Ligand bindings of bovine carboxypeptidase B. II. Affinity chromatography and cooperative ligations

Affinity chromatography was used to characterize the binding properties of carboxypeptidase B with its ligands. The affinity adsorbents employed included arginine directly attached to agarose beads, arginine attached to the same support through hydrophilic and hydrophobic spacers, and immobilized caproylphenylalanine. The enzyme showed marked retardation on all of the arginine columns but only slight retardation on the phenylalanine column. Several hydrophobic ligands in solution enhanced to some extent the enzyme retardation on columns having arginine directly attached to the solid support, while several amino- and guanidino-alkylcarboxylic acids (lysine and arginine analogs) greatly enhanced the enzyme retardation on the phenylalanine column and somewhat enhanced it on the other columns having hydrophobic spacer arms. These observations confirmed that the enzyme has twobinding sites for the soluble and immobilized ligands and that these two sites exhibit cooperative ligations. Binding constants of the enzyme for various soluble ligands were also calculated from their chromatographic effects and the resulting values were interpreted in terms of the cooperative action of the two bindings sites, i.e., one for the primary binding of basic amino acid analogs (SITE I) and the other for hydrophobic compounds (Site II). In this chromatographic study, however, such cooperation of the two sites was obscure when arginine, acylarginine, or alkylarginine was the ligand directing to Site I.