840 15. EFFECTS OF VARIOU'S FACTORS ON INHIBITION 



nate is represented by the differences between the NaCl and NaHCOg 

 curves. The use of a neutral salt to correct for specific buffer effects is often 

 a valuable procedure. If the buffer inhibits, the addition of another inhibitor 

 will create a situation of two inhibitors acting simultaneously, as described 

 in Chapter 10, and the inhibition constants calculated for the added inhi- 

 bitor will not be the true constants for that inhibitor if the buffer effect 

 is neglected. 



The inhibitions produced by phosphate, acetate, bicarbonate, and other 

 buffer anions are frequently due to a competition with a negatively charged 

 substrate for a cationic site on the enzyme. Most enzymes that act upon 

 substrates containing phosphate groups will be inhibited to varying extents 

 by phosphate, because an enzyme cationic group will usually be present at 

 the active center for interaction with the phosphate group and this will 

 be susceptible to inorganic phosphate. In other cases these anions may 

 combine with a metal ion incorporated in the enzyme, as in the complex 

 formed between acetate and the iron atoms of catalase ( Agner and Theorell, 

 1946). 



The binding of ions to proteins in general is often modified by buffers 

 as has been found in the studies on anion complexes with serum albumin by 

 Klotz (1953) and others. These are mainly competitive reactions. The 

 magnitude of the buffer effect depends on the structure of the buffer anion. 

 Often the greatest interference is introduced by large anions and for this 

 reason buffers such as the barbiturates should be avoided whenever possible. 

 Specific buffer effects on proteins can often be demonstrated by electropho- 

 resis as well as by the indirect experiments on anion binding. 



The buffer may affect the ionization of enzyme groups at or adjacent 

 to the active center and thereby alter the binding of substrates or inhibi- 

 tors. The ])K,'s for the ES complexes of fumarase are generally about 0.5 

 unit lower in acetate than in phosphate buffer (Frieden and Alberty, 

 1955) and for the enzyme-malate complexes vary with the phosphate 

 concentration (Massey and Alberty, 1954). The pH curves for both en- 

 zyme activity and inhibition of fumarase are shifted by buffer anions 

 (Massey, 1953 a). As the concentration of phosphate is increased to 5 mM, 

 stimulation of the rate is observed, but beyond this concentration there 

 is progressive inhibition and an increase in K,,^ (Alberty et al., 1954); in 

 a sense, this might be considered as activator inhibition and treated ki- 

 netically in the same way as substrate inhibition. 



Particular attention must be paid to the buffer in inhibition studies 

 involving the heavy metal ions (e.g., Cu++, Pb++, Zn++, Ag+, and Cd++) 

 and other substances readily interacting with certain buffers. Not only 

 may the complexes formed reduce the concentration of free inhibitor, but 

 the complexes themselves may possess activity, indeed may be the true 

 inhibitors in some cases. When two or more buffers are used to compare 

 the effects of inhibitors and determine the specific buffer effects, it is im- 



