The decomposition of peroxynitrite to nitroxyl anion NO؊) and singlet oxygen in aqueous solution) Ahsan Ullah Khan*†, Dianne Kovacic*, Alexander Kolbanovskiy*, Mehul Desai‡, Krystyna Frenkel‡, and Nicholas E. Geacintov* *Department of Chemistry, New York University, New York, NY 10003; and †Department of Environmental Medicine, New York University, New York, NY 10016 Communicated by Michael Kasha, Florida State University, Tallahassee, FL, December 31, 1999 (received for review November 22, 1999) The mechanism of decomposition of peroxynitrite (OONO؊)in reaction of the acidic with the basic reactant and the local aqueous sodium phosphate buffer solution at neutral pH was non-equilibrium conditions made extrapolating this interpreta- investigated. The OONO؊ was synthesized by directly reacting tion to biological conditions at pH 7 uncertain. We decided to nitric oxide with superoxide anion at pH 13. The hypothesis was examine the nature of the reactive species generated in the explored that OONO؊, after protonation at pH 7.0 to HOONO, decomposition of peroxynitrite under more controlled and 1 decomposes into O2 and HNO according to a spin-conserved physiologically relevant conditions. unimolecular mechanism. Small aliquots of the concentrated alka- In this communication we report an important reaction path- line OONO؊ solution were added to a buffer solution (final pH way for the decomposition of peroxynitrite that yields two ؊ Ϫ 1 7.0–7.2), and the formation of O2 and NO in high yields was transient species, nitroxyl anion (NO ) and singlet molecular 1 observed. The O2 generated was trapped as the transannular oxygen. By using well established analytical procedures, an Ϸ peroxide (DPAO2) of 9,10-diphenylanthracene (DPA) dissolved in aqueous alkaline solution (pH 13) of peroxynitrite was allowed carbon tetrachloride. The nitroxyl anion (NO؊) formed from HNO to react in separate but parallel experiments with two chemical (pKa 4.5) was trapped as nitrosylhemoglobin (HbNO) in an aqueous traps, one specific for singlet oxygen and one specific for the Ϫ methemoglobin (MetHb) solution. In the presence of 25 mM nitroxyl anion, NO . Singlet oxygen was trapped as the transan- sodium bicarbonate, which is known to accelerate the rate of nular peroxide (DPAO2) of 9,10-diphenylanthracene (DPA) -decomposition of OONO؊, the amount of singlet oxygen trapped (25–27), and the nitroxyl anion was trapped as nitrosylhemo was reduced by a factor of Ϸ2 whereas the yield of trapping of NO؊ globin (HbNO) in aqueous solutions of methemoglobin ؊ by methemoglobin remained unaffected. Because NO3 is known (MetHb) (28), both with very high yields. The effects of bicar- ؊ Ϫ 1 to be the ultimate decomposition product of OONO , these results bonate ions on the yields of NO and O2 in aqueous solution suggest that the nitrate anion is not formed by a direct isomer- were investigated. .ization of OONO؊, but by an indirect route originating from NO؊ Methods nitrosylhemoglobin ͉ diphenylanthracene endoperoxide Chemicals. Bovine MetHb (Sigma) was crystallized, dialyzed, and lyophilized. DPA (97%), perylene (99%), 2,3-dimethyl-2-butene [tetramethylethylene (TME)] (98%), acetone (spectral grade), eroxynitrite is a potent oxidant formed by the near diffusion- ⅐ potassium superoxide (KO ), and potassium hydroxide (KOH) Pcontrolled reaction of nitric oxide (NO ) and superoxide ion 2 Ϫ were obtained from Aldrich. Angeli’s salt (Na2N2O3) was ob- (O2 ) (1). Both nitric oxide and superoxide are produced by tained from Cayman Chemicals (Ann Arbor, MI). Hydrogen activated macrophages (2, 3), neutrophils (4), and endothelial peroxide (H2O2) (30% aqueous solution) (Certified ACS), so- cells (5, 6). There is evidence that peroxynitrite is formed in dium hypochlorite (NaOCl) (4–6% available chlorine, purified significant concentrations in vivo (7–9) and may contribute to an grade), and acetone were from Fisher. Carbon tetrachloride increased risk for cancer (10), artherosclerosis (11), stroke (12), (Reagent ACS) was obtained from Spectrum Chemical (Gard- and other diseases (13). Peroxynitrite is a stable anion in alkaline nia, CA), and nitric oxide (NO) gas was from Matheson Gas solution (pKa of 6.8); however, once protonated, it decomposes (East Rutherford, NJ). rapidly with a half-life of less than1satphysiological pH at 37°C (14), generating reactive species that readily react with biomol- Peroxynitrite. Peroxynitrite was generated as described (23) by ecules such as lipids (15), amino acids (16), and DNA (17). bubbling NO gas into a deoxygenated 1 M KOH solution at 0°C, Central to the question of the biochemistry of peroxynitrite is the Ϸ and then adding KO2 powder ( 5 mg), in small amounts, until mechanism of decomposition and the identity of the reactive a bright yellow solution was obtained. The pH of the solution was species, a subject of intense research and controversy (18–21). Ϸ13, and the concentration of peroxynitrite was in the range of Speculations about the decomposition mechanisms are largely 70–110 mM based on the absorbance at 302 nm (␧ ϭ 1,670 based on kinetic and thermodynamic considerations (22). It has MϪ1⅐cmϪ1) (29). been shown that bicarbonate ion enhances the rate of disap- pearance of peroxynitrite, leading to the proposal of a nitros- Reagent Solutions. A concentrated solution of MetHb was made operoxycarbonate anion adduct, with a distinctly different chem- in 1 ml of deoxygenated 70 mM phosphate buffer (pH 7.0) and istry from that of OONOϪ (19). We reported previously that the mere acidification of an aqueous peroxynitrite solution resulted in chemiluminescence at Abbreviations: DPA, 9,10-diphenylanthracene; HbNO, nitrosylhemoglobin; MetHb, met- 1,270 nm characteristic of the deactivation of singlet oxygen (23). hemoglobin; TME, tetramethylethylene. 1 †To whom reprint requests should be addressed at: 453 Brown Building, Department of By comparing the intensity of this emission to that of O2 generated by the reaction of hydrogen peroxide (H O ) with Chemistry, New York University, 29 Washington Square, New York, NY 10003. E-mail: 2 2 [email protected]. hypochlorite anion (OClϪ), which is known to be stoichiometric 1 The publication costs of this article were defrayed in part by page charge payment. This (24), it was concluded that the yield of O2 from peroxynitrite article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. was nearly stoichiometric. These results suggested a spin- §1734 solely to indicate this fact. 1 conserved process leading to the generation of O2 from Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073͞pnas.050587297. peroxynitrite. However, the highly exothermic neutralization Article and publication date are at www.pnas.org͞cgi͞doi͞10.1073͞pnas.050587297 2984–2989 ͉ PNAS ͉ March 28, 2000 ͉ vol. 97 ͉ no. 7 Downloaded by guest on September 28, 2021 was further purified by passing through a Sephadex G-25 column (Pharmacia) using 70 mM phosphate buffer as the elutant. The eluted solution was then diluted with additional 70 mM phos- phate buffer to bring the concentration to around 60–85 ␮M. The concentration was determined by absorbance at 406 nm ␧ ϭ Ϫ1⅐ Ϫ1 ( 406 154 mM cm ) (30). Solutions of Na2N2O3 (80 mM) in 10 mM NaOH were prepared just before use and were stored on ice. Solutions of KNO2 (100 mM) were prepared in 70 mM phosphate buffer (pH 7.0). Solutions of NaHCO3 were prepared by adding premeasured amounts of solid sodium bicarbonate, to give a final concentration of 25 mM (31), to individual polypro- pylene centrifuge tubes, to which were added 2-ml aliquots of the MetHb solutions in 70 mM phosphate buffer. ؊ NO Trapping by MetHb Under N2. Experimental procedures were as follows: First, aliquots were taken from an individual sample of the 2 ml of buffered 80 ␮M MetHb solution (with or without sodium bicarbonate), absorption spectra were taken with either a 1-mm path length cuvette for the 400-nm Soret region or 1-cm for the visible region. Next, the solutions were transferred back to the centrifuge tube. To this MetHb solution was then added ␮ one aliquot (from 1 to 10 l) of either peroxynitrite or KNO2 solution, or KOH solution, immediately before the absorption spectrum was recorded. The absorption spectrum of the ͞ MetHb Na2N2O3 solution was recorded 10 min after addition, resulting in the HbNO reference spectrum (without bicarbon- ate). Absorption measurements were made by using a Hewlett Packard 8453 UV-VIS diode array spectrophotometer. After the absorption measurements, the pH of each of the solutions was Fig. 1. Methemoglobin conversion to nitrosylhemoglobin. Shown are ab- measured with a pH meter (Horizon Ecology, Chicago). sorption spectra of solutions of 80 ␮M MetHb in 70 mM phosphate buffer (pH 7.0) at room temperature under N2. Trace a, MetHb solution at pH 7.0; trace Trapping of 1O . 2 In a typical biphasic experiment, 0.50 ml CCl4 b, MetHb solution at pH 7.2; trace c, MetHb solution with 100 mM KNO2; trace solutions containing either 80 mM DPA and 0.8 mM perylene d, MetHb solution, equilibrated absorption spectrum measured Ϸ10 s after (solution A), or 80 mM DPA, 0.8 mM perylene, and 80 mM TME the addition of 160 ␮M ONOOϪ, final pH 7.2; trace e, absorption spectrum of (solution B), were pipetted into 15-ml propylene centrifuge MetHb solution 10 min after the addition of 160 ␮M Angeli’s salt (pH 7.0). tubes; 100-␮l aliquots of 75 mM OONOϪ solution (pH 13) were then pipetted on top of the CCl4 layer, and the two layers were Ϫ mixed thoroughly by vortexing. While continuing the vortexing, and 0.5 ml CCl4 solution, as described above for the OONO a total of 5 ml of 20 mM phosphate buffer (pH 7) (with or decomposition reactions. without bicarbonate) were added drop-wise to this emulsified Results CCl4 solution to initiate the decomposition of peroxynitrite. Trapping of NO؊ from Peroxynitrite by MetHb.