On the Subunit Structure of the Protein of Human Serum High Density Lipoprotein

• Published in: The Journal of biological chemistry Vol. 247; no. 18; pp. 5850 - 5855
• Main Authors: Scanu, Angelo M., Lim, Chang T., Edelstein, Celina
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 25.09.1972; American Society for Biochemistry and Molecular Biology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)44835-7

Fraction IV, separated and purified from delipidated human serum high density lipoprotein of d 1.063 to 1.125 g per ml (HDL2) by gel filtration in 8 m urea, was further studied before and after reduction with β-mercaptoethanol (β-ME), or reduction and carboxymethylation (iodoacetamide). Both the reduced (R-IV) and the reduced and S-carboxymethylated (SC-IV) preparations exhibited a single band by polyacrylamide gel electrophoresis with a molecular weight of about 8,500, a figure corroborated by Sephadex G-200-8 m urea and agarose-guanidine HCl column chromatography. In the absence of the reducing agent, Fraction IV had a molecular weight of about 17,000. Treatment of R-IV (in the presence of β-ME) or of SC-IV by the bifunctional reagent dimethylsuberimidate led to the formation of four components separable by analytical sodium dodecyl sulfate (SDS)-polyacrylamide gel, with an apparent molecular weight of approximately 8,500; 17,000; 25,000; and 33,000. In turn, unreduced and suberimidate-treated IV, exhibited two bands (SDS-polyacrylamide) with an apparent molecular weight of 17,000 and 33,000, respectively. In initial studies, where partially carbamylated preparations were used, fractionation of SC-IV by DEAE-cellulose column chromatography yielded two major and three minor fractions which all reacted with antisera raised in rabbit against whole Fraction IV, and differed mainly from each other in lysine and homocitrullin content. In subsequent studies, which used noncarbamylated products, SC-IV gave a single peak by both Sephadex and DEAE-chromatography, a major component by isoelectric focusing, and exhibited a single band by 8 m urea or SDS-polyacrylamide electrophoresis. This preparation had no histidine, arginine, or tryptophan, and had glutamine as COOH-terminal (carboxypeptidase digestion and hydrazinolysis). It contained no sialic acid, showed no NH2-terminal by either dansylation or by the Edman's procedures, and gave a single precipitin line against specific antisera. It is concluded that Fraction IV, as separated and purified from apo HDL2 by Sephadex chromatography, is made up of chemically very similar, and possibly identical protomers each having the same molecular weight (about 8,500). The data also indicate that in HDL2 these protomers are paired by a single disulfide linkage into dimers of equivalent weight.