3] CONFIGURATION OF GLOBULAR PROTEINS 45 



There is no way of predicting which of these effects will predominate. 

 If ribonuclease is oxidized with performic acid so that all disulfide bonds 

 are broken, it loses its compact configuration even at the isoelectric point. ^^ 

 On the other hand, ovalbumin, which contains just a single disulfide bond 

 in its natural state is one of the proteins most resistant to loss of its com- 

 pact form, whether by acid, base or urea. Finally, serum albumin, with 

 about 16 disulfide bonds, loses its compact configurational more readily 

 than most proteins. Another aspect of the presence of disulfide bonds is 

 that they are hkely to facilitate the reversibility of configurational changes, 

 as Kauzmann** has pointed out. 



It is evident from the preceding discussion that no basic principles of 

 chemistry and no conclusive experimental tests allow us to state precisely 

 what forces maintain a compact configuration for so many isoelectric pro- 

 teins in aqueous solution. Much experimental work is currently going on 

 in many laboratories towards the elucidation of this problem. The most 

 fruitful approach has been to study the conditions under which compact 

 configurations break down. Such breakdown occurs quite generally when 

 the solvent is changed, when an aqueous solution is heated, when acid or 

 base is added, etc. It is usually called denaturation, although some definitions 

 of this term would not include rapidly reversible changes in configuration. 

 A recent general review by Putnam^* and the paper by Kauzmann** earlier 

 referred to provide a summary of results which have been obtained. Unfor- 

 tunately, these results have not as yet progressed to where they can provide 

 an answer to the questions which we have posed. In general, kinetic studies 

 of denaturation predominate. There have been few attempts, however, to 

 characterize the configuration of the denatured form or to determine the 

 thermodynamics of the denaturation process.* The studies have gone far 

 enough, however, to indicate that every protein behaves in an individual 

 way towards various agents which cause configurational change. As a result, 

 it may well be that different proteins possess a compact configuration for 

 quite different reasons. 



The remainder of this paper will summarize recent information obtained 

 by the present author and co-workers, and by others, on configurational 

 changes which accompany the titration of proteins in aqueous solution. The 

 results will serve to illustrate the conclusion that we are still far from a 

 solution of the problems which we set out to solve. 



THE EFFECT OF pH ON CONFIGURATION 



When acid or base is added to an isoelectric protein solution, three things 



* The lack of thermodynamic data results in large part from the fact that denaturation 

 has usually been studied under irreversible conditions. An unfortunate consequence is 

 that we do not know for most proteins whether the observed range of stability of a com- 

 pact configuration reflects conditions under which this configuration is truly the form of 

 lowest free energy, or whether maintenance of compactness is merely a kinetic phenomenon. 



