Interaction of erythroid spectrin with hemoglobin variants: implications in β-thalassemia

Among the few studies, producing contradictory results, done on the interaction of erythroid membrane skeletal spectrin with hemoglobin (Hb), none has been able to provide a quantitative estimate of the association of spectrin with Hb. In this work, studies on the interactions of erythroid spectrin with Hb have been elaborated upon using a novel fluorescence technique. The concentration-dependent change in the fluorescence intensity of fluorescein-conjugated spectrin (F-spectrin) in presence of oxy-Hb indicated binding with a dissociation constant of 20 μM that has been directly evaluated from the increase in the extent of quenching of the fluorescein fluorescence of F-spectrin by reverse titration with the increasing concentrations of different Hb samples isolated from both normal and β-thalassemic patients. The Hb compositions, with major components of the normal HbA, the fetal HbF, and the variant HbA2, of each individual were estimated using the Variant HPLC device of Bio-Rad. Results of the present study indicated that the dissociation constant, Kd, of spectrin binding to Hb decreased from 19.5 ± 2 μM in normal individuals to of 6.5 ± 0.5 μM in the presence of 73% HbA2 along with coeluted variants in the blood samples of patients suffering from β-thalassemia, indicating differential interactions of the Hb variants with spectrin.