Biochemistry 1981, 20, 849-855 849 Wey, C. L., Ahl, P. L., Cone, R. A., & Gaffney, B. J. (1979) Wu, E.-%, Jacobson, K., Szoka, F., & Portis, A., Jr. (1978) Veatch, W. R., & Stryer, L. (1977) J. Mol. Biol. 117, Watts, A., Volotovski, I. D., & Marsh, D. (1979) Biochemistry 1109-1113. Biophys. J. 25, 169a. 18, 5006-5013. Biochemistry 17, 5543-5550. Affinities of Amino Acid Side Chains for Solvent Water? R. Wolfenden,* L. Andersson, P. M. Cullis, and C. C. B. Southgate ABSTRACT: Equilibria of distribution of amino acid side chains, between their dilute aqueous solutions and the vapor phase at 25 “C, have been determined by dynamic vapor pressure measurements. After correction to pH 7, the resulting scale of “hydration potentials”, or free energies of transfer from the vapor phase to neutral aqueous solution, spans a range of -22 kcal/mol. The side chain of arginine is much more hydrophilic than those of the other common amino acids, with an equi- In biological systems, “chemical recognition” usually depends on the structural complementarity of different compounds or functional groups that are attracted to each other by nonco- valent forces. When these interactions occur in an aqueous environment, solvent water must usually be stripped away from the interacting groups. If information were available about the absolute tendencies of these compounds to leave water and enter an empty cavity that neither attracts nor repeals solutes, then it might be possible to draw inferences about specific forces of attraction or repulsion that may be at work in specific cases. The absolute affinity of a compound for an aqueous envi- ronment can be evaluated by determining its vapor pressure over dilute aqueous solutions. From the results, it is a simple matter to calculate a dimensionless equilibrium constant for its transfer from water to a featureless “solvent” of unit di- electric constant, the dilute vapor phase. Measurements of this kind, performed on a variety of organic compounds, suggest that the free energy of interaction between complex molecules and water can usually be approximated as an ad- ditive function of their constituent groups (Butler, 1937; Hine & Mookerjee, 1975). Earlier measurements were confined to relatively volatile solutes that exhibit substantial vapor pressures over water. More sensitive techniques have allowed the recent extension of these measurements to include polar molecules bearing functional groups of biological interest such as the peptide bond (Wolfenden, 1976, 1978). Differences between amino acid residues, in their strength of solvation by water, are likely to be significant in determining the configurations of proteins in solution (Kauzmann, 1959; Tanford, 1962; Perutz, 1965). It would therefore be of interest to have information about the relative affinities of amino acid side chains for solvent water. Even using material of very high specific activity, efforts in this laboratory to detect glycine in the vapor phase over concentrated aqueous solutions have been From the Department of Biochemistry, University of North Carolina, Chapel Hill, North Carolina 27514. Received June 17, 1980. Supported by grants from the National Science Foundation (PCM-7823016) and the National Institutes of Health (GM-18325) and by fellowships from the American-Scandinavian Foundation (to L.A.) and from the Jane Coffin Childs Foundation (to P.M.C.). 0006-296018 110420-0849$01 .OO/O librium constant of - 1015for transfer from the vapor phase to neutral aqueous solution. Hydration potentials are more closely correlated with the relative tendencies of the various amino acids to appear at the surface of globular proteins than had been evident from earlier distribution studies on the free amino acids. Both properties are associated with a pronounced bias in the genetic code. unavailing. This is hardly surprising, since glycine in neutral aqueous solution is present as the uncharged species only to the extent of - 1 part in 200000 (Edsall & Wyman, 1958). Free energies of solvation of charged ammonium and car- boxylate groups are each in the neighborhood of -70 to -80 kcal/mol [Kebarle, 1976; see also Tse et al. (1978)], so that the zwitterionic species of glycine in water can be considered totally nonvolatile. The rare, uncharged species of glycine can be expected, from bond contributions based on correlations of data from the literature (Hine & Moorkerjee, 1975), to exhibit an equilibrium constant of -8 X for transfer from dilute aqueous solution to the vapor phase. Accordingly, the concentration of glycine, at equilibrium in the vapor phase over an aqueous solution containing 1 M glycine, is expected to be no higher than M; much lower values are expected for more polar amino acids. Even if methods more sensitive than those presently available should make it possible in the future to determine free energies for removal of amino acids from water to the vapor phase, it is far from clear that they would serve as good models (even in a relative sense) for the behavior of the various amino acid residues in proteins. It has even been suggested that there may be no significant relationship between free energies of transfer of amino acids from water to organic solvents and their tendencies to appear in internal peptide linkage in the interior rather than at the surface of globular proteins (Janin, 1979). This would seem to indicate either that solvation effects may be less important than originally suspected or that free amino acids are poor models for the relative solvation behavior of amino acids in proteins. Side chains of amino acids in proteins are flanked by peptide bonds, associated with a free energy of solvation of about -10 kcal/mol (Wolfenden, 1978), modest in comparison with the large negative free energies of solvation of ammonium and carboxylate groups mentioned above. The solvent-organizing power of these charged groups is very great and might be expected to affect the relative distribution properties of nearby substituents (Nemethy, 1967; Nandi, 1976). To obtain results that might be more closely comparable with the solvation behavior expected of amino acids in poly- peptides, we decided to examine the behavior of the amino acid 0 1981 American Chemical Society