The reaction of iodoacetate with methionine.

lodoacetic acid and its amide have been widely used in protein chemistry as reagents for sulfhydryl groups. Although both can be expected to undergo other reactions with proteins, for example, with the e-amino groups of lysine residues, the imidazole groups of histidine residues, and the phenolic hydroxyls of tyrosine residues, these reactions arc generally slower than is the one with sulfhydryl groups (I, 2), particularly at neutral or slightly acid pH. The reaction of iodoacetate with the sulfur of methionine residues, however, although little mentioned in the literature that deals with the action of iodoacetate on proteins, might be expected, on the basis of experience with other alkylating agents, to take place readily at neutral pH. For example, experiments carried out in the laboratory of Dr. Max Bcrgmann in 1942 to 1944 (3) with bis(P-chloroethyl)sulfidc (mustard gas), showed that the formation of the sulfonium salt of mcthionine proceeded more rapidly than did reaction of the alkylating agent with either the amino or the carboxyl groups of amino acids. Tocnnies and Kolb (4) in their detailed studies of the formation of various sulfonium salts of methionine, demonstrated that both iodoacetic and bromoacetic acids reacted with methionine at an appreciable rate. The present investigation was prompted by the observation that when proteins that had been reduced with sodium borohgdride were treated with iodoacetate to cover the -SH groups formed (5), quantitative analyses for methionine gave results that were occasionally slightly low. The recoveries of methionine were also sometimes low upon analysis of native ribonuclease that had been treated with iodoacetate under certain conditions (6). In order to ascertain whether sulfonium salt formation might have taken place, the chemistry of the reaction of iodoacetate with methionine has been examined in more detail. Ion exchange chromatography has been used as a means of following the reaction. The ability to detect the alkylation of a methionine residue in a protein will depend in part upon the nature of the products that the resulting sulfonium salt yields when the protein is hydrolyzed in preparation for amino acid analysis. The mechanism of the decomposition of sulfonium salts of methionine has been studied frequently in the past 10 years, as the methyl donating role of sulfonium compounds in mammalian and microbial metabolism has become known (du Vigneaud (7); Challenger (8)). Cantoni’s (9) establishment of the significance of S-adenosyl-L-mcthionine in this connection has recently led to further study of its chemical and enzymatic decomposition by Parks and Schlcnk (lo), Shapiro and Mather (II), and Stekol et al. (12, 13). These studies, taken together with the earlier investigations of