Micellar complexes of human apolipoprotein A-I with phosphatidylcholines and cholesterol prepared from cholate-lipid dispersions

Abstract

Micellar complexes of human apolipoprotein A-I and phosphatidylcholine, with or without cholesterol, were prepared by adding apolipoprotein A-I (apo A-I) to sodium cholate-lipid mixtures. Cholate was removed by dialysis and the apo A-I.lipid complexes were isolated by gel filtration chromatography or by density gradient ultracentrifugation. The lipid mixtures consisted of dipalmitoylphosphatidylcholine or egg yolk phosphatidylcholine in the presence of various molar ratios of cholesterol. The formation of complexes was examined at different phosphatidylcholine (PC)-to-apo A-I ratios, PC-to-cholate ratios, and cholate concentrations. Yields of complexes were maximal when incubation and dialysis were performed near the transition temperature of the PC. Upon lipid binding and complex formation, apo A-I experienced a significant increase in alpha-helix content, and a blue shift in the intrinsic tryptophan fluorescence. In all lipid-protein incubation mixtures, from 600:1 to 75:1, PC/apo A-I (molar ratios), relatively small, stable complexes were present which gave maximum yields at incubation ratios similar to their isolated stoichiometries of 75:1 to 140:1, PC/apo A-I (molar ratios). For the isolated complexes, molecular weights were determined by sedimentation equilibrium to be in the range from 220,000 to 260,000; fluorescence polarization using the hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene showed a broadened and shifted gel to liquid-crystalline phase transition, characteristic of micellar complexes of apo A-I with PC. Complexes prepared using apo A-I, covalently labeled with 5-dimethylaminonaphthalene-1-sulfonyl chloride, had an overall particle rotational relaxation time of 530 ns. On electron micrographs, the complexes, negatively stained with phosphotungstate, appeared as lamellar, discoidal particles.