The objective of this research is to better understand the mechanism of polymerization of deoxy-Hb S using biochemical, biophysical, and recombinant DNA technology, with the long-range goal of finding the means to inhibit the polymerization of deoxy- Hb S. In an attempt to further clarify the mechanism of polymerization of deoxy-Hb S, we will continue to study the role of nuclei prior to polymerization of Hb S and to characterize nuclei using various biophysical techniques including a dynamic laser light-scattering phototmeter which was recently awarded to our laboratory. We will clarify the probability factor for nucleation of various hybrid hemoglobins in the mixtures of sickle and non-sickle hemoglobins, which may be related to clinical severity of patients having with abnormal non-sickle hemoglobins. Recently, we succeeded in the expression of human beta-globin by recombinant DNA engineering techniques. We will now prepare various hemoglobin mutants with amino acid substitutions at the beta 6 position as well as at the intermolecular contact points of deoxy-Hb S polymers. Using these hemoglobins, we will study the effect of various amino acids on the kinetics and thermodynamics of polymerization and on the solubility of deoxy-Hb S. Other recent studies have shown that under certain experimental conditions polymerized products of deoxy-Hb S prepared in high phosphate buffer were similar to those formed in low phosphate buffer. The use of high phosphate buffer is advantageous, since only small amounts of hemoglobin are required for kinetic experiments. To measure the surface hydrophobicity of proteins, we will use a high performance gel filtration column, TSK-GEL-SW, which converts to a hydrophobic interaction column in high phosphate buffer. We will study the surface hydrophobicity of hemoglobins and the relationship between the surface hydrophobicity and solubility of various hemoglobins. We will also clarify the relationship between the strength of hydrophobic interaction and the solubility of deoxy-Hb S. These studies will add to our knowledge of the pathology of sickle cell disease and may contribute to the development of a molecular approach to ameliorating this life- threatening genetic disorder.
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