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

Abstract

Abstract Fraction III, 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 fractionated by ion exchange chromatography (DEAE) in 8 m urea into two major broad components, IIIa and IIIb, in ratios varying from individual to individual and exhibiting a slight difference in mobility by polyacrylamide gel electrophoresis or disc gel electrofocusing. The molecular weight of each of the two fractions was about 27,000, as assessed by sodium dodecyl sulfate-polyacrylamide electrophoresis in the presence or absence of 8m urea. The same value was obtained by agarose-guanidine HCl chromatography. Both IIIa and IIIb reacted similarly against antisera raised against whole III; they contained no half-cystine or sialic acid, had aspartic acid as NH2-terminal (dansylation and Edman procedure), glutamine as COOH-terminal (digestion with carboxypeptidase and hydrazinolysis), but differed slightly in amino acid composition: an established difference was that IIIb contained no isoleucine, whereas IIIa had 1 mole of this amino acid per mole of protein. However, the presence of isoleucine in IIIa was not a constant finding. Upon rechromatography of DEAE, IIIa and IIIb were resolved into three and two components, respectively, each having the amino acid composition of the starting product. After treatment with the bifunctional reagent, dimethylsuberimidate, III exhibited two distinct bands by sodium dodecyl sulfate-polyacrylamide electrophoresis with an apparent molecular weight of 27,000 and 54,000, suggesting a monomer-dimer relationship. Under the same experimental conditions, IIIa or IIIb exhibited only one band with a molecular weight of about 27,000. The results of the present studies were taken to indicate that HDL2 contains at least two polymorphic forms of III having equimolecular weight, but differing slightly in chemical composition and associated together by noncovalent linkage.