Protein Denaturation
The Journal of Physical Chemistry, Vol. 82,No.
14, 1978 1607
Heat Capacity and Volume Changes of Protein Denaturation Jake Bello Department of Biophysics, Roswell Park Memorial Institute, Buffalo, New York 14263 (Received January 5, 1978)
For protein denaturation A V and AC, are estimated for a denatured state in which hydrophobic side chains remain clustered and out of contact with water. From this hypothesis and model peptide data AV is estimated to be small, in accord with published observation. For a protein of 15000 daltons AC, of disordering is estimated at 1000-2000 cal K-l mol, and AC, of solvation at 1000 cal K-l mol-l, for a total of 2000-3000 cal K-' mol-'. The estimated AC,, is of the same magnitude as the observed AC, values for proteins.
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Introduction The denaturation of a protein is a process of disorganization of the compact, dense, ordered native conformation, with concomitant exposure of some fraction of the normally buried groups. The hydrophobic side chains, in particular, are buried in the native conformation, as has been clearly shown by the now numerous X-ray crystallographic analyses, thus confirming expectations based on the solubilities of hydrocarbons in water. It is not uncommon for authors to write of the exposure of hydrophobic side chains on denaturation of proteins. A number of recent investigations of denaturation have given results inconsistent with the expected thermodynamic consequences of exposing hydrophobic groups. Model hydrocarbon data (review by Kauzmannl) predict a large -AV for exposure of buried hydrophobic side chains, of the order of several hundred cm3 mol-l for a typical protein of about 15000 daltons, while experimental results are near zero or even positive (review by Tanford2). Brandts et aL3 have noted that the volume and compressibility changes for denaturation of ribonuclease are not compatible with the results for model hydrophobes, and have suggested high local concentrations of hydrophobic side chains in the denatured protein. Zipp and Kauzmann4 have noted similar results for myoglobin. Aune et al.5have shown that thermal denaturation does not completely unfold proteins. Secondly there are heat capacity anomalies. Solution of model hydrocarbons in water is accompanied by a large positive ACp, of the order of 50 cal K-l mol-l. Denaturation of proteins is also accompanied by a large positive AC,, e.g., 1220 and 2760 cal K-l mol-I for ribonuclease6 and myoglobin,' respectively. While AC, for the solution of a model hydrophobe decreases with increasing pressure, becoming zero at about 5000 kg cm-z, AC, for protein remains large and positive, and even increase^.^ Also, model compound data lead to the prediction that the temperature of maximum stability of a protein should decrease with increasing pressure, whereas it is observed to i n c r e a ~ e These .~ observations, as well as the central fact about hydrophobes, that they are hydrophobic, suggest that we should seek explanations of the volume and heat capacity effects on the basis of a denatured state in which most of the hydrophobic groups are not exposed to water. There is much reason to consider this to be the case for denaturation by heat and by some salts. Badley et ala8 studied the denaturation of lysozyme in aqueous solutions of tetraalkylammonium bromides (up to 1,M) and concluded that only a small additional exposure of hydrophobic groups occurs on denaturation. Nicoli and Benedekg found that heat denaturation of lysozyme increases the hydrodynamic radius from 18.5 to 21.8 A, and not to the 49 calculated for a random coil. Sarfare and Bigelowlo showed that LiBr does not expose all of the 0022-3654/78/2082-1607$01 .OO/O
tyrosyl side chains of bovine pancreatic ribonuclease. Tanford2 reviewed the evidence for thermal and salt denaturation of proteins and concluded that much of the hydrophobic portion remains shielded from solvent. Also, in support of the hydrophobic clustering idea is the observation that denaturation of a concentrated ribonuclease solution by LiCl or NaC104 produces a turbid gel, but that denaturation by guanidinium chloride produces a clear ge1.l' Also, the heating of concentrated ribonuclease produces a turbid gel. The turbidity probably results from intermolecular hydrophobic clusters. Inorganic salts salt out,12and guanidinium chloride salts in, hydrophobe~.'~ (The turbidity or clarity is the relevant point; the gel formation arises from intermolecular disulfide exchange.l') The analysis developed here does not apply to denaturation by agents such as urea and guanidinium chloride which solubilize model hydrophobes and produce denatured states which are random coils or close to random coils. For acid denaturation, complete unfolding appears not to occur in generaL2 I shall consider the hydrophobic groups as clustered together largely out of contact with water, while the hydrophilic groups are more dispersed. The sources of the AC, and AV effects are to be sought in the disordering of the protein and in solvation of unfolded groups. The hydrophobic cluster may be considered the oil drop, since the more rigid wax ball (in K l a p p e r ' ~ 'term) ~ is no longer stabilized by the framework of polar interactions. If the hydrophobic side chains do not become separately dispersed in water when the protein is denatured, no large AV is to be expected from these groups. Although nonexposure of hydrophobes can explain the small magnitude of AV, this hypothesis does not do so uniquely. A small AV is compatible with exposure for several reasons. First, by Klapper's analysis the native protein is a solid-like, high-density particle, the overall volume of which would increase on denaturation. Secondly, B$je and H v i d P and Hvidt16 have pointed out that the partial molar volume of solution of hydrophobes, A P , is strongly concentration dependent, and that our ignorance of the local concentrations of side chains makes uncertain any predictions based on model compounds. Thirdly, for small models the presence of hydrophilic and charged groups has been shown to result in a smaller magnitude of AY0,17-19 Only small changes in volume are expected to arise from solvation of side chain polar groups, most of which are already exposed in the native protein, although some are involved in intramolecular interactions. The most important polar group to become exposed is the peptide group. From the data of Suzuki et a1.20AP for solution of diketopiperazine is +4 cm3 mol-', or +2 cm3 per -CH,CONH- group. This is the resultant of two effects: +AV of fusion and (probably) -AV of solvation. The 0 1978 American Chemical Society
1608
Jake Bello
The Journal of Physical Chemistry, Vol. 82, No. 14, 1978
magnitude may be estimated from the +6.5 cm3 mol-' for the fusion of acetamide,21 which suggests that AV of solvation of the -CHzCONH- group of diketopiperazine is about -3 to -4 cm3 mol-l. However B$je and Hvidt15 have shown that changes in volume on solution of certain polymers are small compared with the corresponding monomers, further supporting the prediction of a small AV contribution from the backbone. We now examine AC, effects accompanying unfolding and solvation. If the hydrophobic side chains are clustered in the denatured protein then no large AC, is to be expected from the hydrophobic groups. Sturtevantzzhas attributed to the hydrophobic effect some 1300 of the 1560 cal K-' mol-l for lysozyme unfolding and the remainder to vibrational changes. This does not square with the effects of pressure on AC, of model hydrophobes and protein^.^ If AC,(hydrophobic) is to be set at some small value, we need other sources of AC, to fill the gap. Tanfordz3has suggested that a large contribution to AC, could "arise from the order-disorder aspect", without giving a magnitude for this contribution. For lysozyme (14 000 daltons) Sturtevantz2has estimated a AC, N 270 cal K-l mol-I for changes in vibrational states on denaturation. We make an estimate of AC,(fusion). Most organic compounds show an increase in AC, on fusion, typically of about 0.2 cal K-' g-l. This provides a quantity of 3000 cal K-l mol-l for a protein of 15000 daltons. We consider exposed residues to be partially "melted" in the native state; accordingly, we reduce the AC, of fusion to 2000 cal K-' mol-'. The assumption of 0.2 cal K-l g-l is risky since some organic compounds show little or no increase. The estimate is strengthened by the results of Yoshida et al.24on poly(oxetane), for which AC, for the transition from semicrystalline to glassy state is 0.2 cal K-' g-l. Also, Wrasid10~~ has found AC, = 0.28 cal K-' g-l for fusion of poly(pheny1ene oxide). Griskey and Fosterz6have found C, of three melted hydrocarbon polymers to be in the range 0.61-0.65, about 0.15-0.2 cal K-l g-l greater than for solid hydrocarbons. Further, AC,(fusion) of acetamide is 11 cal K-' mol-l, or 0.18 cal K-l g-' (see below), or 1100 cal K-l mol-l for 100 backbone residues, with the side chains not included. Thus, AC,(fusion) may be as large as 2000 cal K-l mol-l, and half this may represent a conservative estimate. Another estimate for AC, of fusion is made as follows. From the data of Konicek and Wadso21we estimate C, for liquid acetamide to be 25 cal K-l mol-l. Together with the C, of solid acetamide, 16 cal K-' mol-', we obtain AC,,,, = 9 cal K-l mol-l. C, of liquid acetamide was obtained by extrapolating the C, values of Konicek and Wadso for liquid amides of two types, namely RC(=O)NHCH3 and CH,C(=O)NHR, where R is an unbranched alkyl group of two to four carbon atoms; closely similar values are obtained from both series. Another estimate was made by comparing C, of solid amides where R is tert-butyl with liquid amides where R is n-butyl. For the two amide types the average AC, for the transition solid liquid is +11 cal K-l mol-l. C, values of n-butylamine and tert-butylamine, both liquids, are nearly the same, 44.9 and 45.3 cal K-l mol-l, re~pectively;~' from which we deduce that the C, difference between the amides arises largely from the solid liquid transition, and not from the difference between n- and tert-butyl. Bonner et alnZ8 have estimated C, of liquid acetamide as 29 cal K-l mol-l from a correlation of C, with the number of atoms in a molecule. From this AC,(fusion) for acetamide is 13 cal K-' mol-'. The agreement between the three methods is satisfactory, and we take an intermediate value, 11 cal K-l mol-'. For a 100 +
+
residue protein this term is 1100 cal K-l mol-l for AC, of disordering the backbone, without a contribution from side chains. Taking AC, for disordering side chains as 0.1-0.2 cal K-I g-l, for 100 side chains (about 6000 g mol-l) there is an additional 600-1200 cal K-l mol-l, or a total of about 1700-2200 cal K-l mol-l. This is for disordering without solvation. The two estimates of AC,(fusion) are in fair agreement, in the range of 1000-2000 cal K-' mol-'. Another source of AC, is hydration of hydrophilic groups. Suurkuuskz9measured the partial specific heats of three proteins (lysozyme, chymotrypsinogen, and ovalbumin), and found C," to be 0.37 cal K-l g-l. Since solid proteins have C very near 0.30 cal K-l 8-l (SuurkuuskB Hutchens et a!.%), the excess of 0.07 may be taken as the contribution from hydration of the exterior. This is the surface solvation averaged over the whole protein. The fraction of a native protein which is exposed to solvent lies between about 0.25 and 0.5. Thus, C, of solvation of the exterior is 0.14-0.28 cal K-l g-l, and a middle value of about 0.2 cal K-' g-' is reasonable. Approximately half of the mass of the average residue is the backbone. Assuming that on denaturation one-half of the interior two-thirds becomes exposed (or 5000 g mol-') and that AC (hydration) is also 0.2 cal K-l g-l, the contribution from t k s source is 1000 cal K-l mol-l for the protein. Other reasonable estimates of the proportion of the protein exposed in the native and denatured states will give AC,(hydration) estimates in the range of our estimates. A different analysis leads to a similar magnitude for AC,(hydration). X-ray diffraction analyses show that most charged groups are exposed in the native protein, so that these are expected to contribute little to the thermodynamics of denaturation. However most peptide groups are intramolecularly hydrogen bonded in the native protein. On denaturation, these are presumed to become exposed and hydrated. There will be a AC resulting from the differences between peptide-peptiie and H20-H20 interactions in the native state and peptide-HzO interactions in the denatured state. Bonner et alaz8have estimated the excess C," _of urea to be -5.8 cal K-l mol-l, where excess means the C," less the C," of pure liquid urea. The value of C," of liquid urea was estimated from a correlation between C, and the number of atoms in the molecule. The data of Gucker and Ayres31 on the partial molar heat capacity of urea in water show a strong increase in Cp with concentration, especially at low temperature. A similar effect might occur with the closely connected peptide groups of a protein. For urea at 20 "C, interpolation of the data of Gucker and Ayres31indicates an increase of 9 cal K-l mol-l between infinite dilution and 10 M, a concentration similar to that of peptide groups in the protein. This provides only about 3.2 cal K-l mol-' for solvation of this peptide model. For N-alkylamides, which may be better models for peptide groups, the work of Konicek and WadsoZ7 on N-methylacylamides and N-alkylacetamides, indicates a ACPo N 0, for hydration of the -C(=O)NH group, based on an extrapolation of the amide data to zero alkyl carbon atoms. Since the data of Konicek and Wadso were obtained with liquid amides (no AC, of fusion included), it appears that AC, for the pure peptide group is more positive than for urea. AC, for urea may reflect a substantial disruption of water structure (Finer et al.32),which, by making water more "normal" would lower its C,. The amide group of N-alkylamides may have smaller disruptive effects, and the peptide group in a chain still less, because of lesser access to water, producing a positive AC, of hydration. However, the question of the effect of urea on
c,
The Journal of Physical Chemistty, Vol. 82, No. 14, 1978 1609
Protein Denaturation
water structure is complex and not yet satisfactorily rehydrophobic sequence (which will influence the number of backbone groups which may not become exposed), the solved. (See, for example, the authors cited in ref 33.) Bonner et a1.28estimated the excess of acetamide tightness of the native and denatured conformations, the in water a t 9.2 cal K-' mol-l, where the excess is the exsurface-to-volume ratio, and contributions from polar less C, of liquid acetamide. This provides trapolated groups not considered here. 920 cal K-' mol-' for a 100 backbone unit. Another estimate of the AC (solvation) is made for the unit References and Notes -CH,CHCONH-, t i a t is, the backbone including the (1) W. Kauzmann, Adv. Protein Chem., 14, 1-63 (1959). &CH2. From Konicek and Wadso, again using both series (2) C. Tanford, Adv. Protein Chem., 23, 1-95 (1970). of amides, we obtain for solution at infinite dilution, Ac," (3) J. F. Brandts, R. J. Oliveira, and C. Westort, Biochemistry, 9, 1038-1047 (1970). = 22 cal K-l mol-l. However, the @CH2may be hydro(4) A. Zipp and W. Kauzmann, Biochemistry, 12, 4217-4228 (1973). phobic, and inclusion of its contribution may be contrary (5) K. C. Aune, A. Salahuddin, M. H. Zarlengo, and C. Tanford, J . Biol. to the pressure effects noted above.4 If we assume the Chem., 242, 4486-4489 (1967). P-CH2to remain unexposed, we subtract from 22 the CH3 (6) W. Pfeil, R. P. Hearn, D. P. Wrathall, and J. M. Sturtevant, Biochemistry, 9, 2666-2677 (1970). contribution from Konicek and W a d ~ o , ~14' cal K-' mol-', (7) P. L. Privalov and N. N. Knechinashivili, J . Mol. Biol., 86, 665-684 and from Bonner et al.,2810 cal K-' mol-', leaving 8-12 cal (1974). K-l mol-l for the backbone, or 800-1200 cal K-' mol-' for (8) R. A. Badley, L. Carruthers, and M. C. Phillips, Biochim. Biophys. Acta, 495, 110-118 (1977). 100 residues, in good agreement with the estimate based (9) D. F. Nicoil and G. B. Benedek, Biopolymers, 15, 2421-2437 (1976). on Suurkuusks data. Although the a-CH is, in principle, (10) P. S. Sarfare and C. B. Bigelow, Can. J. Biochem., 45, 651-658 hydrophobic, it is flanked by two hydrophilic peptide (1967). groups, which may largely abolish its hydrophobic char(11) J. Bello, J. fhys. Chem., 82, 243 (1978). (12) E. E. Schrier and E. B. Schrier, J . fhys. Chem., 71, 1851-1860 acter. It would be desirable to obtain the pressure de(1967). pendence of the of model peptides. (13) D. B. Wetlaufer, S. K. Malik, L. Stoller, and R. L. Coffin, J. Am. Chem. The solubility data of Suzuki et al.20on diketopiperazine SOC.,86, 50-58 (1964). (14) M. H. Klapper, Biochim. Biophys. Acta, 229, 557-566 (1971). between 15 and 35 "C indicate a AC, of solution of +49 (15) L. Btje and A. Hvidt, Siopo/ymers, 11, 2357-2364 (1972). cal K-l mol-l. Allowing 20-30 cal K-' mol-' for fusion, AC, (16) A. Hvidt, J . Theor. Biol., 50, 245-252 (1975). of solvation is 19-29 cal K-l mol-', or about 10-15 per (17) S.C a b h i and G. Conti, J. fhys. Chem., 78, 1030-1034 (1974). residue or 1000-1500 cal K-' mol-' for 100 residues. Al(18) R. Zana, J . fhys. Chem., 81, 1817-1822 (1977). (19) J. King, J. fhys. Chem., 73, 1220-1232 (1969). though this is in the range of the other values, it must be (20) K. Suzuki, M. Tsuchiya, and H. Kodono, Bull. Chem. SOC.Jpn., 43, taken with considerable caution since AC, calculations 3083-3086 (1970). require very high accuracy in the solubility data. (21) E. W. Washburn, Ed., "International Critical Tables", 1st ed, Vol. IV, National Research Council, McGraw-Hill, New York, N.Y., 1928, p AC, of denaturation is smaller a t lower t e m p e r a t ~ r e . ~ ~ 14. This could arise from a lesser extent of exposure, as well (22) J. M. Sturtevant, Proc. Natl. Acad. Sci. U.S.A., 74, 2236-2240 as from a smaller AC, for solvation of peptide groups, by (1977). analogy with the smaller AC, of solution of urea at lower (23) C. Tanford, Adv. Protein Chem., 24, 60 (1970). (24) S. Yoshii, M. Sakiyama, and S.Seki, folym. J., 1,573-581 (1970). temperat~re.~~ (25) W. Wrasidio, J . fo/ym. Sci., IO, 1719-1729 (1972). Summing up the AC, contributions, we have AC,(fu(26) R. Griskey and G. N. Foster, SOC.flast. Eng., Tech. Pap., 546-550 sion), 1000-2000 cal K-l mol-'; AC,(solvation), 1000 cal K-' (1968). (27) J. Kdnicek and I. Wadso. Acta Chem. Scad.. 25. 1541-1551 (1971). mol-l, and a total AC, range from 2000 to 3000 cal K-l (28j 0. D. Bonner, J. M. Bednarek, and R. K. Arisman, J . Am. Chem. mol-' for a protein of about 15000 daltons. These SOC.,99, 2898-2902 (1977). quantities, while grossly approximate, are in the range of (29) J. Suurkuusk. Acta Chem. Scand. Sect. B. 28. 409-417 (1974). interest, and do not invoke hydrophobic AC,, except, (30j J. 0.Hutchens, A. G. Cole, and J. W. Stout, J . B i d . Chek., 244, 26-32 (1969). perhaps, for the a-CH group. This does not necessarily (31) F. t. Gucker and F. D. Ayres, J . Am. Chem. SOC.,59, 2152-2155 mean that there is no contribution from the hydrophobic (1937). side chains, but that it appears that AC, can be accounted (32) E'. G.Finer, F. Franks, and M. J. Tait, J. Am. Chem. SOC.,94, 4424-4429 (1972). for with this term being a minor contributor, in keeping (33) S. Subramanian, T. S. Sarma, D. Balasubramanian, and J. C. with the idea that hydrophobic groups are indeed hyAhluwalia. J. fhvs. Chem.. 75. 815-820 (19711. drophobic. For any given protein AC, of denaturation will (34) 6.Acampora &d J. Hermans, Jr., J . Am. Chem. Soc., 89, depend on the proportion of hydrophobic groups and the 1543-1552 (1967).
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