Reaction of human hemoglobin HbA0 and two cross-linked derivatives with hydrogen peroxide: differential behavior of the ferryl intermediate.
Functional concerns regarding hemoglobin-based red cell substitutes have generally centered on two parameters: (a) oxygen binding and delivery properties and (b) stabilization of the hemoglobin tetramer to prevent dimerization. Strategic chemical cross-linking and site-directed mutagenesis have produced proteins that have both physiological oxygen binding characteristics and a markedly prolonged retention time in the circulation. The presence of a large amount of redoxactive iron outside the red blood cell, however, raises some concerns about the potential for toxic side effects, many involving the production or participation of oxygen free radicals. In the present study, HPLC purified human hemoglobin HbA0 and two derivatives, one cross-linked between the lysine 99 residues of the alpha subunits (alpha-DBBF) and the other between the lysine 82 residues of the beta subunits (beta-DBBF) were tested for their susceptibility to oxidation and oxidative damage caused by H2O2. We show that chemical cross-linking resulting in alpha-DBBF induces an increased tendency to form ferryl radical in the presence of H2O2 and stabilizes the radical once formed. The in vitro oxidative modification of alpha-DBBF seen here is a plausible mechanism for some of the in vivo toxicities associated with the infusion of this hemoglobin.
Department of Biochemistry, Microbiology, and Molecular Biology, University of Maine, Orono 04469.
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D001241:Aspirin / Q000031:analogs & derivatives / Q000737:chemistry; D001802:Blood Substitutes / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000737:chemistry; D006441:Hemoglobin A / Q000031:analogs & derivatives* / Q000737:chemistry*; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D007501:Iron / Q000737:chemistry*; D007700:Kinetics; D008956:Models, Chemical; D010084:Oxidation-Reduction; D013053:Spectrophotometry
D001802:Blood Substitutes; D003432:Cross-Linking Reagents; C021630:bis(3,5-dibromosalicyl)fumarate; C066317:ferryl iron; C028881:hemoglobin A(0); D006441:Hemoglobin A; D006861:Hydrogen Peroxide; D007501:Iron; D001241:Aspirin