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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
1R01DK041253-01A1
Application #
3241911
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1990-02-01
Project End
1993-01-31
Budget Start
1990-02-01
Budget End
1991-01-31
Support Year
1
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Albert Einstein College of Medicine
Department
Type
Schools of Medicine
DUNS #
009095365
City
Bronx
State
NY
Country
United States
Zip Code
10461
Hirsch, R E; Rybicki, A C; Fataliev, N A et al. (1997) A potential determinant of enhanced crystallization of Hbc: spectroscopic and functional evidence of an alteration in the central cavity of oxyHbC. Br J Haematol 98:583-8
Hirsch, R E; Witkowska, H E; Shafer, F et al. (1997) HbC compound heterozygotes [HbC/Hb Riyadh and HbC/Hb N-Baltimore] with opposing effects upon HbC crystallization. Br J Haematol 97:259-65
Hirsch, R E; Lin, M J; Vidugiris, G J et al. (1996) Conformational changes in oxyhemoglobin C (Glu beta 6-->Lys) detected by spectroscopic probing. J Biol Chem 271:372-5
Hirsch, R E (1994) Front-face fluorescence spectroscopy of hemoglobins. Methods Enzymol 232:231-46
Hirsch, R E; Friedman, J M; Harrington, J R et al. (1994) Stability of a potential blood substitute, HbXL99 alpha, under high pressure. Biochem Biophys Res Commun 200:1635-40
Hirsch, R E; Vidugiris, G J; Friedman, J M et al. (1994) Alteration of tryptophan fluorescence properties upon dissociation of Lumbricus terrestris hemoglobin. Biochim Biophys Acta 1205:248-51
Hirsch, R E; Harrington, J P; Scarlata, S F (1993) The differential effects of carbon monoxide and oxygen on the pressure dissociation of Lumbricus terrestris hemoglobin. Biochim Biophys Acta 1161:285-90
Hirsch, R E; Lin, M J; Pulakhandam, U R et al. (1993) Hemoglobin oxygen affinity is increased in erythropoietic protoporphyria. Photochem Photobiol 57:885-8
Nagel, R L; Lin, M J; Witkowska, H E et al. (1993) Compound heterozygosity for hemoglobin C and Korle-Bu: moderate microcytic hemolytic anemia and acceleration of crystal formation [corrected] Blood 82:1907-12
Vidugiris, G J; Harrington, J P; Friedman, J M et al. (1993) Absence of ligand binding-induced tertiary changes in the multimeric earthworm Lumbricus terrestris hemoglobin. A resonance Raman study. J Biol Chem 268:26190-2

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