Asymmetry of lipid dynamics in human erythrocyte membranes studied with impermeant fluorophores.

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.