The Heme and Sickle Cell Hemoglobin Polymerization

Abstract

In search of novel control parameters for the polymerization of sickle cell hemoglobin (HbS), the primary pathogenic event of sickle cell anemia, we explore the role of free heme, which may be excessively released in sickle erythrocytes. We show that concentration of free heme in HbS solutions typically used in studies of polymerization kinetics is ∼0.04 - 0.05 mole heme/mole HbS. We show that dialysis of small-molecules out of HbS solutions completely prevents HbS polymerization. We show that that after dialysis, no apo-globin forms and HbS is largely intact. The addition of 100 - 300 μM of free heme to dialyzed HbS solutions leads to nucleation rates and delay times and polymer fiber growth rates faster by two orders of magnitude than prior to dialysis. Towards an understanding of the mechanism of nucleation enhancement by heme, we show that free heme at concentration 66 μM increases by two orders of magnitude the volume of the metastable clusters of dense HbS liquid, the locations where HbS polymer nuclei form. These results suggest that variation of the free heme concentration in the erythrocytes of sickle cell anemia patients may be a major factor for the puzzling complexity of the clinical manifestations of sickle cell anemia. The prevention of free heme accumulation in the erythrocyte cytosol may be a novel avenue to sickle cell disease therapy.