Sickle cell anemia is characterized by a multitude of clinical and cellular abnormalities that arise as a consequence of the presence of the mutant gene produce hemoglobin S (HbS). Although it is likely that many of the attendant cellular defects are acquired, the mechanisms by which they arise are still unknown. The long-term objective of this proposal is to define the mechanisms responsible for the development of membrane abnormalities in SS cells. To achieve this objective we will test the hypothesis that oxidative damage plays a role in the development of membrane and cytoskeletal protein functional abnormalities in SS cells. We will focus on the effects of oxidation on key protein components of the red cell membrane and cytoskeleton, including spectrin, ankyrin and band 3. Protein oxidative damage will be assessed by binding to sulfhydryl-specific chemical probes and by direct analysis of protein amino acids. Additional studies will examine protein function to determine if function has been altered by oxidative modifications. Interactions between spectrin and calmodulin will be studied to determine if abnormal interactions between these proteins plays a role in the generation of irreversibly-sickled cells. Functional studies will determine if enzymes involved in maintaining protein sulfhydryl homeostasis, such as thioltransferase, are damaged in SS cells. The role of the sickling event per se in the development of protein oxidative lesions will be examined in oxygenated-deoxygenated cycling studies. The antioxidant capacity of defined SS cell populations will be measured to determine if oxidative damage results from a decreased capacity of some SS cells to repair oxidative lesions. The role of protein oxidation in the development of morphologic abnormalities will be studied by fluorescent and electron microscopy using sulfhydryl-specific chemical probes. The role of protein oxidation in the development of abnormalities in cell volume regulation will be studied using defined cells with high of low K:C1 cotransport activity. The role of HbS binding to the membrane and iron decompartmentalization in the development of cell morphologic and protein functional abnormalities will be tested in transgenic HbS mice with defined amounts of HbS. Together, these studies may help to elucidate the mechanisms responsible for the development of membrane abnormalities in SS cells and better our understanding of the pathophysiology of this disease and of other hemoglobinopathies characterized by unstable hemoglobins.