Effect of excess α-hemoglobin chains on cellular and membrane oxidation in model β-thalassemic erythrocytes

While red cells from individuals with β thalassemias are characterized by evidence of elevated in vivo oxidation, it has not been possible to directly examine the relationship between excess α-hemoglobin chains and the observed oxidant damage. To investigate the oxidative effects of unpaired α-hemoglobin chains, purified α-hemoglobin chains were entrapped within normal erythrocytes. These "model" β-thalassemic cells generated significantly (P<0.001 ) greater amounts of methemoglobin and intracellular hydrogen peroxide than did control cells. This resulted in significant time-dependent decreases in the protein concentrations and reduced thiol content of spectrin and ankyrin. These abnormalities correlated with the rate of α-hemoglobin chain autoxidation and appearance of membranebound globin. In addition, α-hemoglobin chain loading resulted in a direct decrease (38.5%) in catalase activity. In the absence of exogenous oxidants, membrane peroxidation and vitamin E levels were unaltered. However, when challenged with an external oxidant, lipid peroxidation and vitamin E oxidation were significantly (P<0.001) enhanced in the α-hemoglobin chainloaded cells. Membrane bound heme and iron were also significantly elevated (P<0.001) in the α-hemoglobin chain-loaded cells and lipid peroxidation could be partially inhibited by entrapment of an iron chelator. In contrast, chemical inhibition of cellular catalase activity enhanced the detrimental effects of entrapped α-hemoglobin chains. In summary, entrapment of purified α-hemoglobin chains within normal erythrocytes significantly enhanced cellular oxidant stress and resulted in pathological changes characteristic of thalassemic cells in vivo. This model provides a means by which the pathophysiological effects of excess α-hemoglobin chains can be examined.