Isolation and Characterization of the Cyanogen Bromide Glycopeptide Fragments of cr-1-Antitrypsin”

cy-1-Antitrypsin can be cleaved by cyanogen bromide and separated on a column of Sephadex G-50 into a glycopeptide-containing fraction and four to seven carbohydrate-free peptides. The void volume fractions containing the glycopeptides can be resolved further into four unique glycopeptides (A, B, C, and D) by ion exchange chromatography on DE62 cellulose by elution with increasing concentrations of ammonium bicarbonate. Rechromatography of each glycopeptide peak on DE62 cellulose yields symmetrical peaks upon elution which were characterized by unique amino acid and carbohydrate compositions. Tryptic peptide mapping of each glycopeptide gives the expected number of tryptic peptide spots based on the number of arginine and lysine residues contained in each glycopeptide. The four glycopeptides account for all of the carbohydrate mass of a-1-antitrypsin and 80% of the peptide mass. Glycopeptide A contains 6 residues of N-acetylglucosamine, 2 residues of mannose, 2 residues of galactose, and 3 residues of sialic acid per mol; glycopeptide B contains 6, 2, 2, and 1 residues per mol, respectively, glycopeptide C contains 4,2,2, and 1 residues per mol, respectively, and glycopeptide D contains 3, 1, 1, and 1 residues per mol, respectively. This paper represents the first report of the isolation and purification of milligram quantities of four unique glycopeptide fragments from cy-1-antitrypsin which should prove useful as acceptors in studies concerning the requirements for correct sialic acid addition by human liver sialyltransferase.