Our goal is to obtain the experimental data and mechanistic interpretations required to design the heme pocket in protein-based blood substitutes for optimal gas transport properties and maximum stability to autoxidation and heme loss. Five important properties of potential blood substitutes are: (1) cooperativity and moderately low )2 affinity; (2) discrimination against CO in favor of )2 binding (i.e., KCO/KO2 <200); (3) large association and dissociation rate constants for efficient gas transport in the microcirculation; (4) low rate of autooxidation; and (5) high affinity for heme and stability to denaturation. In another study, we have been examining the 02 and CO binding properties of a series of genetically engineered, heme pocket mutants of pig and sperm whale myoglobin and human hemoglobin. In this project, we propose to measure the autoxidation and heme stabilities of an expanded set of these mutant proteins. Because of complexities due to alpha and Beta subunit differences, tetramer dissociation into dimers, and quaternary conformational changes in human hemoglobin, the myoglobins will be used as model systems to investigate the relationships between oxygen binding parameters and those for iron oxidation and heme loss. The effects of amino acid replacements at positions 64(E7), 68(E11), 67(E10), 45(CD3), 43(CD1), 29(B10), 32(B13), and 107(G8) on the rates of autooxidation will be surveyed. Temperature, pH, and O2 concentration dependences will be measured for native and selected mutant proteins in order to determine the mechanism of this process. Experiments will also be carried out to measure rates of heme binding and dissociation for the same set of proteins. As a test of the utility of this approach, we will then attempt to construct a synthetic myoglobin with optimal O2 affinity, CO discrimination, ligand binding rate constants, and stability to oxidation and heme loss. Autooxidation and heme binding studies will also be carried out with mutants of human hemoglobin containing His(E7) to Gly and Gln and Val(E11) to Ala, Leu, and Ile substitutions in both the alpha and B subunits. the results of these studies and those for myoglobin will be used to design heme pockets in the alpha and B subunits of human hemoglobin with optimal O2 binding parameters and minimal rates of autoxidation and hemin dissociation. The data for the recombinant myoglobins and hemoglobins will also provide more complete structural interpretations of naturally occurring hemoglobinopathies associated with congenital Heinz body hemolytic anemia and elevated levels of methemoglobin.
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