INHIBITION OF ENZYMES 



805 



i.e., reaction of the mercurial is not complete at the time chosen for the 

 readings. Some enzyme SH groups react almost instantaneously with mer- 

 curials and others require 30-60 min at least (see page 809). Kinetic studies 

 should always accompany any titration; to decide arbitrarily that n min- 

 utes of incubation with the mercurial at each concentration is adequate is 

 not a satisfactory procedure. (2) The enzyme may be altered structurally 

 by the mercaptide formation so that new SH groups are progressively ex- 



FiG. 7-21. Titration of yeast alcohol clehydrogenase 

 with 77-]\IB. sliowiny the nonlinearity at low con- 

 centrations of the mercurial. (From Barron and 

 Levine, 19.-i2.) 



posed, in which case there is no clear end-point and the results do not 

 correspond to the original native enzyme. Sometimes the stability of the 

 enzyme can be increased by creating a more physiological environment. 

 (3) The enzyme SH groups may be oxidized during the titration, reducing 

 the number of titratable SH groups. Use of oxygen-free solutions and a 

 nitrogen atmosphere often eliminates this problem. (4) The mercurial may 

 react with other groups or other components of the enzyme system, e.g., 

 in the spectrophotometric titration with p-MB or jj-MPS, causing absorp- 

 tion changes unrelated to SH groups. (5) The mercurial may do something 

 that secondarily alters the ultraviolet absorption, e.g., split off a coenzyme, 

 as demonstrated for NADH: lipoamide oxidoreductase (Palmer and Massey, 

 1962). (6) The presence of substances, especially buffers, reacting with the 



