Asymmetry of lipid dynamics in human erythrocyte membranes studied with permeant fluorophores

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Abstract
The fluorescence anisotropy and mean excited-state lifetime of 1,6-diphenyl-1,3,5-hexatriene, 12-(9-anthroyloxy)stearate, 2-(9-anthroyloxy)stearate and pyrenedecanoic acid in the membranes of intact human erythrocytes, lysate suspensions and ghost membranes were compared. The excited-state lifetime of each lipid fluorophore, estimated by single photon counting, is significantly shorter in the intact erythrocytes as compared to the lysates, owing to nonradiative energy transfer from the lipid fluorophore donors in the membrane to heme acceptors at the endofacial surface of the intact cell. The fluorescence observed in intact cell suspensions is thus weighted in favor of outer leaflet fluorophores, and estimates of the fluorescence anisotropy by steady-state fluorescence polarization indicate that all 4 fluorescent probes experience greater motional freedom in the outer as compared to the inner membrane leaflet. Prior studies of impermeant pyrene derivatives also indicate that the outer leaflet lipids have greater motional freedom.