The selective S-methylation of sulfhydryl groups in proteins and peptides with methyl-p-nitrobenzenesulfonate.

The cysteine residues in proteins and peptides may be quantitatively and selectively converted to the S-methyl derivatives by reaction with methyl-p-nitrobenzenesulfonate. Procedures are described for the reduction and methylation of a variety of proteins at pH 8.6 and 37”, together with the pH rate profile for the methylation of reduced glutathione at 25”. The methylation of S-sulfoinsulin B chain and an S-benzylcysteinyl pentapeptide was performed after removal of the protective groups by mercaptoethanol and sodium in liquid ammonia, respectively. Amino acid analysis of the various S-methylated proteins and peptides revealed no modifications other than the quantitative conversion of cysteine to S-methylcysteine. The specific radioactivity and amino acid analysis of 14C-methylcysteinyl rhodanese isolated after reaction with radioactive reagent revealed that the modifications were confined to the 2 cysteine residues in rhodanese monomer. Methylation of the presumed active site cysteine residue in papain occurred only in 7 M urea and was accompanied by inactivation of the enzyme; papain was refractive to the reagent in the absence of denaturing agents.