The Chromatographic Determination of Cystine and Cysteine Residues in Proteins as S-β-(4-Pyridylethyl)cysteine

Abstract

Abstract Disulfide bonds in proteins and in seed meals were reduced with β-mercaptoethanol to sulfhydryl groups. The generated sulfhydryl groups were then alkylated by treatment with 4-vinylpyridine. The cysteine residues were thus derivatized to S-(4-pyridylethyl)-l-cysteine residues. The cysteine derivative is stable to acid hydrolysis and on amino acid analysis by ion exchange elutes just before arginine. Complete modification of sulfhydryl groups permits quantitative determination of cysteine and cystine content (half-cystine residues) of proteins and of seed meals. The sulfhydryl groups of cysteine are the only groups modified when the alkylation is performed on solutions of reduced proteins at pH 7.5 for 90 to 120 min with an amount of 4-vinylpyridine equivalent to the mercaptoethanol used for reduction. In order to obtain complete modification of the sulfhydryl groups in seed meals, a longer reaction time, 4.5 hours, and a concentration of 4-vinylpyridine equivalent to 3 times the mercaptoethanol concentration were required.