The synthesis, purification, and application of five membrane-impermeant derivatives of pyrene are described. Each probe consists of a membrane-impermeant moiety, either an oligosaccharide or glutathione, linked to pyrene via a connecting arm. Intact human erythrocytes and leaky ghost membranes prepared from them were treated with the probes to label, respectively, the outer membrane leaflet and both leaflets. Motional freedom of the pyrene fluorophores in the membrane was assessed by estimation of the steady-state polarization of fluorescence, the excited-state lifetime, and the excimer/monomer fluorescence intensity ratio. The fluorescence anisotropy of each impermeant derivative was lower in the outer as compared to the inner hemileaflet, whereas the corresponding excited-state lifetimes were similar. Excimer formation was consistently greater in the outer leaflet. The results demonstrate that the impermeant fluorophores experience greater motional freedom ("fluidity") in lipid domains of the outer as compared to the inner leaflet of the human erythrocyte membrane.

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 endothelial 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 four fluorescent probes experience greater motional freedom in the outer as compared to the inner membrane leaflet. The results are in accord with prior studies of impermeant pyrene derivatives, which also indicate that the outer leaflet lipids have greater motional freedom.

Human erythrocyte membranes were enriched or depleted of cholesterol and effects on membrane proteins assessed with a membrane-impermeant sulfhydryl reagent, [35S]glutathione-maleimide. Reaction of the probe with intact cells quantifies exofacial sulfhydryl groups and reaction with leaky ghost membranes permits quantification of endofacial sulfhydryl groups. The mean endofacial sulfhydryl titer of cholesterol-enriched membranes exceeded that of cholesterol-depleted membrane by approximately 45 nmol/mg of protein or 64%. The corresponding exofacial titer of cholesterol-enriched cells was less than that of cholesterol-depleted cells by approximately 0.4 nmol/mg of protein, or 14%. Labeled membranes were examined by autoradiography of sodium dodecyl sulfate-polyacrylamide gel electropherograms to determine the labeling patterns of individual protein bands. Cholesterol enrichment enhanced the surface labeling of Coomassie brilliant blue stained bands 1,2,3, and 5, decreased the labeling of band 6, and did not change significantly that of band 4. The results demonstrate that changes in membrane cholesterol which influence lipid fluidity can alter the surface labeling of both intrinsic and extrinsic membrane proteins.

No abstract

No abstract