KINETICS OF NITRIC OXIDE BINDING TO HEMOPROTEINS

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The hypertensive response to the infusion of hemoglobin has been attributed to the reaction between hemoglobin and the endothelial derived nitric oxide (NO). We explored the interactions of between NO and the ferric form a number of chemically modified. Differences in the initial NO binding to the ferric forms of several chemically modified hemoglobins were observed as well as differences in the slow process in which NO drives the heme reduction. Since NO oxidizes oxyhemoglobin to the ferric hemoglobin (metHb) extremely rapidly in vivo, we hypothesized that a redox cycle between NO and the metHb could be partially responsible for the depletion of NO as a biological transducer, leading to altered vascular tone with variable effects observed for different hemoglobin preparations. The presence of superoxide under ischemic conditions can potentially abate the vasorelaxing effects of NO, by direct reaction with NO leading to the formation of yet another powerful oxidant, peroxynitrite (ONOO-). Reactions of native and chemically modified hemoglobins leads to rapid oxidation of the heme iron to the ferric form and possibly other oxidation products. Time courses obtained from rapid kinetic experiments showed a biphasic loss of the ferrous oxidation state on mixing peroxinitrite with these hemoglobins. Using colorimetric and chemiluminescent immunodetection techniques we were able to show that tyrosine nitration by peroxynitite can occur with both native and modified hemoglobins. Systemic and renal responses to the infusion of unmodified, cross-linked and polymerized cross-linked hemoglobins were studied in anesthetized rats. Unmodified hemoglobin produced significant increase in the mean arterial blood pressure (MAP) throughout the 60 minute infusion. Cross-linked hemoglobin, on other hand, produced a more marked and prolonged increase in MAP over 120 minutes. Only a moderate increase in MAP were observed in rats after 30 minutes infusion with the polymerized protein. The extent of renal insufficiency produced by these proteins, as determined by the Glomarular Filtration rate (GRF) were in the following order, unmodified Hb > polymerized Hb > cross-linked Hb.