Isolation and Sequence of Peptides at the Active Center of Bovine Carboxypeptidase B

The tyrosyl residue at the active center of bovine pancreatic carboxypeptidase B has been labeled with 4-bromoacetamido-1-14C-butylguanidine. The labeled protein was hydrolyzed with chymotrypsin after alkylation of the free sulfhydryl group with iodoacetamide. Two 14C-containing peptides were isolated in 12.5 and 33.6% yields, respectively, by a combination of gel filtration on Sephadex G-25 and ion exchange chromatography on Dowex 50-X4 and 1-X2. Composition, end group analysis, and enzymatic hydrolysis with carboxypeptidase A, aminopeptidase M, and papain suggest the following amino acid sequences for the active center peptides: Peptide A, Thr-Ile-Tyr-Pro-Ala-Ser-Gly-Gly-Ser-Asp-Asp-Trp; Peptide B, Ile-Tyr-Pro-Ala-Ser-Gly-Gly-Ser-Asp-Asp-Trp. The two peptides are derived from the same portion of the enzyme. Comparison with an active center peptide containing tyrosine from carboxypeptidase A revealed certain similarities in the amino acid sequence of the two enzymes.