HETEROGENEITY OF BOVINE CARBOXYPEPTIDASE A .2. CHROMATOGRAPHIC PURIFICATION OF CARBOXYPEPTIDASE A (COX)

Bovine carboxypeptidase A (Cox) has been chromatographically purified on DEAE-cellulose (DE-52) using a complex gradient of both LiCl and β-phenylpropionate. Four fractions having similar specific esterase and peptidase activity have been isolated. Isolation of the carboxyl-terminal peptides obtained by cleavage with cyanogen bromide indicate that peak I is carboxypeptidase AαVal, peak II is carboxypeptidase AαLeu containing a 30% contamination of carboxypeptidase AβVal, and peak III is carboxypeptidase AβLeu. Each of these fractions as well as those isolated from carboxypeptidase A (Anson) are homogeneous by disk gel electrophoresis. As to be expected, the amino acid replacement previously found in the carboxyl-terminal region of carboxypeptidase Aβ and Aγ is also present in Aα. Heat-inactivation studies at 50° reveal that both the chain length of the amino-terminal region of the enzyme and the amino acid replacements characterizing each valine and leucine variant may be important in stabilizing the conformation of the molecule. © 1969, American Chemical Society. All rights reserved.