862 7. MERCURIALS 



(which it seldom is). If the system is in zone B or C, K^ may be a good 

 deal smaller than PI50, and we have seen that in zone C there is no way 

 kinetically of determining K^. The only valid calculation of a true dissocia- 

 tion constant for a mercurial complex with an enzyme, of which I am aware, 

 is that of Madsen and Gurd (1956) for muscle phosphorylase and p-MB. 

 They used an ultracentrifugal method to measure the concentration of free 

 p-MB after equilibration and determined K^ from a plot of 1/r against 

 r/(p-MBy), where r is the molar ratio of p-MB bound to protein and p-MBy 

 is the free mercurial. A value of p^, = 6 was found. It is likely that in 

 most cases in which an enzyme is potently inhibited by a mercurial, a p^, 

 of 6 or less would be found, and in this range it is very difficult to deter- 

 mine the constant by the usual plotting procedures. It will be recalled that 

 W. L. Hughes (1950) found a p^ of 4.46 for the complex of mercaptalbumin 

 and MM (page 759). We may now inquire into what values of K^ would be 

 predicted under ordinary circumstances. Equation 7-3 gives the relation 

 between the experimental constant and the dissociation constant for a mer- 

 curial complex, and if we alter it to correspond to inhibition of enzymes 

 we have 



p^/ = vKi — pKa - p-fi'x , 



where ipK/ is the experimental or apparent dissociation constant. If p^, is 

 taken as 21, \)K„ as 8.7, and ipK^ (for Cl~) as 6.5, all of these being approxi- 

 mations, Y>K/ turns out to be around 5.5. Since pK^ certainly varies from 

 20 to 22, pK^ from 7.5 to 9.5, and p^^, (depending on the ligand) from 6 to 

 9, it is clear that pK/ may vary over a wide range, but at least the values 

 experimentally determined are of the correct order of magnitude. The ex- 

 perimental i)K/ is thus a good deal less than the true p^, because of the 

 competitive effects of H+ and the ligand X~. 



The question of how best to report mercurial inhibitions is a difficult 

 one. First, the concentration-inhibition curves are often very steep (Fig. 

 7-31) so that giving the results of a single concentration may be quite mis- 

 leading. Therefore one can suggest that in all studies a range of mercurial 

 concentration be used, such as to provide different degrees of inhibition, 

 preferably from to 100%. Second, values of K^, which are so useful in 

 other inhibitions, are difficult if not impossible to determine by the usual 

 procedures, especially when the systems are in zones B or C, in which case 

 plgo may vary greatly depending on the enzyme concentration. It is evident 

 that for work with pure enzymes it is best to state the amount of inhibitor 

 present in terms of //moles per milligram of enzyme, or if the molecular 

 weight of the enzyme is known to express this as a molar ratio. However, 

 when impurities are present, and especially when preparations such as ho- 

 mogenates are used, this method is not as useful and even a designation 

 such as //moles of mercurial per milligram of total protein is not very mean- 

 ingful. Third, as we have discussed previously, mercurial inhibition is 



