Studies on Human Serum High-Density Lipoproteins II. Isolation of Subfractions in the Cold

Abstract

The lecithin cholesterol acyltransfer (LCAT) reaction appears to be responsible for the degradation of high-density lipoproteins (HDL). In an earlier study substrate HDL subfractions were isolated and purified in the presence of an SH-blocking agent, DTNB. Difficulties in removing all enzyme inhibition for the subsequent HDL degradation studies argued for the importance of finding an alternate ‘inhibited’ isolation procedure of HDL. HDL with a density of 1.067–1.20 g/ml was isolated and purified in the cold (0 to +4 °C) by repeated preparative ultracentrifugations and by gel chromatography. Five subfractions were obtained in the cold by the stepwise elution on hydroxyl-apatite column chromatography, using phosphate buffers of increasing molarity. Of the five subfractions, three, subfractions Ia (0.001–0.01 mol/1 phosphate buffer), II (0.05 mol/1 phosphate buffer), and III (0.15 mol/l phosphate buffer), appeared to be reproducible from one preparation to the other as judged from polypeptide and relative li...