Phase separation in phosphatidylcholine/anionic phospholipid membranes in the liquid-crystalline state revealed with fluorescent probes.

Abstract

The mixing properties of anionic phospholipids such as phosphatidic acid (PA), phosphatidylserine (PS), and phosphatidylglycerol (PG) with phosphatidylcholine (PC) were examined in the liquid-crystalline state of membranes using extrinsic fluorescent probes incorporated into lipid bilayers. The excimer to monomer (E/M) fluorescence ratio of 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine (PPC) was higher for the PA and PS matrices as compared to that for the PC matrix. When PC was replaced with PA or PS, the E/M ratio of PPC also increased in a concentration-dependent manner. When the concentration of PA or PS was increased in the PC membrane, the fluorescence of 1-palmitoyl-2-[12-[7-nitro-2,1, 3-benzodiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine decreased, indicating the occurrence of lipid clustering. Direct evidence for the PA or PS-induced phase separation in the PC/PA or PC/PS system was provided by the resonance energy transfer between 2-(4,4-difluoro-5-methyl-4-boro-3a, 4a-diaza-s-indacene-3-dodecanoly)-1-hexadecanoly -sn-glycero-3-phospho choline and PPC. The fluorescence polarization of 1,6-diphenyl-1,3, 5-hexatriene further supported the lateral organization of membranes by PA and PS. PA and PS also reduced the polarity of lipid bilayers, as measured by the emission fluorescence of 6-lauroyl-2-dimethylaminonaphthalene. On the other hand, PG had very little effect on the PC matrix, suggesting the ideal miscibility with PC molecules. The results suggest that the mixing properties of PA and PS in the PC matrix are not random but that phase separation occurs in the liquid-crystalline phase of membranes.