Hemolysis of human erythrocytes with saponin affects the membrane structure

Incubation of cells and tissues with saponin makes the lipid bilayer permea ble to macromolecules. Ghosts (membrane preparations) of saponin-lysed eryt hrocytes do not reseal, thus indicating an irreversible damage of the lipid bilayer. We investigated the influence of disturbance of the lipid bilayer on membrane proteins by comparing ghosts of saponin-lysed erythrocytes wit h ghosts of cells lysed in hypotonic buffer. Transmission electron microsco py revealed destruction of the lipid bilayer and emergence of multilamellar buds in saponinlysed ghosts. Freeze-fracture electron microscopy showed re gions with crystalline lipids and an increase in particle-free areas on fra cture faces. The number of protein sulfhydryl groups and the binding of hem oglobin were diminished in saponin-lysed ghosts. A Scatchard plot of hemogl obin binding revealed the decrease of high affinity binding sites. All thes e results indicate an aggregation of band 3 protein also demonstrated by la ser scanning microscopy after incubation of cells labelled with eosin-5-mal eimide with sublytic concentration of saponin. Hemolysis with saponin also affected the interaction between transmembrane proteins and the cytoskeleto n. Dissociation of peripheral membrane proteins by incubation of ghosts in low salt buffer or by blocking sulfhydryl groups was increased and the asso ciation of spectrin with spectrin-depleted vesicles was decreased. The incr eased incorporation of the fluorescent probe Merocyanine 540 into saponin-l ysed ghosts and the increased relative fluorescence quantum yield confirmed the perturbation of lipid bilayer and the changed interaction between memb rane lipids and intrinsic membrane proteins. Our results suggest that perme abilization of the lipid bilayer with saponin to admit the access of antibo dies to the cytoplasmic surface of cells can aggregate transmembrane protei ns and affect the immunocytochemical localization of associated proteins of the cytoskeleton.