements using eq. 9. The difference between the log QBH values for the H- indicator and the acid in the particular If an H - scale is LiCl solution also must be to be defined for LiCl solutions, the indicators used must more closely resemble each other than do formic and acetic a c j d ~ . ~ , 2 ~
H-
=
(20) M. 1.Paul and F. A. Long, Chem. Rev., 57, 1 (1957).
THE THERMAL DENATURATION AKD BGGREGATIOX OF OVALBUAIIS BY JOHN HOLME M i a m i Valley Laboratories, T h e Procter and Gamble Company, Czncznnati 39, Ohio Received September 12, 1962 The techniques of optical rotation, viscosity, ultracentrifugation, electrophoresis, and solubility have been used to study the effect of heat treatment on the properties of ovalbumin in aqueous solutions of p H 5.5, 7.0, and 8.5. The results have been considered as they relate to the possible sequence of events in any denaturation experiment; native denatured -+ aggregated. The monomeric form of the protein which remains after heating has been characterized by viscosity and sedimentation measurements and has been found t o be hydrodynamically equivalent t o the native protein. S o evidence for the existence of a denatured monomeric form of ovalbumin in heated solutions has been found. Aggregation of the protein has been found t o be rapid and extensive with heating over this pH range. The sedimentation constants of the aggregated form were dependent upon the pH (decreasing with increase in p H ) and the ionic strength (decreasing with salt concentration) a t which the aggregate was formed. The aggregation of the protein gave rise t o increases in viscosity and turbidity which were also dependent upon pH (increasing less a t higher pH). The aggregated protein had an electrophoretic mobility a t pH 7.0 and 8.6 greater than that of the original protein.
Introduction The denaturation of ovalbumin has been studied by many workers utilizing many physical techniques. I n general, these reports indicate that denaturation, defined here as an “intramolecular configurational change’’ in accord with present views,1o does occur prior to aggregation of the protein. This conclusion has been given for the urea denaturation of ovalbumin by Frensdorff, et aZ.,3and Steven and Tristram.ll illIany proteins shorn an increase in levorotation when they are exposed to certain environmental conditions. Based upon the relation of a. change in this property to the helix-coil transformation found for certain synthetic polyamino acids,lPthe technique of optical rotation is commonly employed to study protein denaturation. However, recent work by a number of pe~ple’~-l’has pointed out the difficulties in assigning (1) H. A. Barker, J . B i d . Chem., loa, 1 (1933). (2) R. B. Sirnpson a n d W. Kauzrnann, J . Am. Chem. SOC.,76, 5139 (1953). (3) H. K. Frensdorff, M. T. Watson, and W. Kauzmann, i h i d . , 75, 5157 (1953). (4) J. Schellman, R. B. Sirnpson, a n d W. Kauzmann, i b i d . , 75, 5152 (1953). (5) A. S. Tsiperovich. Ukr. Biochem. Zh., 21, 44 (1949). (6) C. F. C. RlacPherson and &I. Heidelberger, J . A m . Chem. Soc., 67, 574 (1945). (7) A. Rothen, Ann. AT. Y . Acad. Sci.,43, 229 (1942). (8) N. F. Burk and D. M. Greenberg, J . B i d . Chem., 87, 197 (1930). (9) F. Haurowitz, F.DiMoia, and S.Tekman, J . Am. Chem. Soc., 74, 2265 (1952). (10) w’. Kauzrnann, Advan. Protein Chem., 14, 1 (1959). (11) F. S. Steven and G. R. Tristram, Biochem. J . , 73, 86 (1959). (12) J. T. Yana and P. Doty, J . Am. Chem. Soc., 79, 761 (1957). (13) G. D. Fasman and E. R. Blout, ibzd., 82, 2262 (1960). (14) E. R. Blout, Proc. Fourth Intern. Cong. Biochem., 6, 37 (1960).
a change in rotation to a particular physical change in the protein and a t this time it appears that much more work is required before the complete significance of rotational changes can be known. The very marked tendency of ovalbumin to aggregate under denaturing conditions has been recognized by viscosity’ and light scattering measurements. l9 Since changes in viscosity can arise from aggregation reactions, it would appear necessary that the absence of any aggregation must be shown before a change in viscosity can be related to an intramolecular change in the protein molecule itself. In order to determine whether a solution of ovalbumin contains native, denatured, and aggregated protein as a result of any given heating experiment, a systematic study was undertaken of changes in optical rotation, viscosity, solubility, ultracentrifugal sedimentation, and electrophoretic properties which accompany heat treatment of ovalbumin under a number of conditions. It was hoped that the process of “denaturation” could be described in such a way that kinetic and thermodynamic treatment of clearly distinguishable features(15) G. E. Perlmann, ibad., 8, 32 (1960). (16) C. Taqford, P. K . De, and U. G. T a g w r t , J . Am. Chem. Soc., 83, 6028 (1960). (17) A. Todd, “A Laboratory Manual of Analytical Methods of Protein Chemistry,” Vol. 2, edited by P. Alexander and R. J. Block, Pergamon Press, Oxford, 1960, p. 245. (18) C. F. C. XalaoPherson, XI. Reideiberger, and D K, LIoore, J . Am. Chem. Soc., 67, 578 (1945). (19) J. F. Foster and R. C. Rhees, Arch. Bzochem. Bzophgs., 40, 437 (1952).
April, 1963
THERMAL DENATURATION AND
intramolecular configurational changes and aggregation-could be made. Experimental Preparation of Protein Solutions.-Crystalline lyophilized ovalbumin20 was used throughout. Reagent grade chemicals were used for the preparation of buffer solutions. Protein solutions were prepared by addition of ovalbumin to the required volume of buffer. TTater solutions of protein were prepared in a similar way with adjustment of the p H to the desired value by addition of hydrochloric acid or sodium hydroxide. Since solution of ovalbumin is incomplete and some "surface denatured" material is formed, all solutions were clarified by centrifugation prior to use. Protein concentrations were determined from semimicro Kjeldahl nitrogen analyses ( % Tu' X 6.3). 2 . Heat Treatment.--Protein solutions were divided into 15 or 20-ml. samples, contained in stoppered testtubes, andimmersed in a water bath maintained at, the desired temperature. At intervals of time ranging up to 150 minutes, a tube was removed and cooled rapidly in ice. 3. Viscosity.-The cooled so!utions were equilibrated in a 30" bath for times of 30 to 120 minutes prior to measurement of viscosities. Flow times of all solutions were determined in an Ostwald-Fenske viscometer (100 bore-flow time for phosphate buffer, 59.4 seconds) at 30 =t0.2". Kinetic energy corrections were small and not applied. Since all solutions were clarified prior to heating and care was taken to prevent "surface denaturation" during handling, particulate matter offered no problem except a t pH 5 . 5 . Here, the particulate material formed during heating was removed pr'or to measurement of viscosity. The very viscous gel-like systems produced at the higher temperatures showed less reproducible values than the solutions treated a t lower temperatures. Reduced viscosities were calculated from flow times relative to the buffer control. Throughout this paper, reduced and intrinsic viscosities will be expressed in units of (g. per 100 ml.)-I. Following the determination of viscosity, aliquots of the solutions were removed For nitrogen analysis, optical rotation, electrophoresis and sedimentation measurements. rotations were determined at 4. Optical Rotation.-Optical 5461 A. in a one decimeler cell with a Rudolph precision polarimeter a t room temperature (26-27'). If the turbidity of a solution was sufficient to prevent measurement of optical rotation, the sample was first clarified by centrifugation and analyzed again for nitrogen content. The unheated solutions containing 1.8% protein gave an average specific rotation of -36 i.2". 5 . Ultracentrifugation .-Sedimentation velocity runs were made in a Spinco Model E ultracentrifuge (at 59,780 r.p.ni. unless otherwise stated) maintained at a constant temperature of about 20" by a Spirico temperature control unit. Areas of peaks were determined with a planimeter from tracings of the fivefold enlarged plates, and corrected for radial dilution. The Johnston-Ogston effects were found to be negligible. Sedimentation rates, S , were corrected for viscosity and density according to the equation
AGGREGATION O F OVALBUMlX 0.301
/
0.25
1.
solvent
[I - .jpl
20, w
- %)I A value of 0.749 cc./g. was used for v. 6 . Electrophoresis.--Protein solutions were subjected to electrophoresis at 1" in a Perkin-Elmer Model 3 8 8 apparatus.
Results Heat Treatment in Phosphate Buffer, pH 'I.O.-The increases in reduced viscosity which occur upon heat treatment of 1.8% ovalbumin in phosphate buffer, pH 7.0, are shown in Fig. 1. Stiff, slightly turbid gels result a t the temperatures of 73 and 75". The turbidity in certain cases rendered optical rotation measurements impossible, so that clarification of these particular solutions was carried out by centrifugation prior to determination of rotation. This clarification procedure yielded firm gel-like residues and clear supernatants of (20) Worthington Biochemical Corporation, Freehold, New Jersey; lot
No. 551 used throughout.
783
700
60'
Time of Hooting (min.).
Fig. 1.-The effect of heat treatment on reduced viscosity of 1.8% ovalbumin in phosphate buffer, pH 7.0, 0.1 ionic strength.
much lower reduced viscosity.
Table I lists the
TABLE I EFFECT OF CLARIFICATION ON PROTEIN CONCENTRATIOK AND VISCOSITY OF OVALBUMIN SOLUTIONS HEATEDIN PHOSPHATE
-
BUFFER,pH 7.0 70 Protein-
I
H e a t treatment
70"-
90min. 150niin. 73"- 30 min. 40 niin. 60min.
Original
1 80 1 80 1 80 1 80 1.80
--qred--
Clarified
Original
Clarified
1 51 1 32 1 26
0.324 0 892 1 250 3 15 15 32
0 072 .057 .077 065
1.07 0 82
.os1
changes in protein concentration and viscosity which occurred upon clarification. The ovalbumin remaining in solution shows viscosity behavior not greatly different from that of the native protein. The intrinsic viscosities of the protein remaining in the clear supernatants obtained from solutions heated a t various temperatures are shown in Table 11. The small increases in TABLE I1 EFFECT OF HEAT TREATMENT IN PHOSPHATE BUFFER,pH 7.0, O N INTRINSIC VISCOSITY OF OVAIBUMIN Sample h1 Xative 0.040 150 min. a t 60" .041 150 min. at 62' .040 150 min. at 65" .050" 150 min. a t 70"-clarified .061" Shown to be polydisperse by ultracentrifugation.
[v]noted after heating a t 65 and 70' might suggest that the molecules had undergone a shape change during heating. Ultracentrifugation of these same solutions, however, revealed the presence of 5-10ojo of material which was much faster sedimenting than monomeric protein. Hence, the increased viscosity cannot be assigned to a configurational change. I n those cases where no aggregate was seen, no increase in intrinsic viscosity was noted. The specific levorotation of ovalbumin was increased only slightly (from a value of - 36 to -41 ') after heating for times from 60 to 150 min. a t temperatures of 50, 60, 65, and 70". A value for [CY] of -43' was obtained for the protein after heating for 40 and 60 miii. a t 73' or 30 min. a t 7.5'. The solutions which received heat treatments for times equal to or longer than 40 min. at 70', 30 min. a t 73', and 20 min. a t 75' requircd clari-
JOHN HOLME
784
0 4
io
io
60
do
Id0
I20
Id0
lk0
Time of Heatlng (min.).
Fig. 2.-The effect of heat treatment on the concentration of monomeric (38) ovalbumin in phosphate buffer.
4-1
A
41 24 B
4I t
0
C
.4
.8 1.2 CONCENTRATION
1.6
2.0
(O/o).
Fig. 3.-Concentration dependence of S Z ~ for , ~ , native and heated ovalbumin. ( A ) Ovalbumin in phosphate buffer, pH 7.0; native ( 0 ) :35 component after 3 hours at 60" ( 0); 3s component after 3 hours at 65" ( 0 ) ; 35 component after 3 hours at 70"clarified( x j. (B)Ovalbuniininacetatebuffer,pH5.5; native(0); 3s component after 2.5 hours a t 55' in acetate, pH 5.5 ( 0 ) ; native ovalbumin in veronal buffer, pH 8.5 ( X ) ; 3s component after 2.5 hours a t 60" in veronal, pH 8.5 (0). (C) Ovalbumin in water, pH 7.0; native ( 0 ) ; 2s component after 2.5 hours at 75' ( 0 j ; 45 component after 2.5 hours at 75' (0 ); native ovalbumin iri ph( sphate buffer. pH 7.0 (- - - -).
Vol. 67
fication before the optical rotations could be measured. These small increases in levorotation would not suggest T-ery extensive changes in the configuration of the protein molecule. Ultracentrifugation of the heated solutions revealed the continuous formation of aggregated ovalbumin during heating, this aggregate having a sedimentation rate of 3 405. The remaining protein was 5t 35 component. The areas of the 3 s peaks were measured and the concentrations of 35 material in the solutions mere calculated using a value of 0.001865 for the speGific refractive index increment of the protein at 5461 A.21 Figure 2 shows the concentration of monomer (3s) as a function of time of heating a t the various temperatures. KO material of sedimentation rate lower than 3s n-as seen in any of the heated solutions. The concentration of the 35 component in each solution was next plotted against the measured sedimentation rate. Figure 3a shows the concentration dependence of the sedimentation constants of native ovalbumin and the 3s material in a number of heated solutions. The data clearly indicate that this hydrodynamic property, XZ~,,,",is unaltered by heating. The relation between S Z Oand , ~ concentration is given by S Z ~=, ~X Z ~ -, ~ ~ k c . Here IC = 0.35 and XZO,~O = 3.53. The results obtained after heating in phosphate buffer, pH 7.0, suggest that if irreversible denaturation (an intramolecular configuratjonal change) occurs, it is not extensive since the intrinsic viscosity and intrinsic sedimentation constant of the monomer are apparently unchanged. The optical rotation reveals the only evidence of denaturation and amounts to only about a 7' increase in levorotation. Effect of Protein Concentration at pH 'I.O.-The changes in viscosity and sedimentation behavior following heat treatment of 1% solutions of the protein revealed similar results to those noted at the higher protein concentration. The increases in viscosity were less, as one would expect, and the rate of disappearance of monomer was slower. The aggregate produced had a sedimentation constant of 3 40s. Changes in optical rotation were very small and of rather poor precision, making them of questionable significance. Effect of pH.-Similar heat treatment of 1.8% solutions '\vas carried out in acetate buffer, pH 5.5, and veronal buffer, pH 8.5, both of 0.1 ionic strength. The rate of aggregation was greater in both cases than at pH 7.0, as is shown in Fig. 4. The changes in viscosity and levorotation which occurred at pH's 8.5 and 5.5 are shown in Fig. 5. A marked difference in physical form exists between solutions heated at pH's 5.5 and 8.5, however. At pH 5.5 heating leads to rapid development of turbidity and actual precipitation of the protein. This precipitate can be readily removed by centrifugation and the clarified solution shows oiil~r slight increases in levorotation (- 7-10') and viscosity (vred increases from 0.035 to 0.048 (g./lOO ml.)-I after 150 min. at 70'). The solution clarified after heating 150 min. at 55' (5-1OOjO aggregate produced) contains essentially only 3s material and the concentration €or this 3 s component is identical dependence of Szo,w with that of native ovalbumin, as shown in Fig. 3b. = 3.41 and k = 0.30. Here, &,,O ( 2 1 ) 0 . 1;. Peilmann and L. G. Lonqsvoith J Am. Chem SOL, 70, 271L' (1948).
April, 1963
THERMAL DEKATURATION AND BGGREGITIOK OF OVALBCMIK
At pH 8.5 marked increases in levorotation and viscosity are produced with but very slight turbidity development. Ultracentrifugation of solutions heated a t 65" a t pH 8.5 revealed the presence of two components, 3s and 20s. The dependence upon coiicentration of the of the 35 component remaining after heating for 150 min. a t 60' was identical \Tith that of native protein as shown in Fig. 3b. Here, X20,wo= 3.39 and 12 = 0.31. Heat Treatment in Water, pH 7.0.--Heat treatment of 1.8% ovalbumin solutions in water, pH 7.0,produced the changes in viscosity and levorotation shown in Fig. 6. At 70" and 75" the viscosities of heated solutions are much less than those obtained in pH 7.0 buffer. The changes in [ai,however, are much greater than those seen a t 70 and 75' in buffer. These results would suggest greater denaturation but less aggregation under such conditions if optical rotation and viscosity were valid means for determining the presence of a denatured species. The ultracentrifuge revealed a single 2s peak for native ovadbumin in water, pH 7.0; a 2s peak for solutions heated at 65'; and two peaks, 25 and 4S, after heating at 70 and 75". Figure 7 reveals that the rate of disappearance of the monomeric protein is somewhat slower in water than in buffer solution, pH 7.0. Figure 3c reveals the concentration dependence of for native 017albumin in water, pH 7.0, along with the corresponding data for the monomer remaining in selected heated solutions. After dialysis against phosphate buffer, pH 7.0,the 2s component possessed a rate of sedimentation equal to that of the native protein in the same buffer. Figure 3c also shows the plot of S20,\$ vs. coiicentration for the 45 component. Here the sharp upward curve at low concentrations reveals the marked effect of absence of electrolyte. This 4s component had a LhW. in phosphate buffer, pH 7.0, of 13, indicating that it is a fairly large aggregate although not as large as those produced in the buffered systems. The results show that the monomer remaining after heating in water is also similar in sedimentation behavior to the native protein. Effect of Heat Treatment on Electrophoretic Behavior.-Electrophoretic examination was made of several of the ovalbumin solutions heated in buffers a t pH 7.0 and 8.5. A new, faster-moving electrophoretic component was continuously produced by heating a t both conditions of pH. The fast peak had a mobility of about 2 units greater than the original protein when heated a t pH 8.5. The mobility difference produced by heating a t pH 7.0 was too small (0.5-1.0 unit) to effect complete resolution of the peaks. I n order to determine whether this faster component represented aggregated ovalburnin or a denatured monomer, heated solutions were centrifuged under conditions calculated to remove aggregate with minimal removal of monomeric protein. Typical results are shown in Fig. 8. Here, electrophoretic patterns are shown of the original protein and of solutions heated 30 min. a t 73" in phosphate, pH 7.0, and 120 min. at 65' in veronal, pH 8.5, before and after centrifugation. These and many other unreported examples show that removal of aggregate (proved by ultracentrifugal examination) was accompanied by removal of the fast electrophoretic component. These results lead to the conclusion that no monomeric form different from the
785
1.8 ..
PH 7.0, 6 5 O
--3 e
I
1.6
-
1.4
-
x
)r
ii.2-
!I
\
1.0
0
0.2 0.4~
u
01 0
20
40
60 80 100 Tlme of Heotlng (mln.1.
120
140
IGO
Fig. $.-The effect of heat treatment on the concentration of monomeric (35) ovalbumin in 0.1 ionic strength buffers. 0.25r
0.151
d 0.10
1
/
I
I*
I
A
/
5
, 65'
$0
eb
Id0
I
1
I
d
0.05
O
'
2b
4'0
20
40
I
80 100 Tlme of Heating (mln.).
60
40
I40
l$O
120
190
160
Fig. 5.-The effect of heat treatment on reduced viscosity and optical rotation of ovalbumin in 0.1 ionic strength buffers. Concentration of 1.8y0ovalbumin.
native in electrophoretic properties is present in the heated solutions. Effect of Removal of Aggregate on Other Solution Properties.-Experiments described in the preceding sections have shown that the removal of aggregate (by centrifugation) lowers the viscosities of heated solutions to values comparable t o those of the unheated samples and quantitatively removes the fast electrophoretic peak. Table I11 indicates the effects of removal of the aggregated protein on these and additional properties of heated protein solutions. It is evident
JOHN HOLME
786
Before Heating
0.154
Vol. 67 Before Centrifugation
After Centrifugation
pH 7.0 ( 3 0 ' a t 73. ) 75-
70' 65'
RED. 0.05
h 70' 40
65'
pH 8.1 ( 12O'at 65. )
L 4
O ! 0
I
I
20
40
I
6'0
TIME
80
I20
100
OF HEATINQ ( M I N
140
I
160
).
Fig. S.-Descending electrophoretic patterns of heated ovalbumin solutions before and after removal of aggregated protein by centrifugation.
that all molecular properties measured here return to values not greatly unlike those of the native protein when the aggregated protein is removed. The fact that the optical rotation sllso returns to values near the original is consistent with the idea that the properties 65' of the aggregate might determine those properties of the solutions which have been chosen as criteria for denaturation (intramolecular change) of the protein. Additional Tests for the Presence of a Denaturated Monomeric Component. a. Tests for Reversible Denaturation.-Optical rotation measurements of 1.8% ovalbumin in phosphate, pH 7.0, and veronal, pH 8.5, were made during heating a t 60, 65, and 70" for times up to 150 min. and during subsequent cooling to room temperature (cooling was allowed to occur slowly in some cases, rapidly in others). No evidence was found that the optical rotations while the solutions were hot exceeded those values reported earlier which were measured at room temperature, indicating the absence of a reversible effect. 0.4 The viscosity of a 2oj, ovalbumin solution was determined during heating a t 60' for times up t o 150 min. KO indication mas found that the reduced viscosity exceeded the values reported earlier for cooled solutions, 1 - 0 thus failing to support a reversible denaturation step. 0 PO 40 60 80 100 120 140 160 Tlme of Hwtlnp (mln.). b. Insolubility of the Isoelectric Point.-Since irreversibly denatured proteins are commonly understood Fig. 7.-The effect of heat treatment on the concentration of to be insoluble under isoelectric conditions, a number monomeric (2s)ovalbumin in water, pH 7.0. of experiments were performed in which solutions TABLE I11 heated in phosphate buffer, pH 7.0, and veronal buffer, EFFECTO F REMOVAL OF AGGREGATE O X PROPERTIES OF HEATED pH 8.5, were dialyzed against water and 0.3 M sodium OVALBUMIN SOLUTIONS chloride solutions or acetate buffer, pH 5.5. The No. of amount of protein which precipitated was determined components from nitrogen analyses and the final supernatants were Protein Sample conon.a [a] llred EL UC examined by ultracentrifugation. In all cases the A. Phosphate buffer, pH 7.0 loss of protein by such precipitation treatments and the 0,036 1 1 1.8% -35.5" Unheated decrease in area of sedimeiiting peaks were equal Heated 90 min. a t 70°b (-43) 1.8 very high (2) 2 (within 5-lOg;b) to the amount of aggregated protein 0,044 1 1 Up t o speed 40,000 r.p.m. 1.1 -33.6 1.1 -34.6 0.036 1 1 30 min. a t 40,000 I?p.m. known exist in the heated solutions. The heating con1.1 0 , 0 3 6 1 1 -27.1 120 min. a t 40,000 r.p.m. ditions examined in such a manner included 2 and 3 hr. B. Verond buffer, p H 8.; a t 65', 2 hr. a t 70", and 2 hr. at 75'. An additional 0.035 1 1 1.7%' -39.0' Unheated 0,317 2 2 fractional precipitation experiment was performed in Heated 2 hr. a t 65' 1.7 -53.4 -45.9 0.293 2 2 1.4 1.5 min. at 40,000 r.p.m. which a solution heated 3 hours at 65" in phosphate 0.063 1 1 -40.2 150 min. a t 40,000r.p.m. 1 . 2 buffer, pH 7.0, was dialyzed against water, then 0.3 Ad Solution too turbid to measure CY a Kjeldahl nitrogen x 6.3. XaC1, clarified and analyzed for protein concentration. Protein concentration calcuor see peaks in electrophorepis. This supernatant was then treated with additional lated from area of sedimentation peaks. Fig. 6.--The effect of heat treatment on reduced viscosity and optical rotation of 1.8% ovalbumin in water, pH '7.0.
April, 1963
THERMAL
DENATURATION A N D AGGREGATIOX O F OVALBUMIN
quantities of sodium chloride to give concentrations up to 1.74 M. No precipitation other than a t 0.3 A!! sodium chloride was encountered until L concentration of 1.74 M salt was reached. Since native ovalbumin acted similarly a t the high concentration of salt, no peculiar properties of the heated protein were recognized. Discussion A denatured protein is one in which the three-dimensional configuration of the molecule is considered to be different from that of the native form; generally the denatured state is believed to be a state of less order. The process of denaturation is one during which this change in configuration occurs and leads to a form of the protein which may then aggregate. In order t o characterize the nature of the configurational change it is essential that the denatured form of the protein be isolated. The properties of this molecule must then be such as to explain the changes in solution properties one sees during the denaturation process which produced this species. The present study has included the application of a number of physical techniques to follow the thermal denaturation and aggregation of ovalbumin. Changes in properties commonly associated with the formation of a denatured protein such as optical rotation, viscosity, electrophoretic mobility and solubility have been measured. It has been found that aggregation of the protein occurs simult~~neously with changes in these properties making it difficult to assign these changes to an intramolecular process. Heat treated solutions which contain aggregated protein have been subjected t o centrifugation to remove the aggregate. The properties of viscosity, optical rotation, sedimentation rate, electrophoretic mobility and solubility of the monomeric form of ovalbumin remaining in the solutions have been found to be essentially the same as those of the native protein. These observations have led to the conclusion that ovalburnin molecules of significantly different configuration than native do not exist in the heated solutions. Other workers ha,ve reported denatured forms of proteins possessing unchanged hydrodynamic properties. Saenkozz and Strachitskii and F ~ r f a r o v areported ~~ this for ovalbumin and serum albumin based on viscosity measurements. Conne112*has reported a denatured monomeric form of cod myosin which cannot be distinguished ultracentrifugally from the native form. The heat treatment of thyroglobulinz5and bovine serum albuminz6leads to formation of monomeric denatured forms which have the same sedimentation properties as the native molecules. I n the last mentioned case the denatured species was recognized by insolubility a t high salt concentrations a t the isoelectric pH after 2 minutes of heating a t 65’ in phosphate buffer, p1-I 7, 0.2 ionic strength. No evidence of such a form of ovalbumin mas found irl the present work. The literature does not reveal many examples of unequivocal proof of the existence of monomeric denatured form of protein possessing markedly different hydrodynamic properties. Configurational changes are inT. V. Saenko, Ukr. Biokhim. Zh., 24, 196 (1952). I
