Purification of carboxypeptidase B from human pancreas

Abstract

Carboxypeptidase B of the human pancreas was purified by chromatography on DEAE-cellulose and CM-cellulose columns. Two forms of the enzyme, named carboxypeptidase B1 andB2, were separated. They have similar mol.wts. (34250±590) as established by polyacrylamide-gel disc electrophoresis and by gel filtration. Carboxypeptidase B2 migrates further toward the anode in disc electrophoresis. When the amino acid content of the enzymes was analyzed, carboxypeptidase B2 had four more glycine and three more aspartic acid residues than had form B1. The amino acid sequence of the human carboxypeptidase B1 differs from that of the bovine enzyme only in two places in the N-terminal 20-amino-acid sequence. The N-terminal amino acid in carboxypeptidases B1 and B2 is alanine. The peptide 'map' of the tryptic digest of carboxypeptidase B1 contained more peptides than did that of form B2. The K(m), the V(max.) and the pH optimum of the cleavage of the peptide substrate hippurylarginine and the ester substrate hippurylargininic acid were similar for both enzymes. CoCl2 accelerated the peptidase activity, and cadmium acetate enhanced the esterase activity, of human carboxypeptidases B1 and B2. Urea and sodium dodecyl sulphate inhibited the enzymes.