762 7. MERCURIALS 



structural effects should be distinguished from preferential reaction of mer- 

 curials with denatured protein in cases in which native and denatured pro- 

 tein exist in equilibrium under nonphysiological conditions (e.g., at low pH's 

 or high temperatures) (Habeeb, 1960). They should also be distinguished 

 from changes brought about in proteins evident after precipitation. The 

 thermal coagulation of serum proteins is enhanced by p-MB and the coagula 

 are firmer, more elastic, and more transparent, the water bound to the clot 

 being around 4 times greater (Jensen, et al., 1950), but this does not pro- 

 vide evidence that protein configuration before coagulation is altered by 

 the mercurial. High Hg++ concentrations weaken keratin fibers so that they 

 break under less tension (Hoare and Speakman, 1963). This would be ex- 

 pected if interchain disulfide bonds are disrupted. Changes in gross protein 

 properties seldom provide information on the more important and subtler 

 localized modifications which are believed to occur. 



In view of the significance of configurational changes in enzyme inhibi- 

 tion, and in the belief that more examples will be postulated and established 

 in the coming years, we may summarize some of the possible mechanisms 

 by which such effects can be brought about. (1) In those cases in which 

 there are equilibria between SH and S — S groups, or where there is a cyclic 

 oxidation and reduction, and in which the S — S bonds contribute to the 

 stability of a local configuration, mercaptide formation may loosen the 

 structure. (2) The SH groups themselves may contribute to the stability, 

 perhaps by hydrogen bonding or the binding of cofactors, so that mercura- 

 tion may again enhance dissolution of the native structure. (3) The intro- 

 duction of a charged group, such as occurs with p-MB or p-MPS, will alter 

 the local electric field, and this may favor instability. (4) Hg++ and or- 

 ganic mercurials which dissociate to form Hg++ can bind to two groups 

 simultaneously and thereby distort protein configuration. (5) Reaction of 

 mercurials with non-SH groups, especially N- and 0-containing groups, 

 may reduce hydrogen bonding between polypeptide chains. It is not ne- 

 cessary that the affinity for these groups be especially high, and there is 

 some indirect evidence that it is often the excess mercurial, above that 

 required to saturate the SH groups, which is responsible for denaturation. 

 There is increasing reason for believing that proteins are not rigid structures 

 but often exhibit a fair degree of flexibility (page 1-199), so that it is reason- 

 able that reversible modifications of the structure may be fairly easily in- 

 duced. 



Estimation and Titration of Protein SH Groups with Mercurials 



The older methods for the determination of SH groups, using nitroprus- 

 side, ferricyanide, iodine, or other reagents, are now considered to be gen- 

 erally unreliable when applied to proteins, due mainly to lack of specifi- 

 city, and, in addition, these methods are often rather laborious. Ampero- 



