716 



6. O-IODOSOBENZOATE 



the slow phase is a further reaction with enzyme groups rather than a sec- 

 ondary inactivation. It has been emphasized several times with other en- 

 zymes, e.g. /3-amylase (Englard et al., 1951) and phosphoglucomutase (Mil- 

 stein, 1961), that the reaction with o-iodosobenzoate is slow. For this reason, 

 many of the results given in Table 6-1 are not comparable, since different 

 times of incubation with the inhibitor were used and usually the times were 

 not given. Unless preincubation of the enzyme with o-iodosobenzoate for a 

 reasonably long period (at least 30 min) is done, it is likely that the inhibi- 

 tions determined are partial and do not accurately represent the true effect 

 of the oxidant on all of the enzyme present. 



Fig. 6-2. Rates of inhibition of ribonuclease by 

 1 raM o-iodosobenzoate at various pH's. The times 

 indicate the duration of the incubation of the en- 

 zyme and inhibitor. (From Ledoux, 1954.) 



Effects of pH 



It has been emphasized that in titrations of protein SH groups a pH near 

 neutrality must be maintained if specificity is desired; below a pH of 7 the 

 oxidizing power of o-iodosobenzoate increases, and oxidation of other pro- 

 tein groups may occur. Also the solubility of o-iodosobenzoate decreases 

 rapidly below pH 7. One might expect, therefore, that the inhibition of en- 

 zymes might decrease as the pH is raised above 7, but just the opposite 

 has been observed. /^-Amylase is inhibited more strongly and more repro- 

 ducibly at pH 7.8 than at pH 7 (Englard et al., 1951 ), succinate dehydrogen- 

 ase is inhibited more at pH 7 than at pH 6 (Stoppani et al., 1953), and 

 ribonuclease is inhibited more rapidly and completely as the pH is increased 

 from 7 to 10, no inhibition occurring at pH 5 (Fig. 6-2) (Ledoux, 1954). The 

 very rapid initial oxidation of ribonuclease at pH's above 8.5 may be due 



