An assay for carboxypeptidases A and B onpolypeptides from protein

Abstract

Summary New assays for carboxypeptidase A and carboxypeptidase B were developedusing polypeptides prepared from proteins as substrates. The substrates were prepared by treating casein with either α-chymotrypsin or trypsin and then reductively methylating the amino groups of the resulting mixtures of peptides with NaBH 4 and HCHO. Reductively methylated chymotrypsin-treated casein and reductively methylated trypsin-treated casein were relatively specific substrates for carboxypeptidase A and carboxypeptidase B, respectively. Hydrolysis of COOH-terminal amino acids was quantitatively measured by reaction of newly formed amino groups with trinitrobenzenesulfonic acid. Controls containing the substrates without additions of the carboxypeptidases gave very low blank values with trinitrobenzenesulfonic acid because of the unreactivity of this reagent with dimethylamino groups. As little as 0.1 μg of either enzymes was adequate for assay. The pH optima for both enzymes were similar to the optima of these enzymes on dipeptides, but the pH profiles were broader. Carboxypeptidase B did not hydrolyze COOH-terminal e- N,N -dimethyllysines, but it rapidly hydrolyzed COOH-terminal homoarginines.