Ascorbate Is a Potent Antioxidant against Peroxynitrite-induced Oxidation Reactions
2000; Elsevier BV; Volume: 275; Issue: 22 Linguagem: Inglês
10.1074/jbc.m909228199
ISSN1083-351X
AutoresMichael Kirsch, Herbert de Groot,
Tópico(s)Vitamin C and Antioxidants Research
ResumoPeroxynitrite (ONOO−/ONOOH) is expected in vivo to react predominantly with CO2, thereby yielding NO2· and CO⨪3 radicals. We studied the inhibitory effects of ascorbate on both NADH and dihydrorhodamine 123 (DHR) oxidation by peroxynitrite generated in situ from 3-morpholinosydnonimineN-ethylcarbamide (SIN-1). SIN-1 (150 μm)-mediated oxidation of NADH (200 μm) was half-maximally inhibited by low ascorbate concentrations (61–75 μm), both in the absence and presence of CO2. Control experiments performed with thiols indicated both the very high antioxidative efficiency of ascorbate and that in the presence of CO2 in situ-generated peroxynitrite exclusively oxidized NADH via the CO⨪3 radical. This fact is attributed to the formation of peroxynitrate (O2NOO−/O2NOOH) from reaction of NO2· with O⨪2, which is formed from reaction of CO⨪3 with NADH. SIN-1 (25 μm)-derived oxidation of DHR was half-maximally inhibited by surprisingly low ascorbate concentrations (6–7 μm), irrespective of the presence of CO2. Control experiments performed with authentic peroxynitrite revealed that ascorbate was in regard to both thiols and selenocompounds much more effective to protect DHR. The present results demonstrate that ascorbate is highly effective to counteract the oxidizing properties of peroxynitrite in the absence and presence of CO2 by both terminating CO⨪3/HO⋅ reactions and by its repair function. Ascorbate is therefore expected to act intracellulary as a major peroxynitrite antagonist. In addition, a novel, ascorbate-independent protection pathway exists: scavenging of NO2· by O⨪2 to yield O2NOO−, which further decomposes into NO2− and O2. Peroxynitrite (ONOO−/ONOOH) is expected in vivo to react predominantly with CO2, thereby yielding NO2· and CO⨪3 radicals. We studied the inhibitory effects of ascorbate on both NADH and dihydrorhodamine 123 (DHR) oxidation by peroxynitrite generated in situ from 3-morpholinosydnonimineN-ethylcarbamide (SIN-1). SIN-1 (150 μm)-mediated oxidation of NADH (200 μm) was half-maximally inhibited by low ascorbate concentrations (61–75 μm), both in the absence and presence of CO2. Control experiments performed with thiols indicated both the very high antioxidative efficiency of ascorbate and that in the presence of CO2 in situ-generated peroxynitrite exclusively oxidized NADH via the CO⨪3 radical. This fact is attributed to the formation of peroxynitrate (O2NOO−/O2NOOH) from reaction of NO2· with O⨪2, which is formed from reaction of CO⨪3 with NADH. SIN-1 (25 μm)-derived oxidation of DHR was half-maximally inhibited by surprisingly low ascorbate concentrations (6–7 μm), irrespective of the presence of CO2. Control experiments performed with authentic peroxynitrite revealed that ascorbate was in regard to both thiols and selenocompounds much more effective to protect DHR. The present results demonstrate that ascorbate is highly effective to counteract the oxidizing properties of peroxynitrite in the absence and presence of CO2 by both terminating CO⨪3/HO⋅ reactions and by its repair function. Ascorbate is therefore expected to act intracellulary as a major peroxynitrite antagonist. In addition, a novel, ascorbate-independent protection pathway exists: scavenging of NO2· by O⨪2 to yield O2NOO−, which further decomposes into NO2− and O2. 3-morpholinosydnonimine N-ethylcarbamide diethylenetriaminepentaacetic acid dihydrorhodamine 123 rhodamine 123 trioxocarbonate (1−) 1-carboxylato-2-nitrosodioxidane Oxoperoxonitrate (1−) (ONOO−) can be formed in vivo from the diffusion-controlled reaction (k = 3.9–19 × 109m−1s−1) between superoxide (O⨪2) 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 (568) Google Scholar). The continuous formation of low amounts of ONOO− can be simulated in experimental systems with the O⨪2 and ⋅NO-releasing compound SIN-1.1 The pathological activity of ONOO− and its conjugated acid (hydrogen oxoperoxonitrate (1−), ONOOH), collectively often referred to as peroxynitrite, may be related to its capability to nitrate tyrosine (3.Kooy N.W. Royall J.A. Ye Y.Z. Kelly D.R. Beckman J.S. Am. J. Respir. Crit. Care Med. 1995; 151: 1250-1254PubMed Google Scholar). On the other hand, there is increasing evidence that peroxynitrite mediates its destructive power via oxidizing protein and non-protein sulfhydryls (4.Radi R. Beckman J.S. Bush K.M. Freeman B.A. J. Biol. Chem. 1991; 266: 4244-4250Abstract Full Text PDF PubMed Google Scholar), membrane phospholipids (5.Radi R. Beckman J.S. Bush K.M. Freeman B.A. Arch. Biochem. Biophys. 1991; 288: 481-487Crossref PubMed Scopus (2058) Google Scholar), low density lipoproteins (6.White C.R. Brock T.A. Chang L.Y. Crapo J. Briscoe P. Ku D. Bradley W.A. Gianturco S.H. Gore J. Freeman B.A. Tarpey M.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1044-1048Crossref PubMed Scopus (662) Google Scholar), and NAD(P)H (7.Kirsch M. de Groot H. J. Biol. Chem. 1999; 274: 24664-24670Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Ascorbate is not believed to be an important scavenger of peroxynitrite because the intracellular concentration of ascorbate (roughly 1 mm, e.g. in human lung (Ref. 8.Slade R. Stead A.G. Graham J.A. Hatch G.E. Am. Rev. Respir. Dis. 1985; 131: 742-746PubMed Google Scholar) and neutrophils (Ref. 9.Washko P. Rotrosen D. Levine M. J. Biol. Chem. 1989; 264: 18996-19002Abstract Full Text PDF PubMed Google Scholar)) is significantly lower than the intracellular concentration of GSH (5–10 mm; e.g. Ref. 10.Meredith M.J. Reed D.J. J. Biol. Chem. 1982; 257: 3747-3753Abstract Full Text PDF PubMed Google Scholar), and also because peroxynitrite reacts ∼5.7 times slower with ascorbate (k(peroxynitrite + ascorbate) = 235m−1 s−1 (Refs. 11.Bartlett D. Church D. Bounds P.L. Koppenol W.H. Free Radical Biol. Med. 1995; 18: 85-92Crossref PubMed Scopus (165) Google Scholar and 12.Squadrito G.L. Xia J. Pryor W.A. Arch. Biochem. Biophys. 1995; 322: 53-59Crossref PubMed Scopus (121) Google Scholar)) than with GSH (13.Koppenol W.H. Moreno J.J. Pryor W.A. Ischiropoulos H. Beckman J.S. Chem. Res. Toxicol. 1992; 5: 834-842Crossref PubMed Scopus (1279) Google Scholar). In view of the facts, however, that (i) in the presence of physiological concentrations of HCO3−/CO2 peroxynitrite preferentially reacts with CO2 (14.Squadrito S.L. Pryor W.A. Free Radical Biol. Med. 1998; 25: 392-403Crossref PubMed Scopus (734) Google Scholar) to produce the radicals CO⨪3 (15.Bonini M.G. Radi R. Ferrer-Sueta G. Ferreira A.M.D.C. Augusto O. J. Biol. Chem. 1999; 274: 10802-10806Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 16.Meli R. Nauser T. Koppenol W.H. Helv. Chim. Acta. 1999; 82: 722-725Crossref Scopus (65) Google Scholar) and NO2· (17.Lehnig M. Arch. Biochem. Biophys. 1999; 368: 303-318Crossref PubMed Scopus (66) Google Scholar) with a yield of about 30–35% (18.Lymar S.V. Hurst J.K. Inorg. Chem. 1998; 37: 294-301Crossref Scopus (166) Google Scholar, 19.Goldstein S. Czapski G. J. Am. Chem. Soc. 1998; 120: 3458-3463Crossref Scopus (167) Google Scholar, 20.Hodges G.R. Ingold K.U. J. Am. Chem. Soc. 1999; 121: 10695-10701Crossref Scopus (123) Google Scholar) and (ii) the reactivity of ascorbate on both radicals is high (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), we hypothesized that ascorbate indeed should be an effective peroxynitrite antagonist under in vivo conditions. Furthermore, ascorbate can often restitute a substrate by rereducing the corresponding substrate radical or radical cation (21.Hunter E.P.L. Desrosiers M.F. Simic M.G. Free Radical Biol. Med. 1989; 6: 581-585Crossref PubMed Scopus (143) Google Scholar, 22.Packer J.E. Slater T.F. Willson R.L. Nature. 1979; 278: 737-738Crossref PubMed Scopus (1199) Google Scholar, 23.Sturgeon B.E. Sipe Jr., H.J. Barr D.P. Corbett J.T. Martinez J.G. Mason R.P. J. Biol. Chem. 1998; 273: 30116-30121Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), i.e. an antioxidative process generally referred to as the repair function. DHR is very sensitive to oxidants derived from peroxynitrite (24.Kooy N.W. Royall J.A. Ischiropoulos H. Beckman J.S. Free Radical Biol. Med. 1994; 16: 149-156Crossref PubMed Scopus (676) Google Scholar) and has thus been used to delineate the antioxidative properties of glutathione peroxidase against peroxynitrite-derived oxidation reactions (25.Sies H. Sharov V.S. Klotz L.-O. Briviba K. J. Biol. Chem. 1997; 272: 27812-27817Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar). We recently observed that peroxynitrite and radicals derived from it rapidly react with NADH (7.Kirsch M. de Groot H. J. Biol. Chem. 1999; 274: 24664-24670Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) to yield O⨪2/H2O2 and NAD+. Using both authentic peroxynitrite and peroxynitrite generated in situ from SIN-1, we studied the inhibitory effects of ascorbate on the oxidation of both NADH and DHR in the absence and presence of CO2. Although ascorbate turned out to be a very potent antioxidant against peroxynitrite-derived oxidations reactions in general, surprising differences were found for its antioxidative functions on DHR and NADH oxidation, respectively. Catalase from beef liver (EC 1.11.1.6), copper-zinc superoxide dismutase from bovine erythrocytes (EC1.15.1.1), NADH, and NADPH were obtained from Roche Molecular Biochemicals (Mannheim, Germany). Manganese dioxide, hydrogen peroxide, DTPA, GSH, cysteine, and HClO4 (suprapure) were from Sigma (Deisenhofen, Germany). Ascorbic acid was from Merck (Darmstadt, Germany). 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 and 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 Dr. K. Schönafinger (Hoechst Marion Roussel, Frankfurt/Main, Germany). Peroxynitric acid (O2NOOH) solutions (1.57 ± 0.02 m) were prepared freshly each day as described by Appelman and Gosztola (26.Appelman E.H. Gosztola D.J. Inorg. Chem. 1995; 34: 787-791Crossref Scopus (55) Google Scholar) with minor modifications. In brief, 0.52 g of NaNO2was dissolved in 1.5 ml of 30% H2O2, and 150 μl of 70% HClO4 was mixed with 1 ml of 30% H2O2. Both solutions were cooled in an ice/H2O/NaCl bath to −17 °C. The following procedure was also performed at this temperature. The nitrite peroxide solution was carefully added to the stirred perchloric acid solution in 5-μl aliquots. After each 250-μl addition of nitrite peroxide solution, an additional 100 μl of 70% HClO4 were further added. The resulting product solution is stable for a few hours at −17 °C and was used within 45 min. 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 DTPA (2 mm)) and purified (e.g. solvent extraction, removal of excess H2O2, N2 purging) as described by Uppu and Pryor (27.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 either 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 g/0.5 g in 10 ml) in the absence and presence of ascorbate (400 mm) 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 of pH by about 0.25 units. Various additives (DTPA, NADH, thiols) were then added. The pH was adjusted to 7.5 at 37 °C, and the solution was again bubbled (2 liters/min) with synthetic air or with the CO2 mixture for 20 min. In the case of CO2 bubbling, the pH had to be readjusted to 7.5. SIN-1 solutions were prepared as 100× stock solutions at 4 °C in 50 mmKH2PO4 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 peroxynitric acid (1 μl of 1.57 m O2NOOH was added to 1 ml of reaction solution) were performed in reaction tubes (1.4 ml; Eppendorf, Hamburg, Germany) by using the drop-tube vortex mixer technique as described previously (28.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 peroxynitric acid were performed in a glove-bag (Roth, Karlsruhe, Germany) under synthetic air. Ascorbate was quantified by reading its absorbance at 265 nm (εM = 14500m−1 cm−1; Ref. 29.Buettner G.R. J. Biochem. Biophys. Methods. 1988; 16: 27-40Crossref PubMed Scopus (443) Google Scholar) after the pretreatment of the stock solution with Chelex 100. SIN-1 and SIN-1C were quantified by capillary zone electrophoresis on a Beckman P/ACE 5000 apparatus as described previously (28.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). Formation of RH was quantified spectrophotometrically at 500 nm (εM = 78,000m−1 cm−1) (30.Haddad I.Y. Crow J.P. Hu P. Ye Y. Beckman J.S. Matalon S. Am. J. Physiol. 1994; 267: L242-L249PubMed Google Scholar). The concentration of the peroxynitric acid stock solution (0.2 μl) was quantified by the amount of O2 released in 100 mm potassium phosphate buffer (1 ml) at pH 11. O2 was determined polarographically with a Clark-type oxygen electrode (Saur, Reutlingen, Germany). NAD(P)H was quantified by reading its fluorescence with excitation at 339 nm and emission at 460 nm (31.Klingenberg M. Bergmeyer H.U. Methods of Enzymatic Analysis. 3rd Ed. VII. VCH Verlagsgesellschaft, Weinheim, Germany1985: 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 ε340 = 6200m−1 cm−1 (31.Klingenberg M. Bergmeyer H.U. Methods of Enzymatic Analysis. 3rd Ed. VII. VCH Verlagsgesellschaft, Weinheim, Germany1985: 251-271Google Scholar). Both methods gave identical results; therefore, only one parameter, the decrease of fluorescence, will be shown here. Superoxide radicals were determined by using the modified ferricytochrome c reduction technique of McCord and Fridovich (32.McCord J.M. Fridovich I. J. Biol. Chem. 1969; 244: 6049-6055Abstract Full Text PDF PubMed Google Scholar). Peroxynitrite (100 μm) was vortexed to the reaction solution in the presence of both NADH (500 μm) and cytochromec 3+ (20 μm) and in the absence and presence of SOD (625 nm, i.e. 100 units/ml). NADH was added in surplus amounts to prevent reaction of (residual) peroxynitrite with SOD and cytochrome c 2+formed. The resulting mixture was stored for 2 min at 37 °C. Cytochrome c 2+ formation was determined by reading its absorbance at 550 nm (Δε550 = 21,000m−1 cm−1) (33.Massey V. Biochim. Biophys. Acta. 1959; 34: 255-256Crossref PubMed Scopus (529) Google Scholar). The difference in cytochrome c reduction in the presence and absence of SOD was used to calculate the amount of trapped O⨪2. The experiments with SIN-1 were carried out as end point determinations after 3–4 h of incubation at 37 °C. After that time, SIN-1 (25–150 μm) was completely degraded, as checked by capillary zone electrophoresis (data not shown). In the absence of HCO3−/CO2 about 80 μm residual NADH was found after incubation of 150 μm SIN-1 with 200 μm NADH (Fig.1 A). Under this condition, ONOOH is apparently the decisive oxidant (7.Kirsch M. de Groot H. J. Biol. Chem. 1999; 274: 24664-24670Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). A 6.2-fold surplus of GSH had to be used to achieve a half-maximal NADH protection (IC50: 1240 ± 20 μm GSH), in good agreement with the fact that ONOOH has been estimated to react ∼7.4 times faster with NADH than with GSH (7.Kirsch M. de Groot H. J. Biol. Chem. 1999; 274: 24664-24670Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 13.Koppenol W.H. Moreno J.J. Pryor W.A. Ischiropoulos H. Beckman J.S. Chem. Res. Toxicol. 1992; 5: 834-842Crossref PubMed Scopus (1279) Google Scholar). Ascorbate was 16.5-fold more effective to prevent NADH oxidation (IC50: 75 ± 2 μm) as compared with GSH. As ONOOH reacts ∼6-fold faster with GSH than with ascorbate (11.Bartlett D. Church D. Bounds P.L. Koppenol W.H. Free Radical Biol. Med. 1995; 18: 85-92Crossref PubMed Scopus (165) Google Scholar, 12.Squadrito G.L. Xia J. Pryor W.A. Arch. Biochem. Biophys. 1995; 322: 53-59Crossref PubMed Scopus (121) Google Scholar, 13.Koppenol W.H. Moreno J.J. Pryor W.A. Ischiropoulos H. Beckman J.S. Chem. Res. Toxicol. 1992; 5: 834-842Crossref PubMed Scopus (1279) Google Scholar), the protection on NADH oxidation exerted by ascorbate in the absence of HCO3−/CO2 cannot be explained by direct reaction with ONOOH. In the presence of HCO3−/CO2, about 60 μm residual NADH was detected after a 4-h incubation with SIN-1 (Fig. 1 B). GSH now had largely lost its capability to protect NADH; a high concentration of ∼15 mm had to be applied to achieve a half-maximal protection. In contrast, the strong protective effect of ascorbate was even further increased in the presence of HCO3−/CO2 (IC50: 61 μm). Similar results were obtained by replacing NADH with NADPH (data not shown). The fact that ascorbate was 249-fold more effective than GSH to prevent NADH oxidation suggests that the CO⨪3 radical should be the main oxidant, because it is known that CO⨪3 reacts 260 times faster with ascorbate than with GSH (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). To provide further evidence for this assumption, a comparison between the IC50/[NADH] ratios and the ratios of the corresponding rate constants, namelyk(NADH+CO⨪3)/k(scavenger+CO⨪3), was made (Table I). The IC50/[NADH] ratios correlated reasonably well with the ratios of the corresponding rate constants, supporting the view that NADH was most likely attacked by the CO⨪3 radical.Table IEffect of scavengers on SIN-1-dependent oxidation of NADHScavengerRate constant of the scavenger with CO⨪3IC50IC50/[NADH]k(CO⨪3+NADH) a,bk(CO⨪3+scavenger)k/m −1 s −1 × 10 −6μmAscorbate1400 (1)61 ± 30.30.5Cysteine46 (1)3000 ± 6015.015.2GSH5.3 (1)15,200 ± 20076.0132.1DTT440cDetermined at pH 11. (1)1050 ± 1105.31.7SIN-1 (150 μM), NADH (200 μm), and various scavengers (0–25 mm) were incubated for 4 h in 50 mmpotassium phosphate buffer (pH 7.5, 0.1 mm DTPA, 37 °C). To find the scavenger concentration necessary to achieve a half-maximal NADH protection, concentrations of scavengers were increased stepwise (Δ[scavenger] 250 μm) from 0 to 5 mm(cysteine, dithiothreitol (DTT; ascorbate (GSH) concentration was increased in 25 μm (2.5 mm) steps to reach a final concentration of 250 μm (25 mm)). NADH was quantified by reading its fluorescence with excitation at 339 nm and emission at 460 nm. Data are means ± S.D. of three experiments performed in duplicate.a k(CO⨪3 + NADH) = 7 × 108m−1 (7.Kirsch M. de Groot H. J. Biol. Chem. 1999; 274: 24664-24670Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), estimated value.b At the IC50 level both reactions proceed with identical velocity: IC50 × [oxidant] × k(scavenger + oxidant) = [target] × [oxidant] ×k (target + oxidant); therefore, IC50 × [CO⨪3] × k (ascorbate + CO⨪3) = [NADH] × [CO⨪3] × k (NADH + CO⨪3), and IC50/[NADH] = k (NADH + CO⨪3)/k (ascorbate + CO⨪3).c Determined at pH 11. Open table in a new tab SIN-1 (150 μM), NADH (200 μm), and various scavengers (0–25 mm) were incubated for 4 h in 50 mmpotassium phosphate buffer (pH 7.5, 0.1 mm DTPA, 37 °C). To find the scavenger concentration necessary to achieve a half-maximal NADH protection, concentrations of scavengers were increased stepwise (Δ[scavenger] 250 μm) from 0 to 5 mm(cysteine, dithiothreitol (DTT; ascorbate (GSH) concentration was increased in 25 μm (2.5 mm) steps to reach a final concentration of 250 μm (25 mm)). NADH was quantified by reading its fluorescence with excitation at 339 nm and emission at 460 nm. Data are means ± S.D. of three experiments performed in duplicate. a k(CO⨪3 + NADH) = 7 × 108m−1 (7.Kirsch M. de Groot H. J. Biol. Chem. 1999; 274: 24664-24670Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), estimated value. b At the IC50 level both reactions proceed with identical velocity: IC50 × [oxidant] × k(scavenger + oxidant) = [target] × [oxidant] ×k (target + oxidant); therefore, IC50 × [CO⨪3] × k (ascorbate + CO⨪3) = [NADH] × [CO⨪3] × k (NADH + CO⨪3), and IC50/[NADH] = k (NADH + CO⨪3)/k (ascorbate + CO⨪3). In the presence of CO2, two radicals, namely CO⨪3and NO2·, are produced from peroxynitrite. Both radicals are expected to rapidly oxidize NADH (7.Kirsch M. de Groot H. J. Biol. Chem. 1999; 274: 24664-24670Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Thus, the question rose as to why NO2· was apparently not involved in the SIN-1-induced oxidation of NADH. Since reaction of peroxynitrite with NADH yields O⨪2 in the absence of CO2 (7.Kirsch M. de Groot H. J. Biol. Chem. 1999; 274: 24664-24670Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), and because formation of peroxynitrate (O2NOO−,t 1/2 = 0.7 s, pK a = 5.9; Ref. 34.Logager T. Sehested K. J. Phys. Chem. 1993; 97: 10047-10052Crossref Scopus (135) Google Scholar) has been observed from reaction of peroxynitrite with various substrates in the presence of CO2 (19.Goldstein S. Czapski G. J. Am. Chem. Soc. 1998; 120: 3458-3463Crossref Scopus (167) Google Scholar, 20.Hodges G.R. Ingold K.U. J. Am. Chem. Soc. 1999; 121: 10695-10701Crossref Scopus (123) Google Scholar), we speculated that NO2· has been transformed into O2NOO− in these experiments. In fact, we found that O⨪2 was formed from the reaction of authentic peroxynitrite (100 μm) with NADH (500 μm) in the presence of CO2 as well (3.8 ± 0.3 μm O⨪2, three experiments performed in duplicate). As there is no specific detector molecule for peroxynitrate presently available, we tried to provide further evidence for the formation of this compound by analyzing parameters that reflect the underlying reaction mechanism. As has been shown by Halliwell (35.Halliwell B. Planta. 1978; 140: 81-88Crossref PubMed Scopus (267) Google Scholar), O2 uptake correlates strictly with NADH consumption when NADH is non-enzymatically oxidized by one-electron oxidants (Scheme I). 2 NADH+2 oneelectron oxidant →products+2 NAD· 2 NAD·+2 O2 →2 NAD++2 O⨪2 2 O⨪2+2 H+ →H2O2+O2 SCHEME IHowever, such a correlation should not exist in the present system when peroxynitrate is nearly stoichiometrically formed as an intermediate (Scheme II). ONOO−+CO2 →CO⨪3+NO2⋅ CO⨪3+NADH →HCO3−+NAD⋅ NAD⋅+O2 →NAD++O⨪2 O⨪2+NO2⋅ →O2NOO− O2NOO− →NO2−+O2 SCHEME IIIn the absence of CO2, 250 μm authentic peroxynitrite generated ∼44 μm O2, in agreement with data from previous reports (28.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, 36.Pfeiffer S. Gorren A.C.F. Schmidt K. Werner E.R. Hansert B. Bohle D.S. Mayer B. J. Biol. Chem. 1997; 272: 3465-3470Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 37.Lymar S.V. Hurst J.K. Chem. Res. Toxicol. 1998; 11: 714-715Crossref PubMed Scopus (60) Google Scholar) (TableII). On the other hand, addition of 200 μm NADH resulted in the consumption of ∼40 μm O2. Thus, O2NOO− is unlikely to be formed in stoichiometric amounts under these conditions. In the presence of CO2, only ∼16 μm O2was formed from the decay of 1 mm peroxynitrite. In sharp contrast to the CO2-free situation, the yield of O2 found in the presence of NADH (200 μm) was only slightly altered although the same amount of NAD+ was formed. This observation strongly supports the intermediary formation of peroxynitrate.Table IIEffects of authentic peroxynitrite on O2 formation and NADH consumptionConditionsPresence of CO2O2ΔO2aReferred to "No additives"; separately calculated for the absence and presence of CO2.Residual NADHμmNo additivesNo225.0 ± 4Peroxynitrite (250 μm)No269.3 ± 3+44.3Peroxynitrite (250 μm) plus NADH (200 μm)No185.3 ± 2−39.7119.2 ± 3No additivesYes210.0 ± 3Peroxynitrite (1000 μm)Yes225.5 ± 3+15.5Peroxynitrite (1000 μm) plus NADH (200 μm)Yes221.2 ± 2+11.2119.4 ± 3Authentic peroxynitrite (1 mm) was added under normoxic conditions in the absence and presence of HCO3−/CO2 (25 mm/5%) or NADH (200 μm) to 50 mm potassium phosphate buffer (37 °C, pH 7.5). O2 was measured polarographically with a Clark-type electrode. After the O2 measurement residual NADH was quantified by reading its fluorescence with excitation at 339 nm and emission at 460 nm. Each value represents the mean ± S.D. of six experiments.a Referred to "No additives"; separately calculated for the absence and presence of CO2. Open table in a new tab Authentic peroxynitrite (1 mm) was added under normoxic conditions in the absence and presence of HCO3−/CO2 (25 mm/5%) or NADH (200 μm) to 50 mm potassium phosphate buffer (37 °C, pH 7.5). O2 was measured polarographically with a Clark-type electrode. After the O2 measurement residual NADH was quantified by reading its fluorescence with excitation at 339 nm and emission at 460 nm. Each value represents the mean ± S.D. of six experiments. The formation of O2NOO− from reaction of NO⋅2 with O⨪2 a prioricannot be regarded as an inactivation process for NO⋅2, because peroxynitrate has been reported to oxidize NADH even at pH 7.1 (38.Goldstein S. Czapski G. Inorg. Chem. 1997; 36: 4156-4162Crossref Scopus (43) Google Scholar). Since the efficiency of this reaction is unknown, we studied the authentic peroxynitrate-derived NADH oxidation and the antioxidative effect of ascorbate on it (Fig. 2). Irrespective of the presence of CO2, about 45 μm NADH was oxidized from reaction of peroxynitrate (1.57 mm) with NADH (200 μm), i.e. only ∼3% of the added O2NOO− was able to oxidize NADH. A virtual identical yield was observed with lower peroxynitrate concentrations (0.25–1 mm, data not shown). Ascorbate protected NADH against the attack of peroxynitrate in a roughly linear manner, and the inhibitory effect was not influenced by the addition of HCO3−/CO2. However, relatively high concentrations of ascorbate were necessary to inhibit peroxynitrate-induced oxidation of NADH (IC50: 1150 ± 60 μm). A comparison of the IC50/[NADH] ratio (= 5.8) with the ratio of the corresponding rate constants (k(NO⋅2 + NADH)/k(NO⋅2 + ascorbate) = 10 × 107m−1s−1 (7.Kirsch M. de Groot H. J. Biol. Chem. 1999; 274: 24664-24670Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar)/1.8 × 107m−1 s−1 (Ref. 39.Forni, L. G., Mora-Adrellano, V. O., Packer, J. E., and Willson, R. L. (1986) J. Chem. Soc. Perkin Trans. II, 1–6Google Scholar) =5.6) strongly indicates that ascorbate protected NADH by terminating NO⋅2. The result that peroxynitrate mediates its oxidative properties via NO⋅2 is in line with data of Goldstein et al. (40.Goldstein S. Czapski G. Lind J. Merenyi G. Inorg. Chem. 1998; 37: 3943-3947Crossref PubMed Scopus (50) Google Scholar). Hence, it can be concluded that after the reaction of NO⋅2 with O⨪2about 97% of the amount of NO⋅2 was inactivated regarding its properties to oxidize NADH. As compared with ascorbate, GSH (0–30 mm) exhibited only a very weak protective effect against authentic peroxynitrate. From the present data, it can be calculated that a 50% protection level should be attainable at ∼88 mm GSH (data not shown). Thus, ascorbate is ∼80 times more effective than GSH on inhibiting peroxynitrate-mediated oxidation of NADH. Formation of peroxynitrate via reaction of ONOOH with DHR is highly unlikely, because the intermediately formed DHR radical dismutates quantitatively into DHR and RH (24.Kooy N.W. Royall J.A. Ischiropoulos H. Beckman J.S. Free Radical Biol. Med. 1994; 16: 149-156Crossref PubMed Scopus (676) Google Scholar) and is therefore not expected to generate O⨪2. In the absence of CO2 peroxynitrite generated in situ from SIN-1 (25 μm) yielded 14.3 ± 0.2 μm RH from DHR (50 μm) after 3 h of incubati
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