44 1. lODOACETATE AND lODOACETAMIDE 



bility to inhibitors varies markedly. Many of them, especially the cathepsins 

 and those simply designated "proteinase" or "protease," are not well char- 

 acterized. Despite the large number investigated, there have been very 

 few quantitative studies of the mechanisms of inhibition. Sometimes it is 

 difficult to determine in certain studies whether the inhibitor acts on the 

 enzyme or on the substrate, since preincubation studies have seldom been 

 done. Small synthetic peptides as substrates are preferable from this stand- 

 point, but it is likely that the inhibition will often be different from that 

 found when proteins are used. In some instances one must consider the 

 reaction of iodoacetate with a natural activator (which may be cysteine or 

 glutathione), as postulated by Michlin and Rubel (1933). 



It was demonstrated several times in the early work that the nitroprusside 

 reaction is reduced following reaction of iodoacetate with proteolytic en- 

 zymes. When papain is inactivated with iodoacetate, one SH group is car- 

 boxymethylated, and thus complete inhibition occurs from the addition of 

 1 equivalent of the inhibitor (Balls and Lineweaver, 1939 a), as is also the 

 case with ficin (Liener, 1961). Studies with papain became confused, due 

 mainly to the report of Ganapathy and Sastri (1939) that iodoacetate reacts 

 readily with oxidized (disulfide) papain, leading them to postulate other 

 groups than SH as important. They also said that the amount of iodoace- 

 tate required for inactivation is much less than to react the SH groups; 

 however, papain apparently contains only one reactive SH group, and five 

 additional half-cystines. More recently, iodoacetate-treated papain has been 

 hydrolyzed and an *S-carboxymethyl-L-cysteine residue detected (Finkle and 

 Smith, 1958). E. L. Smith (1958) has suggested that the SH group in papain 

 is perhaps a "high-energy" group, existing as a thiol ester with an adjacent 

 carboxyl group; this may hinder reaction with certain reagents (e.g., nitro- 

 prusside and porphyrindin) but is claimed to increase reactivity to mercuri- 

 als and alkylating agents. When high concentrations of inhibitor are re- 

 quired for inactivation, one cannot be certain that only SH groups are 

 involved, as pointed out by Maver and Thompson (1946) for lymphosar- 

 coma cathepsin, which is inhibited 21% by 10 mM iodoacetamide. 



Comparisons on a quantitative basis between iodoacetate, iodoacetamide, 

 and o-iodosobenzoate are rare, and thus it is interesting to note the results 

 of Tsuboi et at. (1957) on human erythrocyte tripeptidase (Fig. 1-4). Not 

 only is iodoacetamide rnore potent than iodoacetate, but the inhibition- 

 concentration curves are somewhat different in configuration. This enzyme, 

 unfortunately, is not one of those particularly sensitive to iodoacetate; it 

 would be interesting to know what happens to the iodoacetamide curve at 

 lower concentrations. This enzyme is unusual also in that it is more inhi- 

 bited by o-iodosobenzoate than by the alkylating agents. 



