790 7. MERCURIALS 



reaction or can it originate solely by mercaptide formation at the active 

 center? The results taken all in all tend to signify that inhibition usually 

 occurs upon the initial reaction of the SH groups at or near the active 

 center, this being reversible, and that slower structural alterations proceed 

 as a result of either the mercurial already combined or the continued reac- 

 tion with more mercurial (perhaps with the less available SH groups), these 

 changes becoming more and more irreversible, a progressive inactivation 

 being superimposed on the primary inhibition. Lability may also come 

 about by a displacement of coenzyme or cofactors, since these undoubtedly 

 help maintain configurational integrity, especially in the abnormal state in 

 which isolated enzymes find themselves. This does not imply that all en- 

 zymes behave in this fashion; it is quite possible that in some the structural 

 changes may be primary and the sole cause of the inhibition. Certain en- 

 zymes suffer only the primary inhibition and the stability is not reduced 

 by the mercurial, and indeed stability may be increased, as we have ob- 

 served with papain and pinguinain. The requirement to solve the problems 

 of the relation between inhibition and inactivation, and between both proc- 

 esses and the types of SH group reacted, is for more detailed studies cor- 

 relating the time courses of as many of these changes as possible as they 

 occur after introduction of the mercurial. Another approach might be to 

 do occasional experiments at low temperatures, where inactivation or de- 

 naturation would occur very slowly, in this way possibly separating the 

 primary inhibition from these other changes. Finally, it might be suggested 

 that every effort to create conditions favoring stability of the enzymes be 

 made. One gets the impression that often so little attention is paid to the 

 proper pH, ion concentrations, buffers, and other factors, that the enzyme 

 as studied is in a relatively unstable state and hypersusceptible to any in- 

 hibitors subjecting the normal protein configuration to even minor stress. 



Effects of pH, Ions, and Buffers on Mercurial Inhibition 



The effects of pH on OH"" complexes with mercurials (page 736), on 

 mercaptide formation (page 749), and on reactions of proteins with mer- 

 curials (page 760) have been discussed. The results and the factors which 

 may be involved can be summarized as follows: (1) pH affects the ioniza- 

 tion of the SH groups or the competition between mercurial and H+ for 

 the S~ group, (2) pH alters the concentration of 0H~ and hence the amount 

 of mercurial complexed with this anion, (3) pH influences the protein 

 charge possibly attracting or repelling charged mercurials, (4) pH deter- 

 mines the rate of secondary inactivation or denaturation, (5) pH affects 

 the aggregation state of protein-mercurial complexes (e.g., the degree of 

 dimerization of mercaptalbumin complexes with Hg++), (6) pH affects both 

 the rate and the number of SH groups reacted, and (7) generally there is 

 an increased rate of protein reaction with mercurials as the pH is reduced. 



