KINETICS OF NITRIC OXIDE BINDING TO HEMOPROTEINS

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Intravascular administration of cell-free hemoglobin appears to be strongly correlated with a rise in pulmonary and systemic blood pressure. This hypertensive response has been attributed to the reaction between hemoglobin and the endothelium-derived relaxing factor (EDRF), which is now believed to be nitric oxide (NO). We explored the interactions between NO and the oxidized forms of a number of chemically modified hemoglobins. These hemoglobins are blood substitute candidates due to their lower oxygen affinity and greater stability of their tetrameric structures. Using fast reaction techniques, the stopped flow apparatus, we documented that there are differences in the rate of the initial NO binding to the ferric forms of these proteins and in the subsequent NO- driven reduction step. Since NO oxidizes oxyhemoglobin to ferrihemoglobin (metHb) extremely rapidly in vivo, we hypothesized that a redox cycle between NO and 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. Work is in progress to explore the mechanism(s) of interactions of NO with other hemoproteins to fully understand the degree to which heme pocket chemistry and other structural features determine the binding of NO to hemoproteins. In this regard, we began to study both steady state and transient kinetics of NO reactions with simple and more complex hemoproteins such as myoglobin and catalase, respectively. We are also exploring the reactivity of peroxynitrite (OONO-), a product of nitric oxide reaction with superoxide ions, which is thought to be produced in the vascular wall. This will ultimately contribute to the design of hemoglobin that, along with desirable oxygen binding characteristics, exhibits a suppressed ability to react with NO.