(450g) Metastable Mesoscopic Clusters in Solutions of Sickle Cell Hemoglobin

The primary pathogenic event of sickle cell anemia is the polymerization of the mutant hemoglobin (Hb) S within the red blood cells in deoxy- state in the venous circulation. Polymerization starts with homogeneous nucleation of individual polymer fibers, followed by growth and branching via secondary nucleation. Recent results on the mechanism of homogeneous nucleation revealed the formation of ordered nuclei of the HbS polymers is preceded by a disordered, liquid like precursor clusters of several hundred nanometers in size. To test of such clusters exist in solution of HbS we characterized these solutions in oxy- and deoxy- states with dynamic light scattering. For comparison, we used solution of the normal adult (A) hemoglobin. We found that solutions of all tested hemoglobin variants contain scatterers with characteristic times ~ 102 longer than the Hb molecules. They form within a few seconds of solution preparation. Their size does not increase over 3 hours of monitoring. HAT higher Hb concentration their characteristic c rates are slower. Determination of low shear viscosity in Hb solution revealed they it is equal to the high shear values. We considered several formations feasible in Hb solutions: loose networks of molecules, dense liquid droplets, and amorphous aggregates. The characteristics of the scatters listed above convinced us that these are dense liquid droplet, which are metastable with respect to the dilute Hb solution and have sizes from 100 to 500 nm. We developed a procedure to evaluate their volume fraction from the fraction of their scattered intensity and found that they occupy from 10-5 to 10-3 of the solution volume. We found that increasing the concentration of HbS or HbA leads to increase the sizes of clusters. Remarkably, the volume fraction occupied by the clusters has different behavior: for HbS, the volume fraction decreases at higher HbS concentration, while for HbA it increases. We found that the addition of 2 mM of PEG or 0.5 mM of hemin increase the volume fraction of the dense liquid droplets in HbS solutions by ~ 100?e. Since these concentrations of the two additives lead to 100-fold increase in the HbS nucleation rate, this observation supports the action of the dense liquid droplets as HbS nucleation precursor, and show that additives at low concentrators can severely accelerate the nucleation rate by increasing the volume fraction of the precursor.