12 1. lODOACETATE AND lODOACETAMIDE 



chloroacetate at pH 6.8 and 25^; indeed, it requires 44 hr to block 50% of 

 the SH groups with chloroacetate. The iodopropionates react quite slowly 

 with glutathione, indicating the importance of the inductive effects in 

 promoting reactivity, and suggesting that the effects observed with the 

 iodopropionates are not due to reaction with SH groups (Schroeder et al., 

 1933 b). It has generally been considered that reaction of iodoacetate with 

 proteins is entirely through the cysteine residues with respect to SH groups. 

 However, Gundlach et al. (1959 b) have shown that the carboxymethyl- 

 sulfonium salt of methionine is formed at a rapid rate throughout the pH 

 range 2-8.5, and thus that methionine residues must also be visualized 

 as points of attack. 



The relationship between the pH and the reactivity of SH groups with 

 iodoacetate is a very important matter in determining which groups on 

 enzymes are alkylated (see page 33), and we shall now examine the evi- 

 dence obtained with simple thiols. Rapkine (1933 a, 1936) reported that the 

 reaction of iodoacetate with cysteine and glutathione accelerates as the pH 

 is raised, especially around pH 7, indicating that the ionization of the SH 



pH Relative rate of reaction with cysteine 



5.6 0.14 



6.3 0.28 



7.0 1.00 



7.53 1.40 



8.36 2.10 



groups is the critical factor and that the S" ion is the reactive form. Schroe- 

 der et al. (1933 b) found almost no reaction between iodoacetate and gluta- 

 thione at pH 4.5 but that the rate increases as the pH is raised to 8.5. 

 Similarly with bromoacetate, the reaction with cysteine is very slow at 

 pH 5.5, faster at pH 6.5, and fastest at pH 7.2 (Hansen, 1956 b), while 

 the kinetics of the reaction between chloroacetamide and mercaptoacetate 

 indicate the S" form to be the reactive species (Lindley, 1960). A very in- 

 teresting relationship was found by Smythe (1936) when he compared the 

 relative rates of reaction of thiols with iodoacetate and iodoacetamide at 

 different pH's. As the pH was dropped from 7.1 to 6.1. the rates with both 

 were lower, but the decrease was greater with iodoacetate; thus the ratio 

 of the rates with iodoacetamide and iodoacetate was 1.92 at pH 7.1, and 

 4.02 at pH 6.1 (means for all the thiols tested). It would be interesting to 

 know if at higher pH the difference between iodoacetate and iodoacetamide 

 disappears, which might mean that both react with the S^ form equally, 

 but that iodoacetamide reacts relatively more rapidly with the undissociated 

 SH group. Benesch and Benesch (1957) reported that iodoacetamide reacts 



