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 lipid composition. All the reproducible subfractions contained polypeptides A-I, A-II, C-I, C-II, and the thinline polypeptide (TL). The C-III polypeptide was constantly lacking in sub-fraction II but present in subfractions Ia and III. Subfraction II was characterized by a high relative lysolecithin content and a low ratio between un-esterified and esterified cholesterol (indicating high cholesterol ester content). This apparent susceptibility of subfraction II for the LCAT reaction (in agreement with our earlier data) has directed our attention to this fraction for in vitro incubation studies, now in progress.

Key Words:

• Lipoprotein subfractions