824 7. MERCURIALS 



in the rates of reversal, although it may not markedly influence the equi- 

 librium conditions. 



Despite the lack of data on reversal rates, one obtains the impression 

 that reversal is often a good deal faster than the development of inhibition. 

 Unfortunately those workers who measured the rates of inhibition the most 

 carefully usually did not examine the reversibility or, if they did, remarked 

 only that it occurs, without giving data on the time required; and in other 

 cases no reversal was observed. We are thus in the surprising position of 

 having essentially no information in any one case of mercurial inhibition 

 as to the relative rates of inhibition and reversal. Certainly in some instances 

 the reversal is very rapid, as with 3-phosphoglyceraldehyde dehydrogenase 

 (Velick, 1953) and hexokinase (Sols and Crane, 1954) treated with cysteine 

 after inhibition by p-MB, as previously discussed (page 1-623 and Fig. I- 

 13-8). However, in these cases the rates of inhibition are not known, al- 

 though they are probably quite fast; in both, the enzyme and p-MB were 

 preincubated for an arbitrary time (for hexokinase 15 min at 0°). It would 

 be difficult to compare the rates of reversal by dialysis and reversor — it 

 seems never to have been done experimentally — but dialysis is not a very 

 efficient method due to the fact that only a small fraction of the dissociated 

 inhibitor at any time is able to pass out through the membrane, most re- 

 combining with enzyme, while the presence of a high concentration of re- 

 versor throughout the medium ensures that free inhibitor is rapidly bound. 

 Therefore, one does not know in reversal which step is limiting, or if the 

 reversor can facilitate the dissociation of the mercurial from the enzyme. 

 It is impossible to predict the relative rates of inhibition and reversal theo- 

 retically because we do not know the exact reactions involved, particularly 

 the influence of H+ and complexing ions. There is only one way to solve 

 these problems: to do a few critical and accurate experiments. 



The information to be derived from simple reactivation experiments, 

 especially those in which a high concentration of reversor is used, is not 

 as much or as reliable as many seem to have believed (see page 1-624). If 

 complete reversibility is obtained, this shows that no serious inactivation 

 of the enzyme has occurred; it does not imply that structural changes have 

 not been induced by the mercurial. This information is usually important 

 and must be obtained in any quantitative kinetic studies, but this is all 

 that this type of reversal experiment will provide. Failure to recover the 

 activity can be explained in a variety of ways, previously enumerated (page 

 651). More careful reversal studies, especially determination of rates, the 

 effects of pH and ligand concentration, and the degree of reactivation with 

 low reversor concentrations, would undoubtedly be more informative. 



Several hundred studies on mercurial inhibition have included statements 

 relative to reversibility with thiols. It would serve very little useful pur- 

 pose to list these results. Summarizing all of them one finds that complete 



