Structure of the antitumor protein neocarzinostatin. Purification, amino acid composition, disulfide reduction, and isolation and composition of tryptic peptides

The antitumor protein neocarzinostatin has been purified by repeated CM-cellulose chromatography and Sephadex G-50 gel filtration. The protein possesses 109 amino acid residues in a single chain, crosslinked by two disulfide bonds. Reduction and S-alkylation could not be accomplished in aqueous solution and required the use of dithiothreitol in liquid ammonia, followed by treatment with alkyl chloride. Tryptic digestion of (tetra-S-carboxymethyl)neocarzinostatin afforded five tryptic fragments which were fractionated by preparative paper chromatography and high-voltage paper electrophoresis, purified by Sephadex gel filtration and characterized by amino acid and amino end-group analysis. The total number of amino acid residues of these fragments account for the 109 residues of neocarzinostatin.