Isolation and Characterization of the Cyanogen Bromide Fragments from the High-Density Apolipoprotein Glutamine I

The major protein component of human plasma high-density lipoproteins (HDL), designated apoLP-Gln-I by the presence of carboxyl-terminal glutamine, was isolated from HDL by delipidation and chromatography on Sephadex G-150 and DEAE-cellulose in urea. Homogeneity of the isolated protein was established by amino acid analysis, by polyacrylamide gel electrophoresis in sodium dodecyl sulfate, and by amino- and carboxyl-terminal analyses. By amino acid analysis, the isolated protein contained 242 amino acids including three residues of methionine. ApoLP-Gln-I was treated with a 500 molar excess of cyanogen bromide, resulting in cleavage of more than 95% of the methionine residues. Chromatography of the digest on Bio-Gel P-30 in 25% formic acid yielded three of the fragments, CNBr II, III, and IV, in essentially a pure state. Special pretreatment conditions were required prior to chromatography on Bio-Gel P-30 in order to achieve satisfactory separation of the fragments in good yield. The fourth fragment, CNBr I, was further purified on DEAE-cellulose in 6 M urea. CNBr I contained 92 amino acids, was lacking homoserine or homoserine lactone, and had carboxyl-terminal glutamine, establishing this fragment as the carboxyl terminus of apoLP-GIn-I. The amino terminus of CNBr I was arginine. CNBr II contained 89 amino acid residues, amino-terminal aspartic acid, and carboxyl-terminal homoserine. Since only CNBr II contained amino-terminal aspartic acid, this fragment corresponded to the amino-terminal end of apoLP-Gln-I. CNBr III contained 37 amino acid residues, amino-terminal glutamic acid, and carboxyl-terminal homoserine. CNBr IV had 25 residues, amino-terminal serine, and carboxyl-terminal homoserine. Summation of the amino acid residues of the four fragments accounted for 243 residues, which is in good agreement with a value of 242 from amino acid analyses of the intact protein.