Mechanisms of R -state hemoglobin C crystallization
Feeling-Taylor, Angela R.
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Hemoglobin C, a mutant hemoglobin, forms crystals when oxygenated in the red blood cells of patients with homozygous Hb C disease. The mutation arises at the same beta6 site as the critical mutation forming Hemoglobin S, which forms polymers in the deoxy (T) state. Thus, the beta C Glu → Lys mutation site, coupled with the liganded R-state, appears to influence the aggregation of hemoglobin molecules into ordered structures. An extensive study was conducted in vitro (as a model for intraerythrocytic Hb C crystallization) to determine mechanisms contributing to CO Hb C (R-state hemoglobin) crystallization.;The experimental designs for this thesis are driven by the following hypotheses: (1) crystal growth mechanisms of R-state Hb C are similar to those of inorganic crystals; (2) intratetrameric conformational differences between the R-state forms of Hb A and Hb C promote liganded Hb C crystallization; and (3) creation of a transgenic mouse expressing human Hb C will serve as an animal model for CC Disease.;Temperature is an important parameter that influences Hb C Solubility. In situ studies by Charache et. al. (1967) and Lessin et. al. (1969) showed that temperatures >37°C accelerate the production of intraerythrocytic Hb C crystals grown in hypertonic salt solutions. Similar mixtures at 4°C result in no crystallization. This temperature-dependence of solubility was quantified for Hb C crystals grown in vitro. Supersaturating solutions comprised of CO Hb C and potassium phosphate (buffer and precipitant) at concentrations that varied from 8mg/ml--35 mg/ml and 1.8M--2.0M, respectively were placed inside a miniaturized optical scintillation apparatus. Equilibrium temperatures (i.e., the temperature at which a CO Hb C solution is in equilibrium with the CO Hb C crystal) were determined and the results showed that CO Hb C solutions possess retrograde solubility. Crystals nucleated at high (greater than ambient) temperatures, but dissolved at low temperatures (4°C).;The equilibrium temperatures were plotted to generate temperature-dependent solubility curves of CO Hb C. The supersaturation temperature and saturation temperature, 25°C and 20°C, respectively, were estimated from these curves. The supersaturation temperature was used to initiate nucleation. Once nucleation was established, the temperature was lowered to the saturation temperature to maintain crystal growth (without further nucleation). Single CO Hb C crystals (20 mg/ml Hb C, 1.9M potassium phosphate) grew to 2mm inside X-ray capillaries. (Abstract shortened by UMI.).