Purification of carboxypeptidase B from human pancreas

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Abstract
Carboxypeptidase B [EC 3.4.12.3] of the human pancreas was purified by chromatography on DEAE-cellulose and CM-cellulose columns. Two forms of the enzyme, named carboxypeptidase B1 and B2, were separated. They have similar MW (34,250 .+-. 590) as established by polyacrylamide-gel disc electrophoresis and by gel filtration. Carboxypeptidase B2 migrates further towards the anode in disc electrophoresis. Amino acid analysis showed that carboxypeptidase B2 had 4 more glycine and 3 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 2 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 Km, the Vmax. 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 sulfate inhibited the enzymes.