Selective isolation of C‐terminal peptides from proteolytic mixtures of proteins by affinity chromatography on immobilized anhydrotrypsin and anhydrochymotrypsin

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Abstract
Anhydrotrypsin in an immobilized form shows strong specific affinity for the peptides containing Arg, Lys, or S‐aminoethyl‐Cys at their C‐termini. By taking advantage of this unique property of the adsorbent, we established an efficient chromatographic procedure useful for selective isolation of C‐terminal peptides from protease digests of proteins. The utility was demonstrated in cases of a tryptic digest of Streptomyces subtilisin inhibitor in a reduced and S‐carboxymethylated form (RCM‐SSI) (with C‐terminal Phe) and of a chymotryptic digest of α1‐antitrypsin (with C‐terminal Lys): the C‐terminal peptides were recovered as the breakthrough fraction of the chromatography in the former case and as the adsorptive fraction in the latter. Immobilized anhydrochymotrypsin was also useful for selective adsorption of the C‐terminal peptide from the tryptic digest of RCM‐SSI. It was further found that immobilized anhydrotrypsin exerts strong affinity even for human oxyhemoglobin and its α‐chain (with C‐terminal Arg), but not for the β‐chain (with C‐terminal His) and hemoglobin‐haptoglobin complex. Thus the adsorbent may be applicable also to the isolation of macromolecular ligands and to the analysis of macromolecular interactions.