Oxidative crosslinking, spectrin and membrane interactions of hemoglobin mixtures in HbEβ-thalassemia

The authors have studied the interactions of intact hemoglobin mixtures of HbE and HbA, with the major erythroid membrane skeletal protein, spectrin and tailor-made phospholipids membranes containing aminophospholipids to understand the role of spectrin and phospholipids of erythrocytes in the overall pathophysiology of the hemoglobin disorders. Hemoglobin mixtures were isolated and purified from the peripheral blood samples of HbE carriers and different HbE beta-thalassemia patients, taken for diagnosis. Spectrin binding was studied by fluorescence and oxidative crosslinking, by SDS-PAGE. Membrane perturbation experiments were carried out to study the leakage of the self-quenching fluorophore, carboxyfluorescein, entrapped in the phospholipid vesicles. Hemoglobin mixtures with elevated levels of HbE showed stronger interactions with spectrin reflected in the decrease in binding dissociation constant from 17 to 5 mu M upon increase in HbE% from about 30 to 90% in the hemolysates. The yield of the spectrin crosslinked complexes of such hemoglobin mixtures also increased with increase in HbE levels. Presence of ATP/Mg and DPG were found to decrease the overall yield of such complexes and the binding affinity of hemoglobins to spectrin. HbE rich hemolysates also induced greater leakage of entrapped carboxyfluorescein (CF) from phospholipid membranes containing aminophospholipids. Results from this study indicate the roles of skeletal proteins and aminophospholipids, particularly under oxidative stress conditions to be important in the premature destruction of erythrocytes in hemoglobin disorders, e. g. HbE beta-thalassaemia.