3] CONFIGURATION OF GLOBULAR PROTEINS 43 



Static attraction, do not associate appreciably.^^ However, as in the case 

 of hydrogen bonds, there will be a difference in entropy between the forma- 

 tion of intermolecular ion pairs between small ions and the formation of 

 corresponding bonds between protein side chains of opposite charge. There 

 is no experimental evidence at present which would enable one to arrive 

 at a decision concerning the presence of such intramolecular ion pairs in 

 compact protein molecules or on their contribution to the maintenance of 

 a compact structure. (In calculating the energy of electrostatic interaction 

 earlier in this paper, the absence of ion pair formation was assumed : the 

 closest distance of approach of two charges in the models used is 6-5 Â.) 



Hydrophilic groups at the surface. We have discussed the attractive forces 

 between solvent molecules and the attractive forces between segments of 

 the polypeptide chain, which, acting alone or together, could stabihze a com- 

 pact configuration for protein molecules in aqueous solution. As was pointed 

 out earHer, an additional requirement for the stabihty of such a configura- 

 tion is the presence of a small number of surface sites with strong afiinity 

 for the solvent. For proteins in aqueous solution, the side chain groups 

 which bear charges would seem most likely to fill this requirement. Indeed, 

 there is strong evidence from titration curves that most of these groups are 

 in fact in contact with solvent in every globular protein. Ribonuclease, for 

 example, maintains its compact configuration through most of the accessible 

 pH range (see below). The titration curve^^ shows that every one of the 

 carboxyl, imidazole and amino groups (but not all phenolic groups: see 

 below) which this protein is known to possess is titrated reversibly in this 

 pH range. Moreover, there is no conflict between the observed titration 

 curve and a calculated curve based on {a) intrinsic dissociation constants 

 identical with these found in appropriate substances of low molecular weight, 

 {b) electrostatic interaction calculated with the assumption that charges are 

 as close to the surface as in low molecular weight organic acids. Since any 

 variation in the closeness of charged sites to the solvent results in large 

 changes in electrostatic self-energy,^' the presence of as few as one or two 

 of these titratable groups in the interior of the molecule would at once be 

 detected, either by failure to titrate these groups at all or at least by pro- 

 nounced shifts in their dissociation constants. 



Ovalbumin is another protein which maintains a compact configuration 

 over most of the accessible pH range. The titration curve^^ again requires 

 that essentially all charged sites be at the surface. The same conclusion 

 apphes with somewhat less force to most other proteins. Many of these do 

 not maintain their compact configuration over a sufficient range of pH to 

 allow observation of the actual titration of the majority of charged sites in 

 the absence of configurational change. One can often, however, deduce the 

 presence of titratable groups with normal properties even if they are not 

 titrated directly. It is unhkely that any of the small globular proteins has 



