Our long-range goal is to develop recombinant human hemoglobin (rHb) as the starting material for manufacturing all globin-based O2 delivery Pharmaceuticals. We have established the key requirements for a rHb-based blood substitute: (a) moderate O2 affinity and large O2 dissociation rate constants for efficient transport in capillaries;(b) significantly reduced rates of NO scavenging to prevent hypertensive side effects;(c) resistance to auto- and chemically-induced oxidation to inhibit oxidative stress;and (d) low rates of heme dissociation to increase shelf-life. We have used the mechanisms governing these properties to design second-generation extracellular rHb-based blood substitutes with more efficient O2 transport and little or no hypertensive side effect in pre-clinical animal studies. However, there is a clear need to engineer third-generation, non-vasoactive rHbs with increased resistance to denaturation, enhanced expression yields, and reduced production costs. We propose to solve these problems by: (1) optimizing O2 binding to and NO scavenging by human rHb without compromising globin stability and resistance to degradative reactions;(2) improving the stability and enhancing production of recombinant hemoglobin in E. coli by (a) rational and comparative mutagenesis to increase the resistance of the apoprotein to unfolding and (b) co- expression of the alpha and beta rHb chains with the newly discovered erythroid chaperone, alpha hemoglobin stabilizing protein (AHSP);and (3) facilitating rapid incorporation of exogenously added heme into newly synthesized globins by co-expression of rHb with the heme utilization genes (hug) from Plesiomonas shigelloides and related pathogens. PUBLIC HEALTH: We are developing recombinant human hemoglobin to replace donated blood as the source material for all protein-based oxygen carriers that are being developed for the treatment of severe blood loss and believe that this effort should be a national public health priority. An effective hemoglobin- based blood substitute will alleviate chronic shortages of whole blood, provide emergency treatment following military and civilian disasters, and allow the use of transfusions in developing countries lacking a safe blood banking system.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL047020-18
Application #
7844991
Study Section
Erythrocyte and Leukocyte Biology Study Section (ELB)
Program Officer
Mitchell, Phyllis
Project Start
1991-08-01
Project End
2012-05-31
Budget Start
2010-06-01
Budget End
2012-05-31
Support Year
18
Fiscal Year
2010
Total Cost
$325,569
Indirect Cost
Name
Rice University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
050299031
City
Houston
State
TX
Country
United States
Zip Code
77005
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Schotte, Friedrich; Cho, Hyun Sun; Soman, Jayashree et al. (2013) Real-time tracking of CO migration and binding in the ? and ? subunits of human hemoglobin via 150-ps time-resolved Laue crystallography. Chem Phys 422:98-106
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Mollan, Todd L; Khandros, Eugene; Weiss, Mitchell J et al. (2012) Kinetics of ?-globin binding to ?-hemoglobin stabilizing protein (AHSP) indicate preferential stabilization of hemichrome folding intermediate. J Biol Chem 287:11338-50
Tsai, Ah-Lim; Berka, Vladimir; Martin, Emil et al. (2012) A ""sliding scale rule"" for selectivity among NO, CO, and Oýýý by heme protein sensors. Biochemistry 51:172-86
Ekworomadu, MarCia T; Poor, Catherine B; Owens, Cedric P et al. (2012) Differential function of lip residues in the mechanism and biology of an anthrax hemophore. PLoS Pathog 8:e1002559

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