INHIBITION OF ENZYMES 



807 



the SH groups at the active center becoming unavailable for reaction below 

 30° (Barnard and Ramel, 1962). The presence of substrate may either fa- 

 cilitate reaction of SH groups — as with xanthine oxidase (Fridovich and 

 Handler, 1958) and myosin ATPase (Gilmour and Grellert, 1961) — or 

 protect certain SH groups. The question as to which pH, temperature, and 

 medium should be used, or whether substrate or coenzyme should be pres- 

 ent during the incubation, can only be answered generally by stating that 



REACTION WITH 

 SH GROUPS 



0.25 



020 



0.05 



16 20 



MMOLESiIO^/MG 



Fig. 7-23. Titration of myosin ATPase with p-MB, showing 



the different responses of the EDTA-treated and Ca++-acti- 



vated activity. (From Kiellye and Bradley, 1956.) 



whenever possible one should strive for physiological conditions. It is nec- 

 essary, of course, to vary these factors in many instances in order to study 

 the behavior of the SH groups, but the variation should be from a standard 

 set of conditions designed to provide information relevant to the enzyme 

 in a normal state. 



It is frequently difl&cult to determine with certainty the total number of 

 free SH groups in a native enzyme under standard conditions and especially 

 to relate certain SH groups to the catalytic activity. Thorne and Kaplan 

 (1963) titrated pig heart malate dehydrogenase with p-MB, allowing 1 hr 



