SINGLE-STEP ISOLATION AND RESOLUTION OF PANCREATIC CARBOXYPEPTIDASE-A AND CARBOXYPEPTIDASE-B

Carboxypeptidases A and B have been isolated individually from aqueous extracts of mammalian pancreatic acetone powders by affinity chromatography on [N-(ε-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-poly-acrylamide 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. © 1979, American Chemical Society. All rights reserved.