Single-step isolation and resolution of pancreatic carboxypeptidases A and B

Carboxypeptidases A and B have been isolated individually from aqueous extracts of mammalian pancreatic acetone powders by affinity chromatography on [N-(epsilon-aminocaproyl)-p-aminobenzyl]succinyl-Sepharose 4B (CABS-Sepharose). The affinity ligand was synthesized from DL-benzylsuccinic acid, purified, and characterized by UV absorption and NMR spectroscopy. Both enzymes from the various species were homogeneous by NaDodSO4-polyacrylamide gel electrophoresis and displayed high specific activities. No cross contamination of one enzyme species with the other was found. The ease of synthesis of the ligand from its commercially available precursor, its stability, and the mild elution conditions render CABS-Sepharose an excellent affinity support for the single-column isolation of both carboxypeptidases A and B. The procedures extend the utility of this resin previously demonstrated for carboxypeptidase A from human pancreatic juice [Peterson, L. M., Sokolovsky, M., & Vallee, B. L. (1976) Biochemistry, 15, 2501]. The use of CABS-Sepharose as a general affinity matrix for the isolation of metallocarboxypeptidases is suggested.