MULTIPLE BINDING-SITES OF CARBOXYPEPTIDASE-B - EVALUATION OF DISSOCIATION-CONSTANTS BY QUANTITATIVE AFFINITY CHROMATOGRAPHY

Multiple binding sites for carboxypeptidase B (CPB) were studied by quantitative affinity chromatography. In the presence of 25 mM imidazole at pH 6.4, the enzyme was retarded by a gel to which d-Phe had been covalently bound (agarose-PheOH). Neither agarose-d-Phe methyl ester nor an agarose derivative that contained free carboxylate groups (Affi Gel 201) retarded the protein. d-Phe and N-acetyl-d-Phe but not Gly competitively eluted CPB. The dissociation constants (Dunn, B. M., & Chaiken, I. M. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 2382-2385) for the CPB-d-Phe and CPB-N-acetyl-d-Phe complexes were found to be 9.8 mM and 7.8 mM, respectively. The binding of the enzyme to agarose-PheOH was 7.5 times stronger than to the soluble d-Phe, suggesting that the spacer arm and/or agarose matrix was a contributing factor in the binding process. β-Phenylpropionic acid and ε-aminohexanoic acid induced further retardation of CPB on agarose-PheOH. By using a generalized binding equation, dissociation constants for the binary complexes CPB-ε-aminohexanoic acid and CPB- β-phenylpropionic acid in 25 mM phosphate at 6.4 were calculated as 1.8 and 0.65 mM, respectively. The dissociation of the ternary complexes (CPB-modifier-immobilized d-Phe) into the respective binary complexes plus immobilized d-Phe proceeded with KD's = 0.32 and 0.98 mM, respectively. In the presence of ε-aminohexanoic acid in phosphate buffer, pH 6.4, no induced retardation was observed on a column that contained free carboxylates but no Phe. The retardation induced by ε-aminohexanoic acid increased with decreasing pH. The results suggest that binding is facilitated by the presence of an acidic form of the amino acid side chain on CPB. © 1979, American Chemical Society. All rights reserved.