The central goal of this work is a complete understanding of the gelation process of sickle hemoglobin as a paradigm for biological assembly and as a fundamental component of a debilitating disease. The overall strategy we have adopted is to experimentally separate the complex process of gelation into its constituents. Their separate study allows a more precise understanding of the particular process, and is valuable for in- sights into other assembly systems. Heterogeneous nucleation & growth rates will be measured by observing domain growth. These will be studied as a function of concentration, temperature and solution nonideality. To study diffusion & termination, we will perform static experiments using fluorescent tracers to monitor the spatial density of unpolymerized molecules. To understand the connectivity of the gel, we will preform kinetic tracer experiments. Experiments in high phosphate buffer will reveal whether simple exclusion of polymers adequately describes the density of polymer domains. Homogeneous nucleation rates will be measured by observation of stochastic fluctuations of the formation rate and by decomposition of bulk progress curves. The nonideality contributions to nucleation rate measurements will be assessed by the use of nonpolymerizing components. Solution nonideality is an essential component to describe nucleation in the presence of oxygen, as occurs in vivo. Studies are to be carried out by using continuous photolysis of the CO derivative or rapid temperature jump of the deoxy derivative to induce gelation. The resulting spatial and temporal growth is monitored by light scattering, birefringence intensity, absorption or fluorescence.
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