We propose to examine, at a molecular level, the abnormal rigidity in sickle cell membranes. The effectiveness of the oxidant-defense system in sickle cell membranes under different conditions will be evaluated by measuring the specific oxidation of spin labeled phospholipids in membrane of intact cells and in resealed vesicles. The local membrane lipid fluidity of individual classes of major phospholipids, namely phosphatidylcholine, sphingomyelin, phosphatidylethanolamine and phosphatidylserine in sickle cells will be investigated. Spin labeled phospholipids are to be intercalated, specifically, into either the outer or the inner leaflet of the lipid bilayer in both normal and sickle erythrocytes and simplified vesicle systems, and used as probes to monitor the local environments and dynamics of each class of phospholipid molecules under conditions relevant to sickling and cellular oxidation. Furthermore, the segmental motions of spectrin from sickle cell membranes and of oxidatively cross-linked spectrin will be determined. The effects of hemoglobin on the segmental motions of spectrin will also be examined. The experiments will be carried out under both static and flow conditions to assess the roles of mechanical shear forces on membrane local fluidity properties, and on spectrin segmental motions. Both conventional electron paramagnetic resonance and saturation transfer electron paramagnetic resonance will be used. It is hoped that information on the dynamic properties such as local membrane fluidity of specific phospholipids and the segmental motions in spectrin, and on the effects of oxidants and hemoglobin on these membrane properties in sickle cells under different conditions, including static and flow conditions, will provide not only insight into the mechanisms on irreversibly sickle cell formation, but also the molecular basis for the development efficient membrane drugs for sickle cell anemia.
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