Nonideality and the Nucleation of Sickle Hemoglobin

Abstract

The homogeneous and heterogeneous nucleation kinetics of sickle hemoglobin (HbS) have been studied for various degrees of solution crowding by substitution of cross-linked hemoglobin A, amounting to 50% of the total hemoglobin. By cross-linking hemoglobin A, hybrid formation between hemoglobin A and hemoglobin S was prevented, thus simplifying the analysis of the results. Polymerization was induced by laser photolysis, and homogeneous nucleation kinetics were determined by observation of the stochastic behavior of the onset of light scattering. Heterogeneous nucleation was determined by observing the exponential growth of the progress curves, monitored by light scattering. At concentrations between 4 and 5 mM tetramer (i.e., approximately 30 g/dl), the substitution of 50% HbA for HbS slows the reaction by a factor of 10(3) to 10(4). Using scaled particle theory to account for the crowding of HbA, the observed decrease in the homogeneous nucleation rate was accurately predicted, with no variation of parameters required. Heterogeneous nucleation, on the other hand, is not well described in the present formulation, and the theory for this process appears to require modification of the way in which nonideality is introduced. Nonetheless, the accuracy of the homogeneous nucleation description suggests that such an approach may be useful for other assembly processes that occur in a crowded intracellular milieu.

Mechanisms of Homogeneous Nucleation of Polymers of Sickle Cell Anemia Hemoglobin in Deoxy State

“…The delay time and the nucleation induction time u determined here are inherently different from the nucleation time lag or the 1/10 time determined by others: 28,29,31,32,91 the latter two represent expectancy times until a polymer reaches a point from which light is scattered, or at which the solution turbidity is monitored. As such, they are linked in a complex way to the mean time separating nucleation events in steady state.…”

“…30,31,91 Homogeneous nucleation rates were extracted from the distributions of the time to achieve one-tenth of the final reaction yield, 33 assuming that the primary nucleation of HbS polymers is the only source of stochasticity of the data. The apparent reaction order was 47(^5).…”

Molecular Crowding Limits the Role of Fetal Hemoglobin in Therapy for Sickle Cell Disease

“…The prescription for incorporating such crowding molecules has been given. 8,31 Equation (1) was used for the comparison of theory with previously published, measured tenth times. 22 Temperatures of those data varied from 14.1 8C to 35.5 8C, concentrations varied from 28.6 g/dl to 29.2 g/ dl and the fraction of HbF ranged from 0 to 0.38.…”

Liquid–liquid separation in solutions of normal and sickle cell hemoglobin

“…These tests will require the development of a new method to measure the homogeneous nucleation rates of deoxy-HbS polymers. The existing methods extract the nucleation rates from the delay time for the onset of turbidity in polymerizing samples and rely on assumptions needed to separate the nucleation from the growth and replication of the polymers fibers (44)(45)(46)(47). When temperature is lowered from 42 to 35°C, the smaller of the dense liquid droplets disappear, while the larger ones serve as nucleation centers for HbS spherulites.…”