A flow EPR study of deformation and orientation characteristics of erythrocyte ghosts: Effects of lysing and resealing conditions

Abstract

The effects of various conditions in lysing and resealing the red cell membrane on the degree of ghost deformation and orientation in flow are investigated using the flow EPR and spin-label method. The relatively low deformability of the standard ghost, which is lysed and resealed, respectively, in hypotonic and isotonic NaCl-Tris buffer, is markedly enhanced by the presence of Mg-ATP, chlorpromazine, or Ca2+ ion during resealing. The effect is concentration dependent, and there is an optimal level for each treatment. Chlorpromazine and Ca2+ are also effective when added to the resealed ghosts. Mg2+ ion shows an opposite effect reducing the ghost deformability in flow at all concentrations. An isotonic lysis in NH4HCO3 solution with less osmotic stress substantially raises ghost deformability above that of the standard ghosts. These results are interpreted on the basis of a misalignment between the bilayer leaflets that is probably brought about during hypotonic lysis and its recovery to the nearly normal bilayer state by the agents used during or after resealing. The novel finding of deformability enhancing effect of calcium is assumed to be caused by the electrostatic expansion of the inner layer relative to the outer leaflet. The explanations are supported by the resealed ghost shapes observed before and after the treatments; shape recovery from the monoconcave spheroid toward biconcave discoid is observed in most cases concomitantly with improvements of flow characteristics.