MECHANISM AND KINETICS OF SICKLE CELL HEMOGLOBIN

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Sickle cell disease is characterized by the polymerization of a mutant form of hemoglobin under hypoxic conditions which increases red blood cell rigidity leading to microvascular occlusion. The severity of the disease is most likely partially dependent on the kinetics of polymerization during hypoxic conditions and on the kinetics of depolymerization (melting) upon reoxygenation at the lungs. Although the mechanism and kinetics of polymerization have been extensively studied, much less work has concentrated on polymer melting. The focus of this study is to elucidate the mechanism and kinetics of sickle cell hemoglobin polymer melting. Special attention will be put on investigating the role of ligand (oxygen or carbon monoxide) binding to hemoglobin molecules in the polymer during melting. Other factors that may affect melting kinetics such as the length of the polymers will be studied as well as the link between polymer melting kinetics and changes in sickle cell shape and deformability. This study is not only important in regard to understanding the physics of polymerization and depolymerization but also in regard to understanding sickle cell disease thereby providing potential for a new form of treatment. The proposed work will be accomplished by a novel combination of spectroscopic techniques. A special instrument capable of simultaneously conducting spectral measurements of absorption, linear dichroism, circular dichroism, circularly polarized luminescence, and polarized light scattering is being built for application to this project. The spectral measurements will be accomplished with millisecond resolution so that the amount of sickle cell polymer, the ligation state of the polymer and solution phase molecules, and the quaternary state of the hemoglobin molecules can be simultaneously determined as polymer melting is induced. The use of these spectroscopic techniques (and their combination) in this project could lead to applications in other studies. Special attention will be paid in evaluating polarized light scattering (previously shown to be extremely sensitive to polymer concentration) as a biophysical tool. The novel technology that is providing the ability to perform this unique combination of measurements ensures the potential of the discovery of new phenomena.