June, 1961
Noms
1071
Acknowledgments.-This investigation was supported in part by the National Science Foundation and by Grant H-3394 from the National Institutes of Health, United States Public Health Service.
served for 3 of the 6 tyrosyl groups of ribonuclease by ShugarZ0and by Tanford, et a1.21 An abnormally low pK can be observed for an acid group HA (Le., even lower than that predicted in the previous hydrogen bonding theory2) if the conjugate base A can hydrogen bond to a donor D H and, in EFFECT OF HYDROPHOBIC BOYDING ON addition, the side-chain D H and A groups are so PROTEIN REACTIOYS1 sterically confined by surrounding non-polar groups that, in effect, it is very difficult to rupture the BY HAROLD A. SCHERAGA hydrogen bond. This may account for the abD e p a r t m e n t o f C h e m i s t r y , Cornell C n i a e r s i t y , I t h a c a , T e w Y o ~ k normally low p K of a COOH group in ribonuReceived December 6 , 1960 c1ease.l' Limited Proteolysis.-If hydrophobic interacIn a series of papers considerat'ion has been given to t'he effects of side-chain hydrogen bonds on a tions must' be disrupted in order to liberate a pepvariety of protein reactions such as ionization.2,3 tide fragment, in limited proteolysis, the observed limited proteolysis,4,6 a~sociation,~Jthermody- free energy of hydrolysis will contain a contribunamics of reversible denaturation,*-" kinetics of tion from the free energy required to break the denaturation, l 2 ultraviolet difference spectra, 1 3 etc. hydrophobic bond, making it harder to liberate the In recent years, increasing attention has been peptide fragment. If, in addition, an ionizable given to interactions involving the non-polar side- group is embedded among non-polar side-chains chains of proteins, the so-called hydrophobic in a native protein but is accessible to water aft'er bond.14-16 Calculations of the strength of a hydro- limited proteolysis, t'hen the p K ' s of the group phobic bond, based on a statistical mechanic'al in the native and hydrolyzed proteins will differ. treatment of aqueous hydrocarbon solutions, have This will give rise to a pH-dependence of the been reportedI7; further refinements of this theory standard free energy of hydrolysis. The associaare being carried out to obtain more accurate values tion of the S-pept'ide and S-protein after proteolysis of AFO, AHo, ASo and AVO. Preliminary esti- of ribonuclease by subtilisin22could involve hydromates" of the standard free energy of formation phobic interactions. Such interactions are inof a hydrophobic bond range from -0.5 to -1.5 cluded in a postulated model of ribon~clease.~~ Association.-There are numerous examples of kcal. per mole. These are comparable to the values 0 to -0.8 kcal./mole for a heterologous prot,eiii association reactions, e.g., the association of single, side-chain hydrogen bond. 2 , 3 The purpose fibrin monomer which has been shown to involve of the present note is to summarize qualitatively intermolecular hydrogen bond formation between If ionizthe effects of side-chain hydrophobic bonds on the tyrosyl donors and hist'idyl accept,or~.~>~ able groups are accessible to water in the monomers same protein reactions list'ed above. Ionization.-If an ionizable group is embedded but trapped in hydrophobic regions in the polymers, among non-polar side-chains the effective dielec- then the modified pK's will give rise to changes in tric constant of its environment is lower than that pH on association. In many cases, anomalous of water; also the surrounding water structure is entropies of association have been e n c o ~ n t e r e d ;~ ~ - ~ ~ more ordered than that in ordinary water. As a intermolecular hydrophobic bonding, with it's atresult, the p K of the ionizable group will be ab- tendant change in the water structure,17 could normally Such an effect has been ob- account for these anomalies. Thermodynamics of Reversible Denaturation.(1) This investigation was supported by a research grant (E-1473) Hydrophobic bonds will st'abilize helical portions from the National Institute of Allergy and Infectious Diseases, of the National Institutes of Health, U. S.Public Health Service, and by a of a protein molecule and contribute to t'he standresearch grant (G-14792) from the National Science Foundation. ard free energy of denat'uration. If, in addition, (2) &I.Laskowski, Jr., and H. A. Scheraga, J. Am. C h e m . Soc., 76, an ionizable group is embedded among lion-polar 6305 (1954). side-chains in a native protein but is accessible to 13) G. I. Loeb and H. A. Scheraga, J. P h y s . C h e m . , 60, 1633 (19.56). (4) bI. Laskonski. Jr.. and H. A. Scheraga, J. Am. C h e m . Soc., 78, mater in a reversibly denatured form, then t,he pK's 5793 (1956). ( 5 ) 11. Laskowski. J r . , 6 . Ehrenpreis, T. H. Donnelly and H. A . Scheraga. i b i d . , 82, 1340 (1960). ( 6 ) J. AI. Sturtevant, XI. Laskowski, Jr., T. H. Donnelly and H. -1. Scheraga. ibzd., 77, 6168 (1955). (7) S. Ehrenpreis, E. Sullivan and H. 4. Scheraga, Abstracts of the 133rd A.C.9. meeting, p. 26-C, San Francisco, California, April, 1958. (8) H. A. Sclieraga, J . P h y s . Chem., 64, 1917 (1960). (9) H. -1.Scheraga, R. A. Scott, G. I. Loeb, -4. Nakajima and ,J. Hermans, Jr., ibid.. 65, 6S9 (1961). (10) -4.Nakajiina and H Scheraga, J. Am. C h e m . Soc., 83, 1573 (1061). (11) J. Hermans, .Jr., and H. A. Scheraga, ibid., 83, in press (1961). (12) M. Laskowski, Jr.. and H. A. Scheraga, ibid., 83, 266 (1961). (13) AI. Laskowski, J r . , S. J . Leach and H. A. Scheraga, ibid., 82, 571 (1960). (14) I. hl. Klotz. Science, 128, 815 (1958). ( 1 5 ) W. Kauzrnann, Advances in Protein Chem., 14, 1 (1959). (16) I. X.Klotz, Brookhaven S y m p o s i a in Biology. 13, 25 (1960). (17) G . NBmethy and H. A. Schersga, Abstracts of the 138th A.C.S. meeting, p. 4C, New York, N. Y., Sept. 1960; see also the discussion a t the end of Klotz's paper.16
(18) C. Tanford and J . G . Kirknooil. J . A m . Clrem. Soc., 79, 5333 (1957). (19) C . Tanford, T b d , 79, 5340. 5348 (1057). ( 2 0 ) D. Shugar, Biochem. J.,52, 142 (1952). (21) C. Tanford. J. D. Hauenstein and D. G. R m d s , J . .lm. C h e n S o c . , 77, 6409 (1955). (22) F. M. Richards and P. J. Vitliayathil. J . Bzd. Chem.. 234, 1SXJ (1959). (23) €1. A. Scheraga, .I. -4m. C h e m . Soc.. 82, 3847 (1060). (24) 11. Laskoaski. Jr., and IS. -1. Sclieraga, Abstracts of the 126tli A.C.S. meeting, p. 60-C, New Y o r k , N. P., Sept., 1951. ( 2 5 ) P. Doty and G. E. Myers. Disc. F a r a d a y Soc., 13, 51 (1953). (26) R. F. Steiner, A r c h . Biochem. B i o p h y s . , 44, 120 (1953); 49, 71 (1954). (27) H. Edelhoch, E. Katchalski, R. H. Maybury, W. L. Hughes. Jr., and J. T. Edsall, J . Am. C h e m . Soc., 75, 5058 (1953). (28) J. T. Edsall, R. H. Maybury, R. B. Simpson and R. Straessle i b i d . , 76, 3131 (1954). (29) F. Karush. in "Serological and Biochemical Comparisons of Proteins" ( W . H. Cole. e d . ) , Rutgers Univ. Press, New Brunswick, N. J . , 1958, p. 40.
1072
NOTES
Vol. 65
SELF-DIFFUSION I N LIQUIDS. 111. of the group in the native and denatured forms will differ. This will give rise to a pHdependence of TEMPERATURE DEPENDENCE I N PURE the standard free energy of denaturation. While LIQUIDS such a pH-dependence has heretofore been atBY R. E. RATHBUN AND A. L. BABB tributed to side-chain hydrogen bonding18which has been shown to be involved in several protein de- Department of Chemical Engineering, Unioersity of Wushington, Seattle, Washtngton naturations,lOJ1it appears that a contribution from Received December 0, 1960 hydrophobic blonding is also involved in the case of Self-diffusion coefficients were measured in benribonuclease. l1 In the latter case, a preliminary attempt already has been made to obtain a quanti- zene, carbon tetrachloride, methanol and ethanol over a wide range of temperatures using a capillary ta tive treatment . cell technique with carbon-14 labeled tracers. Kinetics of Protein Denaturation-If a native The apparatus and procedure have been fully deprotein contains hydrophobic bonds which are rup- scribed e l s e ~ h e r e . ~ . ~ tured in the formation of activated complexes, Thg ethanol-l-CE4was purchased from the Volk Radiothen these bonds will contribute to the standard chemical Company. The uniformly labeled benzene waa free energy of activation. If, in addition, the purchased from the Nuclear Chicago Corporation and the carbon tetrachloride-C14 and methanol-C14 were obtained production of activated complexes from native from the Nuclear Instrument and Chemical Corporation. protein requires that ionizable groups emerge from The chemicals used in diluting the tracers to the desired activity and in preparing the non-radioactive bulk solutions a hydrophobic to an aqueous environment, then the were of the highest grade commercially available. The rate will be pHdependent. This pH-dependence benzene, carbon tetrachloride and methanol were reagent can be deduced from the model previously used12 g a d e chemicals aa obtained from Merck and Company, and the ethanol was pure anhydrous ethyl alcohol to account for denaturation kinetics in terms of the produced by the U.S. Industrial Chemical Company. involvement of side-chain hydrogen bonds. Results and Discussion Ultraviolet IDiff erence Spectra.-If a chromophoric group is embedded among non-polar sideIn Tables I, 11,I11 and IV, the measured self difchains in a native protein and becomes accessible fusion coefficients are tabulated and compared to water during a limited or extensive configura- with data available in the literature ( b e n ~ e n e , ~ > ~ * ~ e t h a n 0 1 , ~ * ~and - ~ * ~methtional change of the protein, there will be a pertur- carbon tetrachl~ride,’*~ The new experimental values reprebation of the spectrum of the ~ h r o m o p h o r e . ~ ~ - ~ ~ sent, averages of three determinations (except where Deuterium-Hydrogen Exchange.-LinderstrZmand are reported with the maximum deviaLang has interpreted the slow exchange of deu- noted) tions from the average values. terium and hydrogen in terms of hydrogen bondThe recommended average values reported in ing.36 It is worthwhile devising experiments to Tables I-IV were obtained from the reported difdetermine whether the embedding of a group (hav- fusivities by weighting them in inverse proportion ing exchangeable hydrogens) among non-polar side- to their variances. The method of Davies and Pearson’O was used to convert the ranges of the chains will lead to slow exchange. The examples cited above illustrate the possible reported diffusivities shown into unbiased estiprofound influence which hydrophobic bonds can mates of the population standard deviations from have on protein reactions. An attempt is now which the variances were determined. The recommended average values are reported together with being made to treat these effects quantitatively, as the best values of their standard deviations. was done previously for side-chain hydrogen The self-diffusion coefficient (D = cm.2/sec.) bonds.2-’3 Presumably both hydrophobic and has been related to the viscosity (7 = g./(cm.) hydrogen bonds exist between the side-chains of (sec.)), molar volume (V = ~m.~/rnole), and absonative proteins; if so, they may cooperate in pro- lute temperature ( T = OK.) by the relationl1Pl2 viding stabilization of the native structure, as was D?V‘/3/T = p (1) suggested in the case of ribonuclease.” This work was supported by the Office of Ordnance Research, ADDEDIN PR,ooF.-Some of these problems also U.(1) S. Army. have been discussed by Tanford3’ in a recent paper. (2) P. A. Johnson and A. L. Babb, J. Phys. Chem., 60, 14 (1956). (3) A. P. Hardt, D. K. Anderson, R. Rathbun, B. W. M a r and A. L. Acknowledgment.-I should like to thank George Babb, ibid., 65, 2059 (1959). (4) K. Graupner and E. R. S. Winter, J . Chem. Soc., 1, 1145 (1952). N6methy for helpful discussions of these problems. (5) H. Hiraoka, J. Osugi and W. Jono, Ren. Phys. Chem., Japan, 28, (30) D. B. Wetlaufer, J. T. Edsall and B. R. Hollingworth, J . B i d . 52 (1958). Chem., 233, 1421 (1958). (6) J. R. Partington, R. F. Hudson and K. W . Bagnall, J. chim. (31) E. J. Williams and J. F. Foster, J. A m . Chem. Soc., 81, 865 phys., 66, 77 (1958). (1959). (7) H. Watts, B. J. Alder and J. H. Hildebrand. J. Chsm. Phys., 23, (32) C. C. Bigelow and I. I. Geschwind, Compt. rend. trav. lab. Carla659 (1955). berg, 31, 283 (1960). ( 8 ) H. Hiraoka, Y. Izui, J. Osugi and W. Jono, Rev. Phys. Chem., (33) T . T . Herskovits and M. Laskowski. Jr., J . B i d . Chem., 235. Jupan, 28, 61 (1958). PC56 (1960). (9) A. P. Hardt, Ph.D. Thesis, University of Washington, 1957. S. Yaiiari and F. A. Bovey, ibid., 235,2818 (1960). S. J. Leach and H. A. Soheraga, ibid., 235, 2827 (1960). K. Linderstrgm-Lang, “Symposium on Peptide Chemiatry,” SOC.(London). Spec. Pub. No. 2, 1 (1955). (37) C. Tanford, J . Am. Chem. SOC..8 5 , 1628 (1961).
(34) (35) (36) Chem.
(10) 0. L. Davies and E. 9. Pearson, Suppl. J. Roy. Sfatiaticol SOC. 1, 76 (1934). (11) E. N. d a C. Andrade, Phil. Mog., 11,496, 698 (1934). (12) S. Glaastone, K. J. Laidler and H. Eyring, “The Theory of Rate Processes,“ McGraw-Hill Book Co., New York. N. Y., 1941.
