Pergamon Free Radical Biology & Medicine, Vol. 18, No. l, pp. 85-92, 1995 Copyright © 1994 Elsevier Science Ltd Printed in the USA. All rights reserved 0891-5849/95 $9.50 + .00 0891-5849(94)E0133-2 Original Contribution THE KINETICS OF THE OXIDATION OF BY PEROXYNITRITE L-ASCORBIC ACID DELLAND BARTLETT,* DANIEL F. CHURCH,* PATRICIA L. BOUNDS,* and W. H. KOPPENOL §* Departments of *Chemistry and *Biochemistry and ~BiodynamicsInstitute, Louisiana State University, Baton Rouge, LA, USA; and *Department of Chemistry and Physics, Southeastern Louisiana State University, Hammond, LA, USA (Received 7 March 1994; Revised 3 June 1994; Accepted 6 June 1994) Abstract Peroxynitrite [O=NOO-, oxoperoxonitrate(l-)] is a strong oxidant that may be formed in vivo by the reaction of 02"- and NO'. Oxoperoxonitrate(1-) reacts with molecules in aqueous acidic solutions via pathways that involve the highly reactive hydrogen oxoperoxonitrate either as an intermediate in a first-order reaction or as a reactive agent in a simple second- order reaction. ESR experiments show that hydrogen oxoperoxonitrate oxidizes monohydrogen L-ascorbate by one electron: when mixed at pH ca. 5 and passed through a flow cell within 0.1 s, the two-line ESR signal of the ascorbyl radical anion (all = 0.18 T, g = 2.005) is observed. The overall stoichiometry of the reaction was 1 mol of ascorbate oxidized per mol of oxoperoxonitrate(l-) added. The kinetics of the reaction were studied over the pH range 4.0-7.5 by stopped-flow spectrometry. Hydrogen oxoperoxonitrate,observed between 300 and 350 urn, and the oxoperoxonitrate(l-) anion, at 302 nm, disappear faster than predicted for the first-order isomerization to NO3 . The rate increases from pH 4 to 5.8, and then decreases with increasing pH. The rate variation suggests a bimolecular reaction either between the oxoperoxonitrate(I-) anion and ascorbic acid or between hydrogen oxoperoxonitrate and the monohydrogen ascorbate anion. Although the two pathways are kinetically indistinguishable, the pKa values of ascorbic acid and hydrogen oxoperoxonitrate strongly suggest that the reacting species are hydrogen oxoperoxo- nitrate and monohydrogen ascorbate. The second-order rate constant for this reaction is 235 + 4 M ts-t at 250(7. The enthalpy and entropy of activation are AH* = 9.3 _+ 0.5 kcal/mol and AS* = -16 _+ 2 cal/(mol.K), respectively. Keywords---Ascorbate, Peroxynitrite, Oxoperoxonitrate(I-), Sulfhydryl, Antioxidant defense, Reaction kinetics, Stopped-flow spectrophotometry, Electron spin resonance INTRODUCTION There are a number of studies that indicate that peroxy- nitrite [O =NOO-, oxoperoxonitrate(1-)] is formed in vivo. Ischiropoulos et al) have demonstrated that oxoperoxonitrate(1-) is formed from nitrogen monox- ide and superoxide near activated macrophages. The rate constant for this radical-radical combination reac- tion-6.7 × 109 M-Is -l as determined by flash photol- ysis2--is close to diffusion controlled. Under condi- tions where the concentrations of nitrogen monoxide and superoxide dismutase are comparable, this fast rate would allow nitrogen monoxide to compete for super- oxide with superoxide dismutase. The latter reacts cat- alytically with superoxide at a rate of 2.4 × 109 M-ls-l. 3 The concentration of nitrogen monoxide pro- Address correspondence to: W. H. Koppenol at the Institut ftir Anorganische Chemie, Eidgenrssische Technische Hochschule, CH 8092 ZUrich, Switzerland. duced by neurons and endothelial cells normally is of the order of tens to hundreds nanomolar, much less than the 5 to 40 #M concentration of superoxide dis- mutase in cells. However, under pathologic conditions, higher levels of nitric oxide synthase can be induced. A concentration of 0.5 #M nitrogen monoxide has been measured 4 near the surface of an endothelial cell. Near activated macrophages concentrations as high as 10 /zM appear likely (J. S. Beckman, personal communi- cation). Thus, even in the presence of superoxide dis- mutase, oxoperoxonitrate(1-) can be formed. Oxoperoxonitrate(l-) is known to carry out reac- tions that are injurious to the cell: it initiates lipid peroxidation, 5 and it reacts rapidly with sulfhydryls6 and methionine. 7 It may also play a role in reperfusion injury, where ischaemic endothelium produces super- oxide and nitric oxide when blood flow is reestab- lished. 8 In addition, oxoperoxonitrate(1-) nitrates and hydroxylates aromatic compounds, 9A° a reaction that is catalyzed by metal complexes ~1 and Cu/Zn superoxide 85