Immobilized Anhydrochymotrypsin as a Biospecific Affinity Adsorbent for the Peptides Produced by Chymotryptic Hydrolysis1

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Abstract
Anhydrochymotrypsin immobilized on Sepharose specifically adsorbed various peptides containing L-tryptophan, L-tyrosine, or L-phenylalanine residues at their carboxy-termini. These peptides correspond to the specific products of chymotryptic cleavage of polypeptides. A mixture of the chymotryptic peptides once adsorbed on the column could be effectively separated by eluting them with a pH gradient. This adsorbent, on the other hand, showed no substantial affinity toward the substrate-type peptides that contain the aromatic amino acid(s) within the peptide chain, or toward the C-terminal leucine peptides. By taking advantage of this unique property of anhydrochymotrypsin-Sepharose in combination with reversed-phase high-performance liquid chromatography, we have succeeded in separating the C-terminal peptides from chymotryptic digests of reduced and S-carboxymethylated bovine insulin and from tryptic digests from reduced and S-carboxymethylated Streptomyces subtilisin inhibitor.