The long range goals are to develop O2 delivery pharmaceuticals based on extracellular recombinant hemoglobins (rHb). These next generation rHbs will be designed for optimal O2 transport, minimal interference with vasoregulation, enhanced resistance to denaturation, and increased expression levels in E. coli. Seven required properties are: (a) moderate O2 affinity (P50 5-30 mm Hg); (b) discrimination against CO binding; (c) large rate constants for oxygen binding and release; (d) significantly reduced rates of NO scavenging; (e) resistance to autooxidation and reactions with H2O2 (f) low rates of hemin dissociation; and (g) highly stable apoglobin structures. The mechanisms underlying these properties are being determined using sperm whale myoglobin as a simple prototype for the alpha and beta subunits of human hemoglobin, and the results are being used to develop strategies for solving specific problems in the clinical use and commercial development of extracellular hemoglobins (i.e., the hypertensive side effect and production costs). These mutagenesis studies will provide a database for evaluating more general principles for heme protein engineering. We will also address fundamental physiological questions about O2 transport and NO signaling and examine basic mechanisms of NO scavenging and O2 binding which apply to other key heme proteins, including flavohemoglobin NO dioxygenases.
The specific aims for the next five years are to: (1) design new rHb products with low rates of NO scavenging and more efficient O2 transport properties to eliminate the hypertensive side effect of extracellular hemoglobin; (2) determine the relative importance of P50, oxygen dissociation rate constants, and cooperativity on O2 transport in capillaries in order to define the minimum requirements for efficacy; (3) examine systematically all physiologically relevant reactions of NO with the iron atom in hemoglobin to avoid other potential side effects; (4) improve the in vivo stability and in vitro shelf-life of recombinant hemoglobins by enhancing their resistance to autooxidation, reaction with H2O2, and heme loss; and (5) increase expression yields and lower cost of production in E. coli by enhancing the stability of apohemoglobin.
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