Spin-label detection of sickle hemoglobin--membrane interaction at physiological pH.

The spin-label electron paramagnetic resonance technique has been used to compare the interactions of normal and sickle hemoglobin molecules with human erythrocyte membranes. The sickle hemoglobin molecules show an enhanced binding to membranes when compared to normal hemoglobin (HbA) molecules. Using a simple equilibrium model for hemoglobin--membrane interactions, we obtain an equilibrium dissociation constant for sickle hemoglobin of about half that of HbA at pH 7.4 in 5 mM phosphate at 20 degrees C. The interactions are very low affinity in nature and are stronger at lower pH than at pH 7.4 (Fung, 1981a). The difference between normal and sickle hemoglobin persists at both high (pH 7.4) and low (pH 6.7) pH values. The concentrations of hemoglobin at the saturation level are close to physiological concentrations. Removal of spectrin--actin protein molecules from the membranes causes little change in the interactions, indicating that the remaining membrane proteins play the primary role in hemoglobin--membrane interactions. This observation is further supported by data of spectrin--actin-depleted inside-out vesicle samples. The stronger interaction of sickle hemoglobin than normal hemoglobin with membranes is discussed in relation to the formation of irreversibly sickled cells.