On the Aminoethylation of Proteins

Abstract

The reaction of peptide and protein sulfhydryl groups with ethylenimine to give quantitative yields of S-6-aminoethylcysteinyl residues has been studied.Optimal conditions for tryptic hydrolysis of peptide bonds at the carboxyl terminus of such residues are described for the case of insulin B chain.Separation and identification of peptides obtained in this manner illustrate the applicability of the method to cleavage of polypeptide chains.The susceptibility of S-f-aminoethylcysteinyl residues in peptide linkage to hydrolysis by trypsin was first suggested by Lindley (1), who demonstrated that poly-L-AE-cysteinel served as a substrate for the enzyme.He also showed that some AEcysteinyl residues were produced when reduced wool was allowed to react with -bromoethylamine at pH 11.Tietze, Gladner, and Folk (2), using the same reaction conditions, effected conversion of cysteinyl to AE-cysteinyl residues in reduced insulin.By treatment with trypsin and carboxypeptidase B, free AEeysteine was identified.Weil, Seibles, and Telka (3) attempted by the same method to effect quantitative conversion of cysteinyl residues in a-lactalbumin, but only partial reaction was obtained.It seemed that a potentially useful method of degradation of protein molecules was of limited effectiveness because the alkylation was not complete.Recently a report from this laboratory (4) described a method for achieving essentially quantitative conversion of cysteinyl to AE-cysteinyl residues by reaction with ethylenimine under very mild conditions.In this communication we report further studies of this reaction, including isolation and identification of peptides obtained by tryptic hydrolysis of a polypeptide chain at AE-cysteinyl residues.