Isolation and Characterization of the Tryptic and Cyanogen Bromide Peptides of ApoLp-Gln-II (ApoA-II), a Plasma High Density Apolipoprotein

ApoLp-Gln-II (or apoA-II), one of the two major apolipoprotein components of human plasma high density lipoproteins, was subjected to enzymatic and nonenzymatic digestion with trypsin and cyanogen bromide. The individual peptides were isolated to homogeneity by chromatography on DEAE-cellulose, Cm-Sephadex, Sephadex G-100, Sephadex G-75, preparative thin layer plates, and by peptide mapping. Isolated peptides were assayed for purity by amino acid analysis, thin layer chromatography, polyacrylamide gel electrophoresis, and Edman NH2-terminal analysis. Cyanogen bromide cleavage followed by reduction and S-carboxymethylation produced two peptides, C-III and C-IV, of 51 and 26 residues, respectively. The smaller peptide (C-IV) contained homoserine and homoserine lactone and had NH2-terminal pyrrolidone carboxylic acid. It was the NH2-terminal cyanogen bromide peptide. This peptide contained the single S-carboxymethylcysteine residue. C-III was the COOH-terminal peptide. It contained COOH-terminal glutamine and a penultimate threonine residue. Tryptic digestion of reduced and alkylated apoLp-Gln-II yielded eight major tryptic peptides (T-I-1, T-I-2, T-I-3, T-III, T-IV, T-VI-1, T-VII, and T-VIII) and seven additional tryptic peptides which either contained or were contained in two or more of the eight major peptides. Only single unique NH2-terminal (T-I-2), COOH-terminal (T-VII), S-carboxymethylcysteine-containing (T-VIII), methionine-containing (T-VI-1), and isoleucine-containing (T-I-3) peptides were found. The combined compositions of the tryptic or cyanogen bromide peptides were equal to the composition of S-carboxymethyl-apoLp-Gln-II. These results confirm previous observations from this laboratory on intact and reduced and alkylated apoLp-Gln-II and support the concept that the molecule is composed of two identical chains of 77 residues each linked by a disulfide bond. The data available allow a partial alignment of these peptides and indicate that the disulfide bridge is in the NH2-terminal one-third of the molecule. The first three tryptic peptides in the sequence are T-I-2, T-VIII, and T-VI-1. T-VII is the COOH-terminal tryptic peptide. These assignments have been confirmed by manual and automated Edman degradations of the isolated tryptic and cyanogen bromide peptides performed in parallel with the present studies and published elsewhere (Brewer, H. B., Jr., Lux, S. E., Ronan, R., andJohn, K. M. (1972) Proc. Nat. Acad. Sci. U.S.A.69, 1304–1308).