Enhanced oxidative cross-linking of hemoglobin E with spectrin and loss of erythrocyte membrane asymmetry in hemoglobin Eβ-thalassemia

Oxidative stress to the erythrocytes is associated with formation of large molecular complexes of hemoglobin and the skeletal protein, spectrin. In this work, such complexes are formed with hemoglobin mixtures isolated from patients suffering from HbE beta-thalassemia with elevated levels of the HbE and purified erythroid spectrin in the presence of hydrogen peroxide. The complexes are separated on 4% SDS-PAGE and analyzed by densitometry. The results indicate enhanced formation of complexes with higher amounts of HbE, the most common hemoglobin variant prevalent in Southeast Asia. The binding affinity of spectrin with hemoglobin, in the absence of hydrogen peroxide, was found to increase with hemoglobin mixtures enriched with HbE. The presence of ATP was also found to decrease the overall yield of such complexes. Flow cytometric measurements of phosphatidylserine on the red cell surface also showed a lower degree of membrane asymmetry in HbE beta-thalassemie patients than in normal subjects. The present work shows enhanced formation of high molecular weight cross-linked complexes of hemoglobin derivatives with erythroid spectrin in HbE beta-thalassemia. (c) 2006 Elsevier Inc. All rights reserved.