Our working hypothesis is that successful Hb-based blood substitutes may be generate by cross linking and sulfhydryl modification, with fine-tuning achieved by site-directed mutagenesis that is guided by knowledge of the molecular controls of HB function. This hypothesis is support by our recent discovery that the vasoconstrictive activity of cell-free Hb, attributed to NO scavenging, can be controlled by nitrosylation of its thiol groups. Molecular control functions critical to the design of blood substitutes include the ability of modified Hbs to contribute to oxidative toxicity, redox cycling and NO scavenging/donating reactions. We propose to evaluate these critical functions by spectroscopic measurements of the kinetics and equilibria of heme-and sulfhydryl- ligand binding and novel spectroelectrochemical methods for measuring the heme redox potential. These complementary techniques will be used in parallel to quantify differences between normal and modified Hbs. Our innovative spectroelectrochemical methods allow us to directly compare oxidation and ligand-binding curves and will aid in the design of blood substitutes by differentiating between those aspects of function that are responsive to the redox potential of the active-site iron atoms and those aspects governed by steno features. Hb systems selected for study will enable us to separately test the effects of modifications of the heme pocket, the sulfhydryl groups, and the central cavity. Primary attention will be given to defining the range of functional modifications that result from sulfhydryl modification in combination with chemical cross-linking. Our goal is to trace the molecular pathway from specific sites of modifications to their ultimate functional expression in ligand binding equilibria and kinetics that underlie oxidative toxicity, redox cycling and NO scavenging/donating reactions of cell-free hb.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL058248-04
Application #
6056415
Study Section
Heart, Lung, and Blood Initial Review Group (HLBP)
Project Start
1996-09-30
Project End
2001-08-31
Budget Start
1999-09-01
Budget End
2000-08-31
Support Year
4
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Duke University
Department
Miscellaneous
Type
Schools of Earth Sciences/Natur
DUNS #
071723621
City
Durham
State
NC
Country
United States
Zip Code
27705
Remington, Nicole; Stevens, Robert D; Wells, Randall S et al. (2007) Genetic diversity of coastal bottlenose dolphins revealed by structurally and functionally diverse hemoglobins. Gene 398:123-31
Bonaventura, Celia; Fago, Angela; Henkens, Robert et al. (2004) Critical redox and allosteric aspects of nitric oxide interactions with hemoglobin. Antioxid Redox Signal 6:979-91
Bonaventura, C; Crumbliss, A L; Weber, R E (2004) New insights into the proton-dependent oxygen affinity of Root effect haemoglobins. Acta Physiol Scand 182:245-58
Tsai, Ching-Hsuan; Simplaceanu, Virgil; Ho, Nancy T et al. (2003) Site mutations disrupt inter-helical H-bonds (alpha14W-alpha67T and beta15W-beta72S) involved in kinetic steps in the hemoglobin R-->T transition without altering the free energies of oxygenation. Biophys Chem 100:131-42
Fago, Angela; Crumbliss, Alvin L; Peterson, Jim et al. (2003) The case of the missing NO-hemoglobin: spectral changes suggestive of heme redox reactions reflect changes in NO-heme geometry. Proc Natl Acad Sci U S A 100:12087-92
Bonaventura, Celia; Taboy, Celine H; Low, Philip S et al. (2002) Heme redox properties of S-nitrosated hemoglobin A0 and hemoglobin S: implications for interactions of nitric oxide with normal and sickle red blood cells. J Biol Chem 277:14557-63
Taboy, Celine H; Bonaventura, Celia; Crumbliss, Alvin L (2002) Anaerobic oxidations of myoglobin and hemoglobin by spectroelectrochemistry. Methods Enzymol 353:187-209
Dantsker, David; Samuni, Uri; Friedman, Adam J et al. (2002) Geminate rebinding in trehalose-glass embedded myoglobins reveals residue-specific control of intramolecular trajectories. J Mol Biol 315:239-51
Juszczak, Laura J; Manjula, Belur; Bonaventura, Celia et al. (2002) UV resonance Raman study of beta93-modified hemoglobin A: chemical modifier-specific effects and added influences of attached poly(ethylene glycol) chains. Biochemistry 41:376-85
Bonaventura, Celia; Godette, Gerald; Ferruzzi, Giulia et al. (2002) Responses of normal and sickle cell hemoglobin to S-nitroscysteine: implications for therapeutic applications of NO in treatment of sickle cell disease. Biophys Chem 98:165-81

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