Peroxynitrite is formed in vivo from the reaction between nitric oxide and superoxide. Cell-free hemoglobin solutions intended for use as therapeutic oxygen carriers have revealed vasoreactive toxicity. This toxicity may be mediated, in part, by the reactions of hemoglobin with nitric oxide and peroxynitrite. We propose to investigate the ESR changes that occur during the interaction of peroxynitrite with hemoglobin using rapid-freeze, low-temperature, and spin-trapping methodologies. Rapid-freeze and low-temperature ESR will be used to determine changes to the heme iron component of hemoglobin. Spin-trapping methodologies using DMPO as the spin trap will be used to investigate the potential formation of protein free radicals. The single reduced thiol of hemoglobin can be potentially oxidized to a thiyl radical by peroxynitrite. We hope these studies will give additional insight into the reactions of peroxynitrite in vivo and the affects of blood substitute on vascular biology.