Effects of red cell membrane on the polymerization of sickle hemoglobin

We have investigated the effect of erythrocyte membrane preparations on the rate of polymerization and the solubility of deoxyhemoglobin S. The kinetics of polymerization were monitored by measuring changes in turbidity of hemoglobin S solutions as a function of time in a temperature-controlled cuvette. The solubility of deoxyhemoglobin S, an equilibrium measurement, was determined by measuring the hemoglobin concentration of the supernatant following centrifugation of the polymerized hemoglobin S solution. The solubility of deoxyhemoglobin S was unaffected by the presence of red cell membrane ghosts, inside-out red cell vesicles, or inside-out vesicles enriched in erythrocyte spectrin and actin (Bands 1, 2, and 5) in the hemoglobin solution. Furthermore, inside-out vesicles had no significant effect on the kinetics of polymerization. The addition of erythrocyte membrane ghosts to the hemoglobin solution, in ratios approximating that of the intact red cell, increased the rate of polymerization up to 8-fold. However, when corrections were made for the excluded volume contributed by the membrane ghosts, the enhancement of the rate of polymerization was reduced to less than 3-fold. These results fail to show significant facilitation of intracellular sickling by nucleation sites on the red cell membrane.