PRIMARY STRUCTURE OF BOVINE CARBOXYPEPTIDASE B .2. TRYPTIC PEPTIDES FROM REDUCED AMINOETHYLATED PROTEIN

Conditions have been developed for the quantitative reduction and aminoethylation of bovine carboxypeptidase (Cpase) B. The product, after solution in trifluoroacetic acid and precipitation with water, was susceptible in part to tryptic digestion. The application of three cycles of trifluoroacetic acid treatment followed by digestion with trypsin sufficed to convert approximately 90% of reduced, aminoethylated (RAE) Cpase B into soluble peptides. These were subdivided into three groups by gel filtration over Sephadex G-25. The individual groups were further fractionated by chromatography on columns of Dowex 50-X2 and Dowex 1-X2 to afford ultimately free lysine and 35 peptides (ranging in size up to 34 residues) in a state of purity satisfactory for subsequent sequential degradation. Evidence was obtained that peptides containing each of the 7 aminoethyl-cysteine residues in RAE-Cpase B were isolated. There is also evidence that 23 of the 29 theoretically possible lysine- or arginine-containing peptides were obtained. In summary, nonoverlapping peptides were found to provide a representation of 235 of the 300 residues in Cpase B. © 1968.