The purpose of this work is the elucidation of a complete mechanism for the self-assembly of sickle cell hemoglobin. At present, the initial phase of polymerization is well described by a double nucleation mechanism. Accordingly, there are two major aims of this proposal: (1) The double nucleation mechanism is to be tested. This will be done by stochastic measurements on individual domain delay times; by simultaneous measurements of birefringence and light scattering; by studying depolymerization and repolymerization; by studies in high-phosphate buffer. (2) The structure of polymer domains is to be determined. This will be done by kinetic observation of the growth patterns of individual domains; by determining a structure factor vs time as domains are formed; by modelling the growing domain. The experiments proposed will be carried out on a unique instrument recently constructed at Drexel University. This instrument combines the sensitivity of microscopic techniques for observation of kinetics, with the large dynamic range of laser photolysis as an initiator of the reaction. The system employs imaging detectors to collect kinetic information in two dimensions. The apparatus can also collect information from two wavelengths almost simultaneously. These studies are important for understanding assembly, since the polymerization of sickle cell hemoglobin is the most thoroughly characterized of such reactions. These studies are also of great importance for dealing with sickle cell disease since there remains strong evidence that the disease is kinetically controlled, and that kinetic strategies offer the best approach for therapy. These studies are also important in linking domain properties, which likely cause vaso-occlusion, to molecular properties of the assembly process.
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