718 6. O-IODOSOBENZOATE 



Reactivation of o-lodosobenzoate Inhibition 



If the only action of o-iodosobenzoate is the oxidation of SH groups to 

 disulfide bonds, one might expect some reversal of the inhibition by thiols, 

 and this has been observed with certain enzymes. The inhibition of D-amino 

 acid oxidase is reversed completely by cysteine (Rocca and Ghiretti, 1958) 

 but in most cases only partial reactivation is possible, for example, succinate 

 oxidase by dimercaprol (Thorn, 1959) and by glutathione (Slater, 1949), 

 amylo-l,6-glucosidase by glutathione (Earner and Schliselfeld, 1956), thre- 

 onine aldolase by dimercaprol (Karasek and Greenberg, 1957), and alcohol 

 dehydrogenase by glutathione (Barron and Levine, 1952). It is difficult to 

 interpret partial reactivation since failure to reverse the inhibition com- 

 pletely may be due to a variety of factors. No reactivation by thiols has 

 been reported for a few enzymes: Acid phosphatase cannot be reactivated 

 by cysteine or thioglycolate (Tsuboi and Hudson, 1955 b), nor xanthine 

 oxidase by cysteine (Harris and Hellerman, 1956), nor /^-amylase by gluta- 

 thione or dimercaprol (Englard et al., 1951), nor 5-hydroxytryptophan de- 

 carboxylase by thiols (Buzard and Nytch, 1957). However, these failures 

 cannot be immediately attributed to other actions of o-iodosobenzoate and 

 perhaps the most likely explanation is progressive secondary inactivation 

 consequent to the protein distortion induced by disulfide bond formation. 

 Some failures might also be due to attempting reactivation in the presence 

 of oxygen, which can often oxidize the thiols to disulfides, which in turn 

 can inhibit the enzyme, as pointed out by Slater (1949). 



Variation of the Inhibition with the Substrate Used 



The degree of inhibition of lipase by 1 mM o-iodosobenzoate varies with 

 the substrate (Singer, 1948; Singer and Hofstee, 1948 a). The inhibition by 

 p-chloromercuribenzoate is also dependent on the substrate used and Singer 

 postulated that the mercurial is bound near the substrate site so that it 

 sterically interferes with the binding of the substrates, the interference be- 

 ing greater the larger the substrate molecule. However, how this explanation 

 could apply to o-iodosobenzoate is not clear, since the simple formation of 



