A study of partially liganded sickle hemoglobin polymerization kinetics

Sickle cell anemia is a debilitating disease that results from the polymerization of mutated hemoglobin (HbS) inside the red blood cells (RBCs). Polymerization kinetics, including nucleation rates, have been well studied for pure HbS, as well as in the presence of nonpolymerizing molecules, such as HbF, or co-polymerizing molecules such as HbA. For polymerization of HbS in the presence of O2, CO and NO gases there is only one study, using CO, but that work did not resolve nucleation rates. Since polymerization occurs in the presence of these gases ins RBCs in the body, this is a significant gap in understanding. It has already been established that there is a relatively small amount of HbS with O2 or CO ligands in gelled polymers. How the presence of these ligands affect the rates of formation of homogeneous and heterogeneous nuclei is the focus of this work. Through the use of a photolysis light scattering technique, nucleation rates were measured for HbS solutions partially saturated with CO and then O2. The heterogeneous nucleation rates were found by fitting an exponential function to the scattered light growth curves; and the homogeneous nucleation rates were found by analysis of the stochastic variation of delay times. The amount of CO saturation was measured through a single wavelength optical density measurement, and the O2 saturation was measured through a laser photolyzed spectrum. This experiment obtained the nucleation rates of partially saturated CO HbS from 0% to 60% and partially saturated oxygen HbS from 0% to 44%. It was found that the equilibrium model presented by Sunshine et al (J Mol Biol. 1982 158:251-73) is a reasonable approximation for modeling the behavior of nucleation and that O2 and CO can be modeled as affecting nucleation rates similarly. The preceding analysis required that the Hb allosteric model be incorporated into nucleation theory, which is also described. The knowledge obtained in this study can help describe polymerization of HbS in vivo, which can lead to improved models for describing the time at which RBC sickling occurs during vasculature transit.Ph.D., Physics -- Drexel University, 201