Although the main events occurring during hemolysis are knowing to include elevated plasma levels of heme, the mechanisms of its clearance from the plasma and its detoxification are largely unknown. Elevated plasma heme levels cause depletion of circulating hemopexin (Hx), the protein which binds heme specifically. Hx conserves iron, and we have recently shown that it completely prevents the heme-mediated formation of oxygen radicals that lead to peroxidation of lipids and proteins. Heme not complexed to Hx may initiate and/or sustain oxidative tissue (membrane) damage in acute and chronic hemolysis or during muscle injury, e.g. in myocardial infarctions. The objectives of the proposed work are 1) to characterize the structure/function relationships of Hx employing polyclonal antibodies directed against peptides synthetized according to Hx sequences and, by site-specific mutagenesis, to localize amino acids that are axial ligands for heme in the heme-Hx complex; 2) to delineate the mechanisms but which Hx is induce the mRNA level and to identify those regions on the rat Hx gene which regulate its synthesis, in particular during the acute phase reaction and during heme injections; 3) to express in an eukaryotic system a Hx possessing those characteristics of the native molecule which prevent the heme-canalized oxidations of lipids and proteins. The information obtained should provide a basis for additional therapeutic approaches for preventing tissue damage in states in which heme is released intravasculary.
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