This is a revised application for continued funding for studies of hemoglobin molecules designed for use as possible cell-free O2 carriers (""""""""blood substitutes""""""""). The long-range goal of this project is to redesign the heme pocket of recombinant hemoglobins to optimize O2 affinity, discriminate against CO binding, achieve high rate constants for O2 binding, and increase resistance to autooxidation, hemin loss, and globin denaturation. There are 4 specific aims: 1. Construct myoglobin and human hemoglobin prototypes with optimal O2 affinity and increased resistance to autooxidation and reactions with NO and H2O2. Three types of modifications will be studied: (a) rational design of multiple distal pocket replacements; (b) naturally occurring distal pocket structures found in animal hemoglobins modeled in recombinant myoglobins and hemoglobins; and (c) random mutants will be used to attempt to create a new distal pocket combination with greater resistance to unfolding, tighter heme binding, moderate O2 affinity, and low rates of spontaneous chemically-induced autooxidation. 2. Design and evaluate myoglobin and hemoglobin mutants with increased affinities for hemin but normal ligand binding properties. An apomyoglobin, previously designed and produced by this group (H64Y/V68F) will be used to screen the rates of hemin dissociation from Mb and Hb mutants with substitutions at CD3, E10, F7, the FG corner, and other regions which appear important for stabilizing the bound prosthetic group. 3. Screen apomyoglobin and apohemoglobin mutants for greater resistance to unfolding. Unfolding will be measured by intrinsic tryptophan fluorescence and circular dichroism changes during denaturant titration. 4. Development of hemoglobin and myoglobin reagents for examination of the physiological effects of extracellular hemoglobin and for measuring hemin loss, NO production, and CO levels in biological systems. This laboratory already has developed a library of over 200 single and multiple myoglobin mutants which can be used to design more stable proteins for measuring hemin dissociation and for determining rates of NO and CO production in endothelial cell suspensions and enzyme systems.
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