Phase transition release, a new approach to the interaction of proteins with lipid vesicles Application to lipoproteins

Abstract

To study the interaction of proteins with lipid bilayers, we have developed a new experimental approach based on the release of a water-soluble fluorescent dye from liposomes during scans through the lipid phase transition temperature. The fluorescence of carboxyfluorescein is quenched at high dye concentrations inside the vesicles but appears when the dye is released and diluted into the external medium. This new approach, phase transition release, is here applied to the interaction of serum lipoproteins and apolipoproteins with liposomes (for the most part, small unilamellar vesicles of dipalmitoylphosphatidylcholine). The major findings are these: (i) All of the lipoproteins and apolipoproteins tested induce a smooth, rapid release of carboxyfluorescein, essentially complete within a few seconds. HDL apolipoprotein induces 50% carboxyfluorescein release at a lipid/protein molar ratio of 3 400 : 1, whereas a ratio of 160 : 1 is required for native HDL. (ii) The interaction is all-or-none and irreversible. It involves a sufficient perturbation of bilayer structure to permit equal release of carboxyfluorescein ( M r 373 ) and inulin ( M r 5 500 ). In the case of HDL apolipoprotein, this release accompanies formation of a relatively homogeneous population of vesicular recombinant structures. Only at much higher protein/lipid ratios are the often-studied small, disc-shaped recombinants formed. (iii) Much more dye is released if the transition temperature is approached from below than if it is approached from above. (iv) Phase transition release is seen with multilamellar and reverse phase evaporation vesicles, though with patterns different from those seen with small unilamellar vesicles. (v) A large number of proteins are found not to induce phase transition release, even at concentrations of at least 1000-times those required for the HDL apolipoprotein effect. These include trypsin, chymotrypsin, pronase, bovine serum albumin (crystalline), ovalbumin, rabbit immunoglobulin G (and its F(ab)′ 2 and F c fragments), rabbit immunoglobulin M, hemoglobin, hen lysozyme, synexin, ankyrin, myosin, and microtubule-associated proteins. Tubulin and actin, on the other hand, do induce phase transition release. In addition to its use for analysis of protein-bilayer interaction, phase transition release provides a way of reconstituting relatively water-soluble proteins into vesicles, under quantitative control and without detergent.