Resolution and quantitation of apolipoproteins A-I and A-II from human high-density lipoprotein by size exclusion high-performance liquid chromatography.
Apolipoproteins A-I and A-II, extracted from human high-density lipoprotein (HDL), were resolved and quantified by size exclusion high-performance liquid chromatography on TSK 125 and TSK 250 analytical columns connected in series without the use of chemical denaturants or detergents in the eluent buffer. The columns were pre-equilibrated with a solution containing 0.1 M sodium phosphate, pH 7.2, 0.2 M sodium chloride at a flow-rate of 1 ml/min. Delipidated HDL (1 mg protein per ml) was resolved into two populations of apolipoprotein (apo) A-I: one representing the apo A-I monomer and the other, a self-associated form with a molecular weight of approximately 120,000 daltons. The column eluates were screened for immunoreactivity to apo A peptides, and the identity of each peak was confirmed by sodium dodecyl sulfate--polyacrylamide gel electrophoresis followed by immunoblot analysis. Apo A-I peptides isolated by high-performance liquid chromatography disrupted unilamellar phospholipid vesicles to form smaller phospholipid particles that eluted on gel filtration columns within the size range of HDL. Thus, a rapid method for the isolation and quantitation of non-denatured apolipoproteins from HDL has been developed using size exclusion high-performance liquid chromatography.
D016632:Apolipoprotein A-I; D016633:Apolipoprotein A-II; D001054:Apolipoproteins A / Q000097:blood*; D001345:Autoradiography; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006801:Humans; D008075:Lipoproteins, HDL / Q000097:blood*; D008970:Molecular Weight; D010743:Phospholipids / Q000097:blood
D016632:Apolipoprotein A-I; D016633:Apolipoprotein A-II; D001054:Apolipoproteins A; D008075:Lipoproteins, HDL; D010743:Phospholipids