The present proposal is centered upon the discovery by the applicant (Dr. R.E. Hirsch) that the fluorescence of the tyrosine and tryptophan residues in hemoglobins can be detected by front-face fluorometry, even in the presence of hemes. This fluorescence is sensitive to quaternary structure and aggregation state. The fluorescence of hemoglobins can now be used as a probe to assay conformational change, protein-ligand interaction, intramolecular perturbation, self-assembly, and arrangements of higher- order structures (e.g., polymerization of Hb S). Correlation of normal and mutant hemoglobin structure and function is the subject of continued investigation. Moreover, hemoglobinopathies continue to be a world-wide health problem. In order to understand the pathophysiological mechanism of the disease(s), the structural and functional abnormalities occurring in the mutant molecule must be ascertained. Understanding basic mechanisms of normal vs. abnormal structure and function will serve as groundwork to either correct or treat the pathologies. Front-face fluorometry of normal and variant hemoglobins will provide information of conformational change by site-specificity, extreme sensitivity, and high resolution of sites previously inaccessible by other spectroscopic means.
Specific aims are: 1. To identify the aromatic amino acids contributing to the fluorescence emission of hemoglobins. 2. To determine the physical basis for tryptophan emission in the presence of substantial quenching by the hemes. 3. To characterize the influence of heme structure on the fluorescence of hemoglobin. 4. To characterize the equilibrium and kinetic properties of the alpha1Beta2 interface as a function of ligand-induced quaternary and tertiary structural changes. 5. To develop the intrinsic and extrinsic fluorescence of hemoglobins as a tool to characterize both the degree of and the dynamics of self-assembly in (a) tetramerization; (b)Hb S polymerization; and (c)higher-ordered structures of hemoglobins. The results of these studies will provide a better understanding of the noncovalent interactions of the molecule to generate a highly cooperative structure.
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