The rapid and spontaneous interaction between superoxide (O2-.) and nitric oxide (NO) to yield the potent oxidants peroxynitrite (ONOO-) and peroxynitrous acid (ONOOH), has been suggested to represent an important pathway by which tissue may be injured during inflammation. Although several groups of investigators have demonstrated substantial oxidizing and cytotoxic activities of chemically synthesized ONOO-, there has been little information available quantifying the interaction between O2-. and NO in the absence or the presence of redox-active iron. Using the hypoxanthine (HX)/xanthine oxidase system to generate various fluxes of O2-. and H2O2 and the spontaneous decomposition of the spermine/NO adduct to produce various fluxes of NO, we found that in the absence of redox-active iron, the simultaneous production of equimolar fluxes of O2-. and NO increased the oxidation of dihydrorhodamine (DHR) from normally undetectable levels to approximately 15 microM, suggesting the formation of a potent oxidant. Superoxide dismutase, but not catalase, inhibited this oxidative reaction, suggesting that O2-. and not hydrogen peroxide (H2O2) interacts with NO to generate a potent oxidizing agent. Excess production of either radical virtually eliminated the oxidation of DHR. In the presence of 5 microM Fe+3-EDTA to insure optimum O2-.-driven Fenton chemistry, NO enhanced modestly HX/xanthine oxidase-induced oxidation of DHR. As expected, both superoxide dismutase and catalase inhibited this Fe-catalyzed oxidation reaction. Excess NO production with respect to O2-. flux produced only modest inhibition (33%) of DHR oxidation. In a separate series of studies, we found that equimolar fluxes of O2-. and NO in the absence of iron only modestly enhanced hydroxylation of benzoic acid from undetectable levels to 0.6 microM 2-hydroxybenzoate. In the presence of 5 microM Fe+3-EDTA, HX/xanthine oxidase-mediated hydroxylation of benzoic acid increased dramatically from undetectable levels to 4.5 microM of the hydroxylated product. Superoxide dismutase and catalase were both effective at inhibiting this classic O2-.-driven Fenton reaction. Interestingly, NO inhibited this iron-catalyzed hydroxylation reaction in a concentration-dependent manner such that fluxes of NO approximating those of O2-. and H2O2 virtually abolished the hydroxylation of benzoic acid. We conclude that in the absence of iron, equimolar fluxes of NO and O2-. interact to yield potent oxidants such as ONOO-/ONOOH, which oxidize organic compounds. Excess production of either radical remarkably inhibits these oxidative reactions. In the presence of low molecular weight redox-active iron complexes, NO may enhance or inhibit O2-.-dependent oxidation and hydroxylation reactions depending upon their relative fluxes.

MATERIALS AND METHODSGlutathione (GSH), hypoxanthine (HX), superoxide dismutase (SOD), catalase, and diethylenetriaminepentaacetic acid (DETAPAC) were purchased from Sigma. Stock solutions were prepared as follows: 50 mM HX was made in 0.08 mM NaOH, 10 mM DETAPAC in milliQ water, and 10 mM GSH in milliQ water. These stock solutions were diluted in PBS (pH 7.4) to the reported final concentration. XO was purchased from Boehringer Mannheim. DEA/NO was a generous gift from Dr. Joseph Saavedra. Stock solution of DEA/NO were prepared as described previously (10), and stock concentrations were determined by UV absorption at 250 nm, using an extinction coefficient of 8000Reaction Method-A solution containing 0.5 mM HX and 50 M DE-TAPAC in PBS was prepared and referred to as the HX solution. Typically, GSH or DAN was added to the solutions, followed by catalase and/or SOD, and when appropriate, XO was followed by DEA/NO. Samples were incubated for 1 h at 37°C. At the end of this time DEA/NO was completely decomposed and 50 M allopurinol was added to inhibit the activity of XO. The solutions were then analyzed as described below. Under these assay conditions, 1 milliunit/ml XO produced a flux of O 2. and H 2 O 2 of approximately 1 nmol/min each as described previously (9). Determination of Decomposition of DEA/NO and Activity of XOThe rate of the decomposition of DEA/NO is typically determined by the loss of its characteristic absorbance at 250 nm. However, the HX solution strongly absorbs in this region and does not allow an accurate determination of the kinetics of decomposition. We have recently described a method used to determine the amount of NO released from an NO donor, which involves monitoring at 500 nm where HX solution does not absorb (11). In a solution containing 0.5 mM HX and 50 M DETA-PAC, 0.5 g/100 ml of sulfanilamide and 0.03 g of N-(1-naphthyl)ethylenediamine dihydrochloride (NEDD)/100 ml of PBS was dissolved as reported previously (11). Monitoring the appearance of the characteristic azo dye band at 500 nm, the rate constant for decay of DEA/NO was

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