42 CHARLES TANFORD [3 



hydrogen bonds, but in a later paper,^^ this prediction was withdrawn. For 

 formation of phenol-carboxylate hydrogen bonds in proteins, Laskowski 

 and Scheraga^^ h^ve calculated a large loss of rotational entropy which is 

 of the same order of magnitude as the probable loss of translational entropy 

 in the tyrosine-acetate reaction mentioned above. 



Perhaps even more important is the fact that hydrogen bonds in proteins 

 can occur in conjunction with other intramolecular forces, such as hydro- 

 phobic forces. For instance, when several amino acid residues, adjacent in 

 sequence, are all non-polar, then they will all tend to be located in the 

 interior of the molecule and will carry with them the part of the peptide 

 chain to which they are attached. This part of the polypeptide chain, being 

 removed from contact with water, will presumably tend to form peptide 

 hydrogen bonds, and the ability to form such bonds will contribute to the 

 stability of the hydrophobic bonding.* 



That hydrogen bonds are in fact present in the compact forms of globular 

 proteins is strongly suggested by several kinds of experiments. The deter- 

 mination of optical rotatory power, for example by Yang and Doty,^^^ 

 indicates that globular proteins in aqueous solutions are partially in the 

 form of helices. This almost certainly reflects the presence of peptide hydro- 

 gen bonds. The deuterium-hydrogen exchange experiments of Linderstrom- 

 Lang and Hvidt^^-^^ show that some exchangeable hydrogen atoms are 

 more slowly exchanged than others. This result could arise if peptide or 

 amide groups (for example) were in the hydrophobic interior of the molecule 

 without hydrogen bonding, but it is usually assumed that if such groups are 

 so located they will also tend to be hydrogen-bonded. Finally, it will be seen 

 below that in at least two proteins the stability of the compact form towards 

 pH suggests the presence of hydrogen bonds or intramolecular ion pairs 

 between titratable side-chain groups. It is of interest in this connection that 

 the titration curves of some simple dicarboxylic acids ^"^ with bulky non- 

 polar side chains indicate the presence of weak intramolecular bonds between 

 — COOH and —COO". 



Intramolecular ion pairs.] The attraction between positive and negative ions 

 leads to close association between them in the crystalhne state and to the 

 formation of ion pairs in non-aqueous solvents. In water, however, the free 

 energy of hydration is sufficiently negative to prevent ion pair formation, 

 at least between univalent ions. Even guanidinium and acetate ions, the 

 association of which would be favored by hydrogen bonds as well as electro- 



* Hydrogen bonds can be important even if they do not contribute to thermodynamic 

 stability by blocking kinetic pathways to more extended configurations. For example, 

 the activated state for unfolding could be one in which intramolecular hydrogen bonds 

 are ruptured, but in which new hydrogen bonds to water are not yet formed. 



f This new term is suggested as more descriptive than the term 'salt bridge' which has 

 sometimes been used in the past to describe this kind of bond. 



