Abstract
A simple and rapid method has been developed for the separation of apolipoproteins in high density lipoprotein (HDL) fractions by high performance liquid chromatography (HPLC) with gel permeation columns.
HDL was isolated by sequential ultracentrifugation. 10μl of the HDL fraction was added to 200μ of 0.1M sodium phosphate buffer (pH 7.0) containing 0.1% SDS and was incubated at 60°C for 5min. This mixed solution was applied to a HPLC apparatus (HLC 805, Toyo Soda). HPLC conditions were as follows: column, G3000SW (7.5mm I.D.×600mm: TSK GEL, Toyo Soda); eluent, 0.1M sodium phosphate buffer containing 0.1% SDS; flow rate, 0.33ml/min, applied volume, 200μl.
The HPLC pattern monitored by A280 showed two completely separated peaks. It was revealed that the two separate peaks corresponded to apo A-I and apo A-II by several experiments. And quantitation of apo A-I by our method was found to correlate well with that by a single radial immunodiffusion assay.
