808 



7. MERCURIALS 



for reaction at 25°, and could obtain no reliable end-point. As the molar 

 ratio of I : E is increased there is no marked effect on the activity, except 

 for a slight stimulation, until after a value of 5 is exceeded, and then there 

 is a progressive loss of activity as the ratio is elevated, nearly complete inhi- 

 bition occurring at a value of 21.6. It is impossible to interpret these data in 

 terms of relating SH groups to activity. Indeed, it is likely that the enzyme 

 is structurally altered so that SH groups normally not accessible are second- 

 arily unmasked, since good titrations can be determined with the urea- 



100 



2 4 6 8 10 



MOLES p-MB/MOLES ENZYME 



Fig. 7-24. Titration of muscle phosphorylase a with 

 p-MB. (From Madsen and Cori, 1955.) 



denatured enzyme. The aspartate: or-ketoglutarate transaminase from pig 

 heart contains a total of 7 SH groups; when 2 moles of p-MB per mole of 

 enzyme are added, this reacting with 1-2 SH groups, the activity is reduced 

 by 50% (Turano et al., 1963). Such data again are uninterpretable and it is 

 impossible to conclude that SH groups are related in any way to the cat- 

 alysis. Di Sabato and Kaplan (1963) titrated the lactate dehydrogenases 

 from a variety of sources with both Hg++ and p-MB. The total number of 

 SH groups per mole of enzyme varied from 17 to 27 but generally inactiva- 

 tion occurred when 4 moles of mercurial were bound for each mole of en- 

 zyme. It is likely that no major configurational changes occur because no 

 alterations of fluorescence, sedimentation constant, or rotatory dispersion 

 and no immunological changes could be detected, and furthermore cysteine 

 could essentially completely reverse the inhibition. They felt that certain 

 SH groups are part of the active site rather than being vicinal, since statis- 



