APPARENT REVERSAL OF DENATURATION OF PROTEINS
203
(10) PHILPOT, J. S. L.: Nature 141, 783 (1938). R. A., AND CHOBOT, R . : J . Biol. Chem. 92, 569 (1931). (11) STULL,A., COOKE, A,, COOKE, R . A,, AND CROBOT, R . : J. Allergy 3,341 (1932). (12) STULL, A , , SHERMAN, W., AND HAMPTON, S . F.: J. Allergy 12, 117 (1941). (13) STULL, T . , AND PEDERSEN,K . 0.: The Ultracentrifuge. Oxford University Press, (14) SVEDBERG, London (1940). (15) SVENSSON, H . : KolloidLZ. 87, 180 (1939);90,141 (1940). (16) TISELIUS, A.: Trans. Faraday Soo. 93, 524 (1937).
THE DENATURATION O F PROTEINS AND ITS APPARENT REVERSAL. 111' HANS NEURATH, GERALD R. COOPER,
AND
JOHN 0. ERICKSON
Departmnt of Biochemistry, Duke University School of Medicine, Durham, North Carolina Received October $8, 1941
A number of investigations, purported to demonstrate protein denaturation to be reversible, have been published in recent years. In view of the fundamental importance of the problem for considerations of protein structure, it appears appropriate to undertake a critical examination of the data at hand, and to attempt to decide whether and to what an extent denaturation can be reversed at all. In consideration of the limited space available for the contributions to this Symposium, the discussion will be confined to aspects of the problem as they have evolved from the inyestigations reported in the preceding papers of this series (18, 19) and from previous work on reversible protein denaturation. In the face of limited experimental evidence, such a discussion cannot be entirely free of speculations. Denaturation may be defined as some intramolmular rearrangement of the protein giving rise to definite changes in chemical, physical, and biological properties (1). This rather loose definition is given a clearer meaning by stating the most important changes which have been recognized to accompany denaturation. These are (1) a loss of biological specificity, (8) a decrease in solubility, (3) a loss of crystallizing ability, (4) an apparent increase in the number of specific groups, such as sulfhydryl, disulfide, or phenolic groups, and ( 5 ) an increase in the relative viscosity of the solution of a protein and a corresponding decrease in its diffusion constant, indicative of an increase in apparent molecular asymmetry. A protein having undergone all these changes may be said to be denatured. I t is probably denatured if it has undergone any one of the last three changes, but it is questionable whether denaturation has occurred if any one of the first two changes alone has taken place. A more precise definition of denaturation, as of any chemical reaction, necessitates not only qualitative Presented at the Eighteenth Colloid Symposium, which waa held at Cornel1 University, Ithaca, New York, June 19-21, 1941.
204
E. NEURATH, 0. R. COOPER AND J. 0. ERICICBON
but also quantitative considerations. While a protein may be either biologically active or inactive, crystalline or amorphous, there appears to be a gradation in the changes of such properties as apparent molecular asymmetry, the number of detectable specific groups, or solubility. T h p is clearly indicated by the variations in the increase of the number of detectable sulfhydryl groups following denaturation by heat, urea, or guanidine hydrochloride (12), aa well aa by the findings that the apparent molecular asymmetry of a protein increases in a like manner with increasing concentration of a given denaturing agent (8, 18, 19, 20). If there are varying degrees in the effects produced, then there must be considered to exist varying degrees of denaturationa. The term “denaturation” remains ambiguous, unless the nature and magnitude of the changes that have occurred can be recognized. For the purpose of discussion, one may confine the considerations to the maximum changes which a denaturing agent under a given and well-defined set of experimental conditions is capable of producing. This, then, may be regarded aa the final (denatured) state, and that in which the protein exieted prior to denaturation aa the initial (native) state. In order to demonstrate reveraal of denaturation it must be shown that the protein waa denatured in the first place, and that all the reactions which accompany denaturation are themselves reversible. If some reactions are found to be reversible and others irreversible, this indicates that the internal structure of the “reversed”* denatured protein is no longer identical with that of the native, and denaturation is actually irreversible. Reversible denaturation by heat haa been studied by Northrop (21) and h o n and Mirsky (3) for trypsin, by Kunitz and Northrop (15) for chymotrypsin, by Herriott (13) for pepsinogen, and by h o n and Mirsky (2),Miller (16), and Hewitt (14) for serum albumin. Anson and Mirsky (4) have investigated the reversible denaturation of hemoglobin by sodium salicylate and the reversible denaturation of serum albumin by urea (5). In these studies, reversal of denaturation was mainly followed by measurements of change8 in biological activity and in solubility. In the last two casea denaturation was followed slso by spectroscopic measurements and by estimations of reactive protein disulfide groups, respectively. Recently the authors have investigated the apparently reversible denaturation of crystalline serum albumin and amorphous pseudoglobulin of normal horse serum by urea and guanidine hydrochloride (18,19). The extent of denaturation waa followed by measuring the increase in relative viscosity and the de-
’
We consider intramolecular changes ea the main criterion for denaturation. Thb definition excludes the reversible splitting of native protein molecules into smaller units, or reactions with groups on the surface of the molecules which leave their internal structure intact. * The term “reversibly” denatured is employed here mainly in order to conform to the terminology of the literature and not as an accurate description of the process. In the present usage, “reversibly” denatured protein denotes that fraction which, following reveraal of the conditions used for denaturation, approximates most closely the properties of the native protein.
205
APPARENT REVERSAL OF DENATURATION OF PROTEINS
crease in the diffusion constant, as produced by varying concentrations of the denaturing agents. Apparently reversed denatured protein was compared with the native with respect to crystallizing ability, solubility, molecular size and shape, and electrophoretic properties (22, 23). The results of some of these measurements are summarized in table 1. Diffusion measurements on the urea-denatured solutions indicated that, with both serum albumin and pseudoglobulin, all molecules were affected to the same extent by the denaturing agent. Such a uniform mode of action is probTABLE 1 Molecular comtants of native, denatured, and reversibly denatured serum albumin and pseudoglobulin fractions (18, 19)
!!?!
= limiting slope of the specific viscosity versus concentration curves; D' = diffusion
-
constant, corrected for the viscosity of the solvent; (b/a)h = apparent ratio of the molecular axes, assuming 33 per cent hydration; M anhydrous molecular weight, calculated from diffusion and viscosity data
~
I.p M (b/a)n *io? c -~ ___
yo?
Native. . . . , . .
4.30
7.0
3.3
71,900
Native. . . . . . .
In 8 M urea. .
22.25
4.13
13.3
77,800
In 8 M guanidine hydrochloride. . . .
31.55
3.74 I 16.7
df
4.75
5 . 2 170,000
In 5 M urea.. 14.0
3.73
9.3 1 6 2 , ~
86,800
I n 8 M urea.. 28.0
2.97
15.4 170,000
Reversibly denatured by 8 M urea.. .
4.69
4.7
Reversibly denatured by 8 M urea. . .
4.10
7.17
3.1
70,3M)
Irreversibly denatured by 8 M urea
5.90
6.74'
4.7
M,W
* Limiting
Wah
--6.80
5.90
190,000
value.
ably typical for protein denaturation, although it cannot always be recognized. Thus, biological activity or spectroscopic properties follow the all-or-none principle and are devoid of gradation. Changes in solubility or in the number of detectable sulfhydryl groups, however, may be expected to follow the same pattern observed for the changes in molecular asymmetry and are probably manifestations of the primary changes in internal structure. The experiments on heat inactivation of trypsin, chymotrypsin, and pepsinogen, and on heat denaturation of serum albumin, indicate that denaturation affected only a fraction of the protein, leaving the remainder in the undenatured state. The discrepancy between these results and those obtained in the denaturation of the
206
H. NEURATH, Q. R . COOPER AND J. 0. ERICKSON
serum proteins by urea or guanidine hydrochloride may be ascribed to the fact that, in the latter studies, the protein was exposed to a given concentration of the denaturing agent long enough for the reaction to go to completion; in the former studies, however, the protein was exposed to the denaturing agent for a short time. For the conditions to be comparable, the protein would have to be heated, for instance, at each of a series of temperatures over a long period of time, and it is then that heat denaturation would probably exhibit the uniform behavior observed in the urea studies. If the denaturation were quantitatively reversible, reversal of the conditions imposed for denaturation should result in a return of all protein molecules to the
PSEUDOGLOBULIN
I
2
4
6
8
1
0
M O L A R I T Y OF UREA OR GUANIDINE HCL
MOLARITY OF UREA OR GUANIDINE HCL
FIG.1 FIQ.2 FIG.1. The fraction of total serum albumin irreversibly denatured after dialysis, plotted against the molarity of urea or guanidine hydrochloride a t which denaturation occurred. Full circles refer to urea, triangles to guanidine hydrochloride (18). FIG.2. The fraction of total pseudoglobulin irreversibly denatured after dialysis, plotted against the molarity of urea or guanidine hydrochloride a t which denaturation occurred. Open circles refer to urea, full circles to guanidine hydrochloride (19).
condensed configuration. However, it has been found that, with both serum albumin and pseudoglobulin, removal of the denaturing agent by dialysis resulted in the precipitation of a fraction of the protein under conditions where the native protein was soluble (18, 19). The size of this fraction increased with increasing concentrations of the denaturing agent originally present, as seen in figures 1 and 2 for serum albumin and pseudoglobulin. These results may be correlated with those obtained from the viscosity and diffusion measurements. There each concentration of the denaturing agent produced a definite change indicative of a corresponding increase in molecular asymmetri. If we picture the increase in molecular asymmetry as being due to Some sort of unfolding of the protein molecule, we may conclude that, the greater the extent of unfolding, the smaller the fraction which returns to the condensed
APPARENT REVERSAL OF DENATURATION OF PROTEINS
207
configuration. The sigmoidal shape of the curves shown in figures 1 and 2 suggests a probability factor governing the return from the extended to the condensed configuration. The statistical nature of the phenomenon was also indicated by subjecting the reversed protein once again to denaturation. About the same distribution between irreversibly and apparently reversibly denatured fractions was found as was observed in the analogous studies on the native protein. The incomplete reversal of the molecules of the irreversibly denatured fraction to the condensed configuration is undoubtedly due to irreversible secondary reactions of either intermolecular or intramolecular nature. While with pseudoglobulin molecular-weight or shape studies on the irreversibly denatured fractions were inhibited by gel formation, it was found that the average molecular weight of the irreversibly denatured serum albumin did not differ very greatly from that of the native. Here, intramolecular changes appear to be the main cause for irreversibility. The relatively low viscosity of the irreversibly denatured serum albumin, as compared with that of the denatured in the presence of urea (table l),suggests that removal of the denaturing agent produced internal strains in the molecules which caused them to recoil to a less extended configuration. The difference in the quantitative distribution between reversed and irreversed fractions found for serum albumin on the one hand, and pseudoglobulin on the other, is indicative of the difference in internal structure that exists between these two proteins. Denaturation of both proteins is reversible to the extent that the reversed denatured molecules exhibit about the same size and shape as the native molecules from which they were derived (table 1). Yet the intrinsic structure of the native proteins was changed upon reversible denaturation. With serum albumin this was evidenced by a decreased tendency to crystallize, a higher solubility in sodium sulfate (18),and differences in electrophoretic mobilities (23). In the case of the amorphous pseudoglobulin, the observed spread in electrophoretic (22) and solubility (19) patterns was about equal for the native and the reversibly denatured protein (figure 3). However, differences have been observed in respect to electrophoretic mobility (22). In the light of these findings, denaturation of these proteins under the conditions described must be considered as an irreversible process. The changes in relative dimensions of the molecules, involved in apparent reversal of denaturation by concentrated urea solutions, may be interpl;$ed as a long-range elasticity comparable in magnitude to that seen with keratin or myosin (6). This indicates that the denatured molecules must still possess a high degree of internal cohesion, since otherwise they would not exhibit the tendency to recoil to a condensed state. Also, the relative dimensions of the denatured molecules, as calculated from viscosity measurements, are of much lower order of magnitude than those they would exhibit if they were isolated, fully extended polypeptide chains (8). The question of the nature and site of the attractive forces responsible for the contraction is at present still a matter of conjecture and touches the fundamentals of protein structure.
208
H. NEURATE, 0. R. COOPER A N D J. 0. ERICKBON
If the mode of foldingof the polypeptide chains in the protein molecule were determined solely by the properties of the main chains and uninfluenced by the
Fro. 3. Electrophoresis curves of native pseudoglobulin (top of figure) and “reversibly” denatured pseudoglobulin (bottom of figure) at pH 7.6 in solutions of 0.1 ionic strength. D denotes migration into the protein solution. Curves taken from reference 22.
nature of the side chains, one would expect, for all proteins, a specific and universal structure which conceivably could be restored upon reversal of denatura-
APPARENT REVERSAL OF DENATURATION OF PROTEINS
209
ion. If, however, the internal structure is also guided by attractive forces operating between the side chains, strict reversal would be improbable, in view of the large number of ways in which the side chains can interact. The importance of the side chains as a structure-determining factor has been emphasized by Bull (g), and their function in reversible contraction is clearly illustrated, for instance, by comparison of the physical properties of silk and wool (6). This point has been stressed recently also by Astbury4,who has shown how, in a newly derived type of folded polypeptide chains, the side chains are crowded together in groups of three, thus producing most effective conditions for mutual attraction. Apparently, side-chain attraction plays a dominant r61e in protein structure. It remains to be decided whether the structure of the denatured protein is very specific or whether the transformation from the native condensed configuration to the extended denatured configuration involves a significant change in entropy (10). In the former c m , protein denaturation could be strictly reversible, whereas in the latter it would be improbable that the specific configuration of the native protein could be restored from a much less specific state of the denatured protein. This problem has been approached by Mirsky and Pauling for the reversible heat inactivation of trypsin (17). From the data of Northrop (21) and of Anson and Mirsky (3) they calculated a value of 100 E.U. for the entropy difference between the active (native) and inactive (denatured) state which corresponds to a value of 1020 for the number of accessible configurations of the inactive protein. While Mirsky and Pauling consider this as indicative of a much less completely specified state, devoid of a uniquely defmed configuration, it appears that the magnitude of the changes involved in this particular reaction is of a surprisingly low order for a compound of high molecular weight. For a rough estimate we may represent a protein molecule by a crude model, containing a great number of vibrational frequencies vi. These we shall a w e to be classical oscillators at the temperature in question. We shall blame the changes in entropy, occurring as a result of the inactivation, entirely on the changes in vibrational frequency, to see what changes in frequency are needed to account for a given entropy change. It is not inferred that changes in vibrational frequency are the only ones occurring during heat inactivation; this over-simplified concept is introduced merely for an estimate of the maximal effects. The relation between entropy change and frequency may then be expressed a s 6 (11)
Here A8 is the entropy change per molecule, k the Boltmann constant, f the number of internal degrees of freedom, T the temperature, h the Pbnck con4 We are greatly indebted to Dr. Aetbury for sending UB the manuscript of a paper on thin subject. The authors are greatly indebted to Dr. F. London for disouMionn of this sspeot of the problem.
210
H. NEURATH, G . R. COOPER AND J. 0. ERICKSON
stant, v' the vibrational frequency in the final state, and v the vibrational frequency in the initial state. If we define the change in frequency as
- 6v
= v'
-v
then
For an entropy change per mole of A S = 100 E.u., and a value off about6 10,000, -6viIvi becomes about 0.005; that is to say, the changes in vibrational frequency do not exceed 0.5 per cent. This extremely low value does not change appreciably even if the value for A S were in error by as much as 100 per cent. If, therefore, the heat inactivation of trypsin involves such a small extent of opening up in protein structure, the reversal of this process is hardly surprising. In fact, one wonders whether the protein has been denatured at all or whether heat treatment under the conditions employed in these experiments did not merely inactivate the enzyme without denaturing the protein. The observations reported by Northrop (21) indicate this heat denaturation to be a somewhat peculiar reaction. T h y , inactivation is reversible in the pH region 1 to 7 if the protein is heated to 100'C. for not more than 5 min., whereas inactivation is irreversible at higher pH values. Inactivation at temperatures between 50' and 70°C. is also irreversible, at pH regions over which it is reversible a t higher or lower temperatures. These facts emphasize the need of further quantitative measurements on the effect of heat on the physical and chemical properties of the enzyme' in order to ascertain whether denaturation, in the sense discussed in the introduction to this paper, has occurred a t all. Attention may be drawn also to the studies of Bawden and Pirie (7) on tomato bushy stunt virus, in which it was shown that, from preparations which had been completely inactivated by exposure for 30 min. to a temperature of 55"C., inactive cr-tals could be obtained which were indistinguishable in shape from those of active preparations. Here, inactivation and denaturation apparently were not related to one another. The experiments by h s o n and Mirsky (2) on the reversible denaturation of serum albumin by heat or by acid acetone have been criticized by Hewitt (14) on the ground that there was insufficient evidence for the protein having been denatured under the conditions chosen in their work. Assuming denaturation to have occurred, reversal would be conceivable if the heat treatment were to involve entropy changes of the same order of magnitude as those observed for trypsin. This is quite possible, as the heat treatment was carried out in both cases under similar conditions, i.e., for a short period of time and in acidified solutions. In these cases, the heat-treated protein would still be in a rather specified state, and upon cooling might return to the specific structure in which Assuming about 3500 atoms per molecule. Experiments on the effect of heat on certain physical and chemical properties of globular proteins are under way in this laboratory. 6
7
APPARENT REVERSAL OF DENATUUTION OF PROTEINS
211
it existed originally. The entropy changes involved in denaturation by urea or guanidine hydrochloride are probably of a considerably higher order of magnitude. The drastic changes in molecular shape produced by these denaturing agents testify to this assumption, and under these conditions denaturation is essentially irreversible. SUMMARY
Certain aspects of the problem of reversible protein denaturation, including the meaning of the term “denaturation”, the criteria for its reversal, and the significance of these phenomena in relation to protein structure, have been discussed. No absolute evidence for strictly reversible denaturation is available. - Denaturation of serum proteins by concentrated urea or guanidine hydrochloride solutions are concluded to be irreversible processes. The authors are indebted to the Rockefeller Foundation, the Lederle Laboratories, Inc., Pearl River, New York, and the Duke University Research Council for support of this work. REFERENCES
(1) ANSON,M. L.: In The Ckrnislry of the Amino Acids a d Proteins, edited by C . L. A. Schmidt,, pp. 407 ff. Charles C. Thomas, Springfield, Illinois (1938). (2) ANSON,M. L., A N D MIRSKY, A. E.: J. Gen. Phyaiol. 14,725 (1931). (3)ANSON,M. L.,IND MIRSKY,A. E.: J. Gen. Physiol. 17, 393 (1933-34). (4) ANSON,M. L.,A N D MIRSKY,A. E.: J. Gen. Physiol. 17,399 (1933-34). (5) ANSON,M.L., AND MIRSKY,A. E.: J. Gen. Physiol. 19,427 (1936). (6) ASTBURY, W.T.: Trans. Faraday Soc. 34, 377 (1938);Compt. rend. trav. lab. Csrbberg, SBr. chim. 22,45 (1938). ASTBURY, W. T.,AND DICKINSON, s.: PrOC. Roy. SOC. (London) B129, 307(1940). (7) BAWDEN, F. C., AND PIRIE, N. W.: Biochem. J. 34, 1278 (1940). (8) BULL,H . B.: J. Biol. Chem. 139, 39 (1940). (9)BULL,H.B.: In Advances in Enzymology, pp. 1 ff. Intemcience Publishers, Inc., New York (1941). (10) EYRINO,H., A N D STEARN, A. E.: Chem. Rev. 24, 253 (1939). R. H.,AND GUOGENHEIM, A. E.: Statistical Thermdynarnics. Cambridge (11)FOWLER, University Press, Cambridge (1939). (12)GREENSTEIN, J. P.: J. Biol. Chem. 126, 501 (1938);128,233 (1939);130,519 (1939). (13) HERRIOTT,R. M.:J. Gen. Physiol. 21, 501 (1937-38). L.F.: Biochem. J. 28, 575 (1934). (14) IIEWITT, (15)KUNITZ,M., AND NORTHROP, J. H.: J. Gen. Physiol. 18,433 (1934-35). (16)MILLER,B. F.: J. Exptl. Ned. 68,625 (1933). (17) MIRSKY,A. E., AND P A ~ L I K L.: G , Proc. Natl. Acad. Sci. U. 5.22,439 (1936). H., COOPER,G.R., AND ERICKSON, J. 0.: J. Biol. Chem., in press. (18) KEURATH, (19)NEURATH, H., COOPER,G. R., AND ERICKSON, J. 0.: J. Biol. Chem., in press. (20) NEIJXATE,H., AND SAUM,A. M.: J. Biol. Chem. 128,347 (1939). (21) NORTHROP, J.H.: J. Gen. Physiol. 18,323 (1932-33). (22) SHARP,D.( I . , COOPER, G. R., AND NEURATH, H.: J. Biol. Chem., in press. (23) SHARP,D. G., COOPER,G. R., ERICKSON, J. O., AND NEURAIH. H.: Unpublished experiments.
