The long-term objectives of the proposal are to study the equilibrium and kinetics of ligand binding and protein assembly in hemoglobins and other systems. (1) The enzymatic depletion of oxygen in a special stopped-flow cuvette will allow continuous determinations of oxygen equilibria in diverse hemoglobins. Dual and multi-wavelength spectrophotometry will be used to obtain spectra of intermediates during the deoxygenation process for use in global fittings for Adair constants. A practical aim is to develop a rapid and convenient procedure for determining oxygen binding constants in mutant and engineered hemoglobins. Hemoglobins to be studied include human Hbs, Hbs that are naturally T-state, dimeric Hbs, and the high molecular weight bracelet Hb from Lumbricus. Stopped-flow and laser photolysis studies will be carried out on a number of the Hbs to determine the kinetics of conformational changes, and for the dimeric systems, to obtain a complete kinetic description in terms of the MWC model. (2) EXAFS studies will be continued on oxy, CO, and deoxy forms of model R and T state Hbs, as well as on di-Br heme Hbs locked into R and T states to quantitate structural changes at these sites to compare with concomitant changes at the Fe. (3) The kinetics of biotinylated probes, protein and nucleotides binding to avidin will be measured by fluorescence anisotropy stopped-flow. The chemistry will be developed to provide a new procedure for preparing chains of diverse Hbs. The kinetics, stoichiometry, and Tm's related to the binding of fluorescently-labeled oligonucleotides to model DNA's will be studied by changes in fluorescence anisotropy. These procedures may eventually provide a convenient first step for the human genome project, provide an alternative to blotting techniques, and, with amplification, allow rapid detection of viral DNA, of possible relevance for AIDS research. (4) Hot-atom chemistry involving isotopes of Br will be used to develop an alternative to photoaffinity determinations of biological structures. (5) NMR and light-scattering stopped-flows will be used to study protein assembly and dissociation and to correlate aggregation state with ligand binding. (6) New procedures will be explored for integrating systems of differential equations for aid in modeling kinetic processes.
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