Formation of Peroxynitrite from Reaction of Nitroxyl Anion with Molecular Oxygen
2002; Elsevier BV; Volume: 277; Issue: 16 Linguagem: Inglês
10.1074/jbc.m108079200
ISSN1083-351X
AutoresMichael Kirsch, Herbert de Groot,
Tópico(s)Electron Spin Resonance Studies
ResumoPeroxynitrite (ONOO−/ONOOH) is generally expected to be formed in vivo from the diffusion-controlled reaction between superoxide (O2⨪) and nitric oxide (⋅NO). In the present paper we show that under aerobic conditions the nitroxyl anion (NO−), released from Angeli's salt (disodium diazen-1-ium-1,2,2-triolate,−ON=NO2−), generated peroxynitrite with a yield of about 65%. Simultaneously, hydroxyl radicals are formed from the nitroxyl anion with a yield of about 3% via a minor, peroxynitrite-independent pathway. Further experiments clearly underline that the chemistry of NO− in the presence of oxygen is mainly characterized by peroxynitrite and not by HO⋅ radicals. Quantum-chemical calculations predict that peroxynitrite formation should proceed via intermediary formation of⋅NO and O2⨪, probably by an electron-transfer mechanism. This prediction is supported by the fact that H2O2 is formed during the decay of NO− in the presence of superoxide dismutase (Cu(II),Zn-SOD). Since the nitroxyl anion may be released endogenously by a variety of biomolecules, substantial amounts of peroxynitrite might be formed in vivo via NO− in addition to the "classical" ⋅NO + O2⨪pathway. Peroxynitrite (ONOO−/ONOOH) is generally expected to be formed in vivo from the diffusion-controlled reaction between superoxide (O2⨪) and nitric oxide (⋅NO). In the present paper we show that under aerobic conditions the nitroxyl anion (NO−), released from Angeli's salt (disodium diazen-1-ium-1,2,2-triolate,−ON=NO2−), generated peroxynitrite with a yield of about 65%. Simultaneously, hydroxyl radicals are formed from the nitroxyl anion with a yield of about 3% via a minor, peroxynitrite-independent pathway. Further experiments clearly underline that the chemistry of NO− in the presence of oxygen is mainly characterized by peroxynitrite and not by HO⋅ radicals. Quantum-chemical calculations predict that peroxynitrite formation should proceed via intermediary formation of⋅NO and O2⨪, probably by an electron-transfer mechanism. This prediction is supported by the fact that H2O2 is formed during the decay of NO− in the presence of superoxide dismutase (Cu(II),Zn-SOD). Since the nitroxyl anion may be released endogenously by a variety of biomolecules, substantial amounts of peroxynitrite might be formed in vivo via NO− in addition to the "classical" ⋅NO + O2⨪pathway. Peroxynitrite (ONOO− 1The abbreviations used are: ONOO−oxoperoxonitrate(1-)ONOOHhydrogen oxoperoxonitrate(1-)peroxynitriteONOO−/ONOOHSIN-13-morpholinosydnonimineN-ethylcarbamideAngeli's saltdisodium diazen-1-ium-1,2,2-triolateDHRdihydrorhodamine 123RHrhodamine 123CO3⨪trioxocarbonate(1-)BAbenzoic acidDAN2,3-diaminonaphthaleneNAT2,3-naphthotriazoleDEA-NONOatesodium 2-(N,N-diethylamino)-diazenolate-2-oxideO2⨪superoxide/ONOOH) can be formedin vivo from the diffusion-controlled reaction (k = 3.9–19 × 109m−1 s−1) between superoxide (O2⨪) and nitric oxide (nitrogen monoxide, ⋅NO) (1.Ross A.B. Mallard W.G. Helman W.P. Buxton G.V. Huie R.E. Neta P. NDRL/NIST Solution Kinetics Database 3.0. NDRL/NIST, Gaithersburg, MD1998Google Scholar, 2.Kissner R. Nauser T. Bugnon P. Lye P.G. Koppenol W.H. Chem. Res. Toxicol. 1997; 10: 1285-1292Crossref PubMed Scopus (569) Google Scholar). The pathological activity of ONOO− is closely related to its reaction with CO2 (3.Squadrito S.L. Pryor W.A. Free Rad. 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Cancer Surv. 1989; 8: 363-384PubMed Google Scholar) and carcinogenicN-nitrosoamines (23.Challis, B. C., and Kyrtopoulos, S. A. (1978) J. Chem. Soc. Perkin Trans. I 299–304Google Scholar) are additionally expected to be produced by peroxynitrite. oxoperoxonitrate(1-) hydrogen oxoperoxonitrate(1-) ONOO−/ONOOH 3-morpholinosydnonimineN-ethylcarbamide disodium diazen-1-ium-1,2,2-triolate dihydrorhodamine 123 rhodamine 123 trioxocarbonate(1-) benzoic acid 2,3-diaminonaphthalene 2,3-naphthotriazole sodium 2-(N,N-diethylamino)-diazenolate-2-oxide superoxide A further source of endogenous peroxynitrite may be the nitroxyl anion (NO−). This anion has been reported to be generatedin vivo from reduction of ⋅NO by Cu(I),Zn-SOD (24.Murphy M.E. Sies H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10860-10864Crossref PubMed Scopus (288) Google Scholar), hemoglobin (25.Gow A.J. Stamler J.S. 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FEBS Lett. 1996; 382: 223-228Crossref PubMed Scopus (249) Google Scholar, 31.Arnelle D.R. Stamler J.S. Arch. Biochem. Biophys. 1995; 318: 279-285Crossref PubMed Scopus (542) Google Scholar), although this reaction is less well understood. Donaldet al. (32.Donald C.E. Hughes M.N. Thompson J.M. Bonner F.T. Inorg. Chem. 1986; 25: 2676-2677Crossref Scopus (87) Google Scholar) have proven that the photochemical decomposition of Angeli's salt, a chemical NO− donor compound (33.Angeli A. Angelico F. Gazz. Chim. Ital. 1903; 33: 245-252Google Scholar), in fact yields peroxynitrite under aerobic conditions (Reaction 1). This photochemical process is probably the reason why NO− has often been referred to as a peroxynitrite-yielding compound, even in textbooks of inorganic chemistry (34.Wiberg N. Holleman-Wiberg Lehrbuch der anorganischen Chemie. Walter de Gruyter & Co., Berlin1995Google Scholar).NO−+O2→ONOO−REACTION 1Only very low yields of nitrated products have been observed from NO−-induced reactions (35.Ohshima H. Celan I. Chazotte L. Pignatelli B. Mower H.F. Nitric Oxide-Biol. Chem. 1999; 3: 132-141Crossref PubMed Scopus (67) Google Scholar, 36.vanUffelen B.E. van der Zee J. de Koster B.M. vanSteveninck J. Elferink J.G.R. Biochem. J. 1998; 330: 719-722Crossref PubMed Scopus (89) Google Scholar). From these facts it was concluded that during thermal decomposition of Angeli's salt only a small amount of peroxynitrite is generated (36.vanUffelen B.E. van der Zee J. de Koster B.M. vanSteveninck J. Elferink J.G.R. Biochem. J. 1998; 330: 719-722Crossref PubMed Scopus (89) Google Scholar). Recently, two research groups (37.Miranda K.M. Espey M.G. Ludwick N. Kim S.M. Jourd′heuil D. Grisham M.B. Feelisch M. Fukuto J.M. Wink D.A. J. Biol. Chem. 2001; 276: 1720-1727Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 38.Reif A. Zecca L. Riederer P. Feelisch M. Schmidt H.H.H.W. Free Rad. Biol. Med. 2001; 30: 803-808Crossref PubMed Scopus (35) Google Scholar) apparently disproved the capability of NO−to generate peroxynitrite under more physiological conditions. They reported, for example, that typical peroxynitrite-mediated reactions,e.g. nitrosation reactions, could not be observed (37.Miranda K.M. Espey M.G. Ludwick N. Kim S.M. Jourd′heuil D. Grisham M.B. Feelisch M. Fukuto J.M. Wink D.A. J. Biol. Chem. 2001; 276: 1720-1727Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) and that NADPH could be oxidized by NO− under hypoxic conditions (38.Reif A. Zecca L. Riederer P. Feelisch M. Schmidt H.H.H.W. Free Rad. Biol. Med. 2001; 30: 803-808Crossref PubMed Scopus (35) Google Scholar). Unfortunately, these experiments were performed in the presence of organic buffer compounds (Good's buffer) which are known to effectively react with peroxynitrite (39.Lomonosova L.L. Kirsch M. Rauen U. de Groot H. Free Rad. Biol. Med. 1998; 24: 522-528Crossref PubMed Scopus (60) Google Scholar, 40.Kirsch M. Lomonosova E.E. Korth H.-G. Sustmann R. de Groot H. J. Biol. Chem. 1998; 273: 12716-12724Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Thus, the formation of peroxynitrite by NO− very easily may have been masked. In the present study, we demonstrate that in the presence of molecular oxygen NO− indeed mainly yields peroxynitrite and that NO− additionally produces HO⋅ radicals via a minor, peroxynitrite-independent pathway. Furthermore, we present a key experiment which suggests the intermediacy of O2⨪during the NO−-mediated formation of peroxynitrite. Catalase from beef liver (EC 1.11.1.6), copper-zinc superoxide dismutase from bovine erythrocytes (EC1.15.1.1), and NADH were obtained from Roche Molecular Biochemicals (Mannheim, Germany). Manganese dioxide, benzoic acid, and hydrogen peroxide were from Sigma (Deisenhofen, Germany). Angeli's salt came from Alexis-Deutschland (Grünberg, Germany). DHR and DAN were obtained from Molecular Probes (Leiden, The Netherlands). Commercially available mixtures of oxygen 5.0 and nitrogen 5.0 (20.5% O2, 79.5% N2, "synthetic air") and commercially available mixtures of oxygen 5.0, nitrogen 5.0, and carbon dioxide 4.6 (20.5% O2, 74.5% N2, 5% CO2) were purchased from Messer-Griessheim (Oberhausen, Germany; 5.0 and 4.6 mean purities of 99.999 and 99.996%, respectively). SIN-1 and its decomposition product SIN-1C were generously provided by Drs. K. Schönafinger and J. Pünter (Aventis, Frankfurt/Main, Germany). NAT was synthesized as described by Wheeler et al. (41.Wheeler G.L. Andrejack J. Wiersma J.H. Lott P.F. Anal. Chim. Acta. 1969; 46: 239-245Crossref Scopus (17) Google Scholar). Peroxynitrite (oxoperoxonitrate(1-)) (0.73 m) was prepared by isoamylnitrite-induced nitrosation of hydrogen peroxide (0.12 mol of isoamylnitrite, 100 ml of H2O2 (1m) plus diethylenetriaminepentaacetic acid (2 mm)) and purified (e.g. solvent extraction, removal of excess H2O2, N2-purging) as described by Uppu and Pryor (42.Uppu R.M. Pryor W.A. Anal. Biochem. 1996; 236: 242-249Crossref Scopus (227) Google Scholar) and stored at −79 °C. All other chemicals were of the highest purity commercially available. Care was taken to exclude possible contamination by both bicarbonate/carbon dioxide and transition metals. Doubly distilled water was bubbled (2 liters/min) with synthetic air at room temperature for 20 min. This water was used for synthesis of oxoperoxonitrate(1-), NaOH (0.01–0.5 n) and for all other solutions. Phosphate buffer solutions (50 mm) were treated with the heavy metal scavenger resin Chelex 100 (0.3/0.5 g in 10 ml) by gently shaking for 18 h in the dark. After low-speed centrifugation for 5 min, the solutions were carefully decanted from the resin. The resin treatment resulted in an increase in pH by about 0.25 units. Various additives (DHR, NADH, and benzoic acid) were then added. The pH was adjusted to 7.5 at 37 °C and the solutions were again bubbled (2 liters/min) with synthetic air or with the CO2 mixture for 20 min. In the case of CO2bubbling, the pH had to be readjusted to 7.5. SIN-1 and Angeli's salt solutions were prepared as ×100 stock solutions at 4 °C in 50 mm KH2PO4 and in 10 mm NaOH, respectively, and used within 15 min. SIN-1 was added to 1 ml of phosphate buffer and incubated in 12-well cell culture plates (volume of each well 7 ml, Falcon, Heidelberg, Germany). Under HCO3−/CO2-free conditions, these plates were placed in an air-tight vessel (10 liters). During the first 15 min of each experiment, these vessels were flushed (5 liters/min) with synthetic air in a warming incubator (Heraeus, Hanau, Germany). In the presence of HCO3−/CO2 the plates were placed in an incubator for cell culture (37 °C, humidified atmosphere of 95% authentic air and 5% CO2, Labotect, Göttingen, Germany). The experiments with authentic peroxynitrite (2 μl of 25–125 mm ONOO− in 0.5n NaOH was added to 1 ml of reaction solution) and with Angeli's salt (1 ml of reaction solution) were performed in reaction tubes (2.0 ml, Eppendorf, Hamburg, Germany) by using the drop-tube Vortex mixer technique as described previously (40.Kirsch M. Lomonosova E.E. Korth H.-G. Sustmann R. de Groot H. J. Biol. Chem. 1998; 273: 12716-12724Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Under HCO3−/CO2-free conditions, the experiments with authentic peroxynitrite and Angeli's salt were performed in a glove-bag (Roth, Karlsruhe, Germany) under synthetic air. Peroxynitrite, SIN-1, and Angeli's salt (each 50–600 μm)-dependent hydroxylation of BA (5 mm) were employed. After vortexing, the samples were kept for 2 min (in the case of peroxynitrite), 4 h (in the case of SIN-1), and 30 min (in the case of Angeli's salt) at 37 °C, respectively. The product formed was measured by reading its fluorescence with excitation at 290 nm and emission at 410 nm (37.Miranda K.M. Espey M.G. Ludwick N. Kim S.M. Jourd′heuil D. Grisham M.B. Feelisch M. Fukuto J.M. Wink D.A. J. Biol. Chem. 2001; 276: 1720-1727Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Formation of RH was quantified spectrophotometrically at 500 nm (εM = 78,000m−1 cm−1) (43.Haddad I.Y. Crow J.P. Hu P. Ye Y. Beckman J.S. Matalon S. Am. J. Physiol. 1994; 267: L242-L249PubMed Google Scholar). Angeli's salt (50–600 μm)-dependent nitrosation of DAN (200 μm) was employed. After vortexing, the samples were incubated for 30 min at 37 °C. NaOH (0.5 m) was added (5:1, v/v, final pH 11–11.5). The product formed, i.e. NAT, was quantified by reading its fluorescence with excitation at 375 nm and emission at 415 nm (44.Miles A.M. Wink D.A. Cook J.C. Grisham M.B. Methods Enzymol. 1996; 268: 105-120Crossref PubMed Google Scholar). Standard calibration curves were prepared from known amounts of NAT. NADH was quantified by reading its fluorescence with excitation at 339 nm and emission at 460 nm (45.Klingenberg M. Bergmeyer H.U. Methods of Enzymatic Analysis. 3rd Ed. VII. VCH Verlagsgesellschaft, Weinheim1985: 251-271Google Scholar). Standard calibration curves were prepared from known amounts of NAD(P)H. Additionally, the oxidation of NAD(P)H was followed photometrically at 340 nm using Δε340 = 6200m−1 cm−1 (45.Klingenberg M. Bergmeyer H.U. Methods of Enzymatic Analysis. 3rd Ed. VII. VCH Verlagsgesellschaft, Weinheim1985: 251-271Google Scholar). Both methods gave identical results, therefore, only one parameter, the decrease of fluorescence, will be shown here. Hydrogen peroxide was quantified by two techniques. In peroxidase assays, horseradish peroxidase-catalyzed formation of a colored product was measured. 4-Aminoantipyrine and 3,5-dichloro-2-hydroxybenzenesulfonic acid were used as peroxidase substrates. The quinoneimine dye formed from these substrates was measured spectrophotometrically at 546 nm (46.Ioannidis I. de Groot H. Biochem. J. 1993; 296: 341-345Crossref PubMed Scopus (129) Google Scholar) (peroxidase assay). Alternatively, H2O2 was quantified by the amount of O2 released upon addition of catalase (1,000 units/ml) (catalase assay). O2 was determined polarographically with a Clark-type oxygen electrode (Saur, Reutlingen, Germany). Both methods gave identical results, therefore, only one parameter, the peroxidase assay, will be shown here. The nitrate yields from decomposition of Angeli's salt (100 and 200 μm) were quantified by the use of nitrate reductase in conjunction with the Griess assay. The Griess assay was carried out as described elsewhere (46.Ioannidis I. de Groot H. Biochem. J. 1993; 296: 341-345Crossref PubMed Scopus (129) Google Scholar). Density functional theory andab initio calculations were performed with the Gaussian 98W (Revision A.9) suite of programs (47.Frisch, M. J., Trucks, G. W., Schlegel, H. B., Gill, P. M. W., Johnson, B. G., Robb, M. A., Cheeseman, J. R., Keith, T., Petersson, G. A., Montgomery, J. A., Raghavachari, K., Al-Laham, M. A., Zakrzewski, V. G., Ortiz, J. V., Foresman, J. B., Cioslowski, J., Stefanov, B. B., Nanayakkara, A., Challacombe, M., Peng, C. Y., Ayala, P. Y., Chen, W., Wong, M. W., Andres, J. L., Replogle, E. S., Gomperts, R., Martin, R. L., Fox, D. J., Binkley, J. S., Defrees, D. J., Baker, J., Stewart, J. P., Head-Gordon, M., Gonzalez, C., and Pople, J. A. (2000)Gaussian 98W Revision, A9 Ed., Gaussian Inc., Pittsburgh, PAGoogle Scholar). Geometries were fully optimized to stationary points, using the CBS-QB3 methodology in the density functional theory calculations and single-excitation CI calculations and second-order M⊘ller-Plesset (48.Møller C. Plesset M.S. Physiol. Rev. 1934; 46: 618-622Crossref Scopus (13521) Google Scholar) (MP2) calculations with the 6–311+G(d) basis set on the ab initio level. Calculation of UV-VIS absorption spectra was performed by single point energy calculations on the CBS-QB3-optimized structure using the protocol of the time-dependent density functional theory method (49.Bauernschmitt R. Ahlrichs R. Chem. Phys. Lett. 1996; 256: 454-464Crossref Scopus (5081) Google Scholar). Aqueous solvent interactions were evaluated with the PCM (50.Miertus S. Scrocco E. Tomasi J. Chem. Phys. 1981; 55: 117-123Crossref Scopus (8186) Google Scholar) procedure incorporated in Gaussian 98W. To verify whether an electron transfer between NO− and O2 would be thermodynamically feasible, geometry optimizations and frequency calculations were done using the MP2 approximation. Molecular interactions were then evaluated with the IPCM (51.Foresman J.B. Keith T.A. Wiberg K.B. Snoonian J. Frisch M.J. J. Phys. Chem. 1996; 100: 16098-16104Crossref Scopus (1190) Google Scholar) procedure. To prove whether ONOO− is formed during the decay of NO− in the presence of oxygen, we attempted to identify peroxynitrite by UV-visible spectroscopy. As the half-life of ONOO− is short at physiological pH values and since UV light induces the decomposition of Angeli's salt to peroxynitrite (32.Donald C.E. Hughes M.N. Thompson J.M. Bonner F.T. Inorg. Chem. 1986; 25: 2676-2677Crossref Scopus (87) Google Scholar), 8 samples of 5 mm Angeli's salt from the same stock solution were incubated in parallel runs in the dark at 37 °C at pH 12.25 (Fig. 1A). The decay of Angeli's salt is slow at these experimental conditions (t1/2 ∼12 h), thus, reaction times of several hours were necessary to monitor the significant changes of the optical density. The initial absorbance at the beginning of the experiment was 0.35 ± 0.05 at 302 nm. With increasing reaction time, the optical density at 302 nm increased continuously to reach a maximum value of 0.88 ± 0.08 after 6 h, followed by a further decrease of the absorption at longer reaction periods. After 24 h the extinction value had dropped to 0.4 ± 0.05, clearly showing that a relatively long-lived intermediate has been formed during the decay of Angeli's salt. The absorption at 302 nm had decayed completely when the reaction solution was briefly (20–30 s) bubbled with CO2 after 5 h of incubation (data not shown). These observations strongly indicated the intermediary formation of ONOO−. In fact, when the initial UV spectrum was subtracted from the one observed after 4 h of incubation, the resulting difference spectrum exhibited an absorption spectrum with a maximum at 302 nm (Fig. 1, B and C), similar to what has been reported for peroxynitrite (52.Kortüm G. Finckh B. Z. Physikal. Chem. 1940; 48: 32-49Google Scholar). This was verified by comparison with the UV spectrum of authentic ONOO− (Fig. 1C). The scatter of the difference spectrum at shorter wavelengths (λ< 295 nm) derives from the strong absorption of Angeli's salt in this wavelength region. The absorbance at 302 nm has been attributed to the cis-conformer of peroxynitrite (53.Tsai, J.-H. M., Hamilton, T. P., Harrison, J. G., Jablowski, M., v. d. Woerd, M., Martin, J. C., and Beckman, J. S. (1994) J. Am. Chem. Soc. 116, 4115–4116Google Scholar,54.Hughes, M. N., and Nicklin, H. G. (1968) J. Chem. Soc. A 450–452Google Scholar). This is excellently supported by time-dependent density functional theory calculations (Table I), which show that thetrans-conformer of ONOO− should absorb at longer wavelengths (λmax = 374 nm). Thus,cis-ONOO− is produced from the NO−-donating compound Angeli's salt during its decay in aerobic solution.Table IQuantum chemically calculated UV-visable absorption of ONOO−isomersCompoundUV-visible absorptionAbsorption coefficient, ɛOscillator strength, fExperimentCalculationnmcis-ONOO−3023031670 ± 501-aRef. 54.0.0928trans-ONOO−4510.00053740.08963200.00052,4-cyclo-ONOO−3520.00033100.00032590.00261-a Ref. 54.Hughes, M. N., and Nicklin, H. G. (1968) J. Chem. Soc. A 450–452Google Scholar. Open table in a new tab The above observations qualitatively prove the formation of peroxynitrite from NO−; the yield of this reaction remained to be established. To this end, the potential of Angeli's salt to oxidize both DHR and NADH were compared with those of the⋅NO/O2⨪releasing compound SIN-1. Increasing concentrations (0–25 μm) of SIN-1 as well as of Angeli's salt stimulated the oxidation of both DHR and NADH (each 50 μm) in a linear fashion (Fig. 2, A and B). While SIN-1 oxidized DHR and NADH with yields of ∼100 and 85%, respectively, Angeli's salt was found to be significantly less effective, oxidizing DHR and NADH with yields of ∼60 and 65%, respectively. Thus, the production of peroxynitrite from Angeli's salt is only 65 ± 5% of that from SIN-1. This fact implied that in the absence of suitable targets (i.e. HEPES, DHR, and NADH) the formation of nitrate from Angeli's salt should be in the same range. In fact, about 65 μm nitrate was formed during the decay of 100 μm Angeli's salt at pH 7.5 irrespective of the presence of CO2 (Fig. 2C). Interestingly, Angeli's salt-derived formation of nitrate was maximal at physiological pH values. Control experiments revealed that nitrate was not a contaminant of the applied Angeli's salt because nitrate could not be detected after decomposition of Angeli's salt (100 μm) at pH 12.25 in the presence of 20 μmCu2+(data not shown). Consequently, Angeli's salt had consumed oxygen with an efficiency of about 65%. To verify this, the oxygen uptake induced by Angeli's salt (50–200 μm) was determined polarographically (Table II). As expected, the amount of O2 consumed by NO−was found to be around 65% of the employed amount of Angeli's salt. Thus, the nitroxyl anion yields peroxynitrite with a yield of about 65%. Our oxygen uptake experiments are in disagreement with data of Miranda et al. (37.Miranda K.M. Espey M.G. Ludwick N. Kim S.M. Jourd′heuil D. Grisham M.B. Feelisch M. Fukuto J.M. Wink D.A. J. Biol. Chem. 2001; 276: 1720-1727Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), who observed a 1:1 stoichiometry between NO− (or rather Angeli's salt) and O2. However, it should be remembered that the experiments of Mirandaet al. (37.Miranda K.M. Espey M.G. Ludwick N. Kim S.M. Jourd′heuil D. Grisham M.B. Feelisch M. Fukuto J.M. Wink D.A. J. Biol. Chem. 2001; 276: 1720-1727Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) were performed in the presence of HEPES, which is known to effectively react with peroxynitrite, thereby further increasing the uptake of O2 (40.Kirsch M. Lomonosova E.E. Korth H.-G. Sustmann R. de Groot H. J. Biol. Chem. 1998; 273: 12716-12724Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar).Table IIYields of Angeli's salt-derived O2 consumptionConditionsO2ΔO22-aReferred to "no additives."Yield2-bYield (%) = ΔO2 × 100/[Angeli's salt].μm%No additives225.0 ± 4Angeli's salt (50 μm)190.4 ± 8−34.669.2Angeli's salt (100 μm)154.8 ± 5−70.270.2Angeli's salt (150 μm)127.9 ± 8−97.164.7Angeli's salt (200 μm)96.7 ± 12.5−128.364.22-a Referred to "no additives."2-b Yield (%) = ΔO2 × 100/[Angeli's salt]. Open table in a new tab Since only ∼65% of the generated NO− was converted into peroxynitrite, the question arose whether other (reactive) intermediates were formed from the remaining 35% of NO−. Recently, two research groups (55.Stoyanovsky D.A. Clancy R. Cederbaum A.I. J. Am. Chem. Soc. 1999; 121: 5093-5094Crossref Scopus (30) Google Scholar, 56.Ohshima H. Gilibert I. Bianchini F. Free Rad. Biol. Med. 1999; 26: 1305-1313Crossref PubMed Scopus (80) Google Scholar) reported that NO− released from Angeli's salt should generate hydroxyl radicals. As peroxynitrite is known to produce HO⋅ radicals (57.Richeson C.E. Mulder P. Bowry V.W. Ingold K.U. J. Am. Chem. Soc. 1998; 120: 7211-7219Crossref Scopus (152) Google Scholar, 58.Beckman J.S. Beckman T.W. Chen J. Marshall P.A. Freeman B.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1620-1624Crossref PubMed Scopus (6731) Google Scholar) with an efficiency of about 28% (9.Hodges G.R. Ingold K.U. J. Am. Chem. Soc. 1999; 121: 10695-10701Crossref Scopus (124) Google Scholar, 59.Gerasimov O.V. Lymar S.V. Inorg. Chem. 1999; 38: 4317-4321Crossref Scopus (97) Google Scholar), it is unclear whether the HO⋅ radicals detected by these groups may have derived exclusively from peroxynitrite. To check on this important point, the Angeli's salt (0–500 μm)-induced hydroxylation of benzoic acid (5 mm) was studied and compared with that of authentic peroxynitrite (0–500 μm) (Fig. 3, A and B). In the absence of CO2 peroxynitrite-dependent hydroxylation of BA increased in a linear manner with increasing concentrations of peroxynitrite. In the presence of CO2, however, peroxynitrite-mediated hydroxylation of BA was inhibited by about 99%. The effect of CO2 to strongly suppress peroxynitrite-derived formation of hydroxyl radicals is in full agreement with recent reports (57.Richeson C.E. Mulder P. Bowry V.W. Ingold K.U. J. Am. Chem. Soc. 1998; 120: 7211-7219Crossref Scopus (152) Google Scholar, 60.Lemercier J.N. Padmaja S. Cueto R. Squadrito G.L. Uppu R.M. Pryor W.A. Arch. Biochem. Biophys. 1997; 345: 160-170Crossref PubMed Scopus (111) Google Scholar). Moreover, this effect of CO2 offers the possibility to distinguish between HO⋅ released from peroxynitrite and HO⋅ radicals released from other sources. Similar to peroxynitrite, Angeli's salt-mediated hydroxylation of BA increased with increasing concentration of Angeli's salt in the absence of CO2 although not in a strictly linear fashion (Fig. 3B). The efficacy of Angeli's salt to hydroxylate BA decreased with increasing concentration compared with authentic peroxynitrite. This result again is in disagreement with observations by Miranda et al. (37.Miranda K.M. Espey M.G. Ludwick N. Kim S.M. Jourd′heuil D. Grisham M.B. Feelisch M. Fukuto J.M. Wink D.A. J. Biol. Chem. 2001; 276: 1720-1727Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) who found that Angeli's salt was much more effective in hydroxylating BA than authentic peroxynitrite. Again, the usage o
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