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Direct observation of phospholipid arrangement in biomembranes by fluorescence spectroscopy

The spontaneous transfer of pyrenedecanoyl-Iabeled phospholipids from small unilamellar vesicles to human erythrocyte ghost membranes was determined by a spectroscopic method. The fluorescence emission spectra of unilamellar vesicles containing 1 -acyl-2-pyrenedecanoyl- or 1 -O-(1'-alkenyl- -2-pyrenedecanoyl-sn-glycero-3-phospholipicjs of the choline and ethanolamine type show very high excimer to monomer fluorescence intensity ratios. Upon "dilution" of the fluorescent lipids into the erythrocyte acceptor membranes the excimer intensity decreased and the monomer intensity Increased. The time course of the monomer fluorescence increase revealed faster uptake of pyrene-labeled ether lipids (plasmalogens) as compared to the diacyl analogs. Significant amounts of pyrene-labeled diacyl phospholipids were incorporated into ghosts only in the presence of phospholipid transfer proteins. After incubation with pyrenephospholipid vesicles under appropiate conditions erythrocyte membranes showed almost exclusively pyrene monomer fluorescence Indicating random distribution of the fluorescent phospholipid probe within the membrane. Monomer fluorescence lifetimes of pyrene phospholipids In the erythrocyte membranes as determined by multifrequency phase fluorometry are very heterogeneous. In contrast, a single exponential decay ((tau)=103 ns) was observed for the monomer fluorescence of the pyrene phospholipids in unilamellar egg yolk phosphatidyicholine vesicles (label/lipid = 1/300 (mol/mol)). Thus, the fluorogenic phospholipids experience a much more heterogeneous environment in the biological membrane compared with a protein-free lipid bilayer system.