A novel procedure for the separation of high-density lipoprotein subspecies by affinity chromatography

Plasma high-densitjj lipoprotein (HDL) consists of a number of structurally and functionally distinet subspecies. Flotation procedures permit the separation of HDL, and HDL, (density ranges 1.063-1.125 and 1.125-1.21 g/ml, respectively), whilst apolipoprotein (apo) E-rich and apo E-poor subfractions have been obtained by heparin-Sepharose affinity chromatography (Weisgraber & Mahley, 1980). Although HDL has been resolved into five subfractions by gradient gel electrophoresis (Blanche et a/., 1981) and by gel filtration (Clifton et a/., 1987), methods for the physical isolation of these subfractions have not been reported. This communication describes the separation and characterization of subfractions of HDL, by cation exchange on an affinity column. HDL was separated from the plasma of normal, healthy male subjects by ultracentrifugatiron (40 h at 10.5 000 g) at density I .2 1 g/ml after the removal of very-low-density and low-density lipoproteins by centrifugation for 18 h at density 1.063 g/ml. The HDL was applied to a 1 c m x 12.5 cm column containing heparin-Sepharose (Griffin et a/., 19880) in 5 mM-Tris/HCI, 50 mM-NaCl, 25 mM-MnCI,, pH 7.4, and the non-bound fraction I was eluted with this buffer. The bound HDL species were eluted by stepwise addition of (1)