VIEWS: 2 PAGES: 7 POSTED ON: 1/23/2013
Biochem. J. (1993) 290, 51-57 (Printed in Great Britain) 51 Peroxynitrite-induced luminol chemiluminescence Rafael RADI,*ttThomas P. COSGROVE,* Joseph S. BECKMAN* and Bruce A. FREEMAN*t§ Departments of * Anesthesiology, t Biochemistry and § Pediatrics, University of Alabama at Birmingham, Birmingham, AL 35233-6810, U.S.A. Vascular endothelial cells, smooth muscle cells, macrophages, plex with luminol, yielding luminol radical and 02'-. Luminol neutrophils, Kupffer cells and other diverse cell types generate radical reacts with 02'- to form the unstable luminol superoxide (02'-) and nitric oxide ('NO), which can react to endoperoxide, which follows the light-emitting pathway. form the potent oxidant peroxynitrite anion (ONOO-). Neither 'NO nor O2;- alone were capable of directly inducing Peroxynitrite reacted with luminol to yield chemiluminescence significant luminol chemiluminescence in our assay systems. which was greatly enhanced by bicarbonate. The quantum These results suggest that ONOO- can be a critical unrecog- chemiluminescence yield of the ONOO- reaction with luminol nized mediator of cell-derived luminol chemiluminescence re- in bicarbonate was approx. 10-3. Chemiluminescence was ported in previous studies. In addition, it is shown that bicarb- superoxide dismutase-inhibitable, indicating that 02- was a key onate can participate in secondary oxidation reactions after intermediate for chemiexcitation. 02'- appears to be formed reacting with ONOO-. secondarily to the reaction of a bicarbonate-peroxynitrite com- INTRODUCTION protein thiols , deoxyribose  and membrane phospholipids Vascular endothelial cells, smooth muscle cells, macrophages, . In addition to its role in oxidation reactions, ONOO- can neutrophils, Kupffer cells and a growing list of other cell types also nitrate free or protein-associated tyrosine . generate superoxide (02'-)  and nitric oxide ('NO) . Different Luminol chemiluminescence has been widely used to detect the mediators stimulate the simultaneous production of 'NO and production of reactive oxygen species (i.e. 02-- H202 and 'OH) 02--' including interferon y , calcium ionophores [2,4], from enzyme, cell and organ systems [17-19] and has been useful lipopolysaccharide [5,6] and phorbol esters [4,7]. Thus con- for examining the kinetics and reaction mechanisms of oxygen- comitant 'NO and 02'- generation may be enhanced in a variety radical processes [17,20]. In order to yield light, luminol has of pathophysiological situations such as ischaemia-reperfusion, to undergo a two-electron oxidation and form an unstable acute inflammatory processes, atherosclerosis, bacterial infec- endoperoxide. This luminol endoperoxide decomposes to an tions and sepsis. excited state, 3-aminophthalic acid, which relaxes to the ground Both 'NO and 02- are free-radical species that rapidly react state by emitting photons [18,21]. In most cases of luminol with each other in aqueous solution at pH 7.4, yielding per- chemiexcitation in biological systems, 02 -is a key intermediate oxynitrite anion (ONOO-) (k2 = 3.7 x 107 M-1 s-1) . Several [22,23], but alternative pathways ofchemiexcitation not requiring observations support the in vivo formation of ONOO-. First, the 02- have been described [17,24]. half-life of endothelial-derived 'NO is doubled in the presence of Luminol chemiluminescence is observed during the respiratory superoxide dismutase (SOD), suggesting that 02- is involved in burst of macrophages and neutrophils, which has been commonly its degradation . Second, inhibitors of 'NO synthase increase attributed to the production of 02- and H202 . Nevertheless, detectable 02-- release from macrophages . Third, decompo- there are instances where more potent oxidation reactions are sition of the syndominine, SIN-1, to products including 'NO and needed to explain observed chemiluminescence yields . For 02'- yields a species with 'OH-like reactivity, inferring the example, myeloperoxidase liberated from macrophage granules intermediate formation of ONOO- [11,12]. Finally, ONOO- has greatly enhances the reactivity of H202 towards luminol by been directly detected as a product of phorbol myristate acetate- forming an oxohaem oxidant species . Also, 'NO-derived activated rat lung alveolar macrophages, which produce intermediates contribute to luminol chemiluminescence in 100 pmol of ONOO-/min per 106 cells . phorbol ester-activated Kupffer cells and may react via ONOO- Peroxynitrite (PKa = 6.8 ) is an unstable species at . physiological pH (t1 < 1 s), protonating to peroxynitrous acid Herein we report that ONOO-, a newly described biological (ONOOH) which spontaneously decomposes to 'NO2 and 'OH oxidant of emerging significance [26-28] induced SOD- in 20-30 % yield: inhibitable luminol chemiluminescence. Furthermore, it is shown that bicarbonate can participate in secondary oxidative reactions after reacting with ONOO-. *NO+02'- ONOO- ONOOH -*'NO2+*OH (1) EXPERIMENTAL The remaining ONOOH will directly isomerize to nitrate (NO3-, [1 1]). Materials Peroxynitrite may be an important mediator of free-radical- Cysteine, cystine, uric acid, mannitol, dimethyl sulphoxide dependent toxicity [1 1-14] because of its strong oxidizing proper- (DMSO), horseradish peroxidase type VI and tetranitromethane ties towards different biomolecules, including protein and non- were obtained from Sigma. Potassium superoxide, cis-dicyclo- Abbreviations used: SOD, superoxide dismutase; DMSO, dimethyl sulphoxide. t Permanent address: Department of Biochemistry, Faculty of Medicine, University of the Republic, Montevideo, Uruguay CP11800. 1 To whom correspondence should be addressed. 52 R. Radi and others hexano- 1 8-crown-6 and tetrafluoroborate nitronium were from bicarbonate buffer at pH 10.5 (Figure 1). The proportion of Aldrich. H202 and luminol (5-amino-2,3-dihydro-1 ,4-phthal- chemiluminescence yield in phosphate buffer, with respect to azinedione) were obtained from Fluka and Cu/Zn SOD was bicarbonate buffer, was increased by lowering pH. At pH 7.5, generously provided by Grunenthal GmBH. the luminol chemiluminescence yield of ONOO- reaction in Peroxynitrite was synthesized in a quenched-flow reactor as phosphate buffer became 15 % of that in bicarbonate buffer. The previously described [11,14,15]. Fresh solutions of potassium addition of H202 (10, 50 and 200,#M) to reaction mixtures superoxide (0.1 M) were prepared daily in DMSO and 18-crown- containing 400 ,tM luminol and 100 ,tM ONOO- in either 6 as described elsewhere . A 1.7 mM 'NO solution was bicarbonate or phosphate buffers at pH 10.5 did not have a prepared by extensive bubbling of 'NO gas in anaerobic deionized significant effect on chemiluminescence yield or the time course water contained in a gas sampling tube. of light emission (results not shown). The quantum yield of ONOO--induced chemiluminescence (quanta/molecules of ONOO- consumed) was determined at Inactivation of Cu/Zn SOD SOD inactivation by H202  was performed by incubating 2.0 ,ug/ml SOD (2100 units/mg) with 1.0 mM H202 in 50 mM 12 120 sodium pyrophosphate, pH 10.0, at 25 °C for 30 min, with residual H202 removed by Sephadex G-25 chromatography. 0) SOD activity was determined by measuring the inhibition of 10 cytochrome c3+ reduction by xanthine plus xanthine oxidase . 0 0 I E SOD was approximately 86 % inactivated by this method. Z 8- ._ 0 C U) (A tn U) r- Chemiluminescence measurement C 6- ._ s ._ Chemiluminescence studies were performed in a SLM DMX- 1000 fluorimeter by closing the excitation window and maximally X 4- U) x 0) opening the emission slit width to 16 nm. Reactions were initiated 0 CD by injection of ONOO- directly to cuvettes with continuous stirring through a syringe adapted to the instrument. Light 2 emission was simultaneously recorded by computer interface. Reported chemiluminescence is the integrated light emission from a 5 s time interval, unless otherwise stated. When studying 0- 300 400 50( the effect of different oxygen tensions on chemiluminescence Wavelength (nm) yield, reactions were performed with continuous oxygen, air or Figure 1 Chemiluminescence spectra for luminoiONOO- reactions nitrogen bubbling at 1-2 ml/min in a 3 ml cuvette volume. Peroxynitrite (100 ,uM) was added to reaction mixtures containing 400 ,uM luminol in 50 mM bicarbonate (O or phosphate (0), pH 10.5, at 25 °C. Maximal light intensities obtained at different emission wavelengths are reported. Results are means+S.E.M. Spectrophotometric analysis Absorbance measurements and spectra were performed with a Gilford Response spectrophotometer. Luminol oxidation 25 was measured by absorbance decrease at 350 nm (e = 7200 M-1' cm-') and 300 nm (e = 6800 M-1 * cm-'). For ONOO- concentrations utilized in this paper, there was no significant 20- interference of the ONOO- decomposition products N03- and NO2- on luminol oxidation determination, because of the extremely low absorption coefficients of these species in O15- the 300-350 nm region compared with luminol (NO2-, £350 = 23 M-lcm-' and 6300 = 10 M-l cm-l; NO3-, £350 = 0 and 6300 = 9 M-l cm-'). Data reported herein are the average of three independent x 10 determinations performed in a one-day experiment. Each ex- periment was repeated on a minimum of 4 different days and similar results were obtained with a maximum 10 % variation. 5 RESULTS 0 40 80 120 160 Luminol chemiluminescence spectra and quantum yield Time (s) Addition of peroxynitrite stimulated luminol chemiluminescence Figure 2 Luminol chemiluminescence as a function of ONOO- concentrafton in both bicarbonate and phosphate buffer. Emission spectra were Reactions were started by addition of 10 ,uM (A), 25 /uM (A), 50 ,uM (0) or 100 ,uM (0) similar for both buffers, with Aem maximum = 425 nm (Figure 1), ONOO- to reaction mixtures containing 400 ,sM luminol in 50 mM bicarbonate, pH 10.5, at which was used as the Aem for further studies. Maximal light- 25 °C. kd values were 0.020 s-1, 0.024 s-1, 0.022 s-1 and 0.021 s-1 for 10, 25, 50 and emission intensity in phosphate buffer was 0.3-0.5 % of that in 100 ,tM ONOO- respectively. Results are means+ S.E.M. Peroxynitrite and chemiluminescence 53 500 and was quantified as the first-order rate constant kd [17,20]. Chemiluminescence intensity was proportional to ONOO- con- centration, with the calculated kd independent of ONOO-. Light- emission decay in bicarbonate buffer was zero order with respect 400- to luminol in concentrations up to 1 mM. No further in- crease in 100 ,uM ONOO--induced chemiluminescence yield was obtained with concentrations of luminol greater than 400 ,uM. 300- Alternatively, chemiluminescence was strongly influenced by pH, C with kd increasing at lower pH (Figure 3). Addition of a second portion of ONOO- to reaction mixtures after chemiluminescence .2' 200- ceased resulted in similar chemiluminescence yields. Thus quenching by ONOO- and luminol reaction byproducts did not artifactually influence the ONOO--induced luminol chemilumi- 100 nescence. Chemiluminescence decay rates and peak intensities depended on bicarbonate concentration, with kd directly proportional to bicarbonate (Figure 4a). Total chemiluminescence yields (area 0 10 20 30 below the curve) were similar for all bicarbonate concentrations Time (s) at a particular pH (Figure 4b). In contrast, the potassium Figure 3 Luminol chemiluminescence as a function of pH phosphate concentration of phosphate-buffered systems did not influence the rate of luminol chemiluminescence decay. No Reactions were started by addition of 100 uM ONOO- to a reaction mixture containing 400 IM chemiluminescence was observed after addition of tetranitro- luminol and 50 mM bicarbonate, pH 10.0 (-), 9.5 (A), 8.5 (0) and 7.5 (+) at 25 °C. kd methane (200 ,uM), nitronium tetrafluoroborate (200 ,uM), values were 0.17 s-, 0.45 s-1, 1.11 s-1 and 1.20 s-1 respectively. Chemiluminescence was nitrate (100 ,uM), nitrite (100 1sM), nitric oxide (up to 200 ,uM) or integrated every 0.2 s. potassium superoxide (up to 2.5 mM) to 400 ,M luminol in 50 mM bicarbonate, pH 10.5, at 25 'C. Luminol chemilumi- nescence from O2;- only occurs when 02-- reacts with luminol pH 10.5 with luminol in excess of ONOO- (400,uM luminol, radical, the formation of which requires prior reaction with 100 ,M ONOO-). Quantum yields were approx. 10' in bi- stronger oxidants. carbonate buffer and < 10-s in phosphate buffer. A secondary Net luminol oxidation was measured spectrophotometrically standard of persulphate/H202 was used as our reference system at 350 and 305 nm and was ONOO- concentration-dependent . (Table 1), with about 5-8 and 3-6 mol of ONOO- consumed per mol of oxidized luminol at pH 7.5 and 10.5 respectively. Luminol oxidation yields were greater at pH 10.5 than at pH 7.5, were Time course of luminol chemlluminescence greater at lower ONOO- concentrations and were similar for Luminol chemiluminescence induced by ONOO- reached peak bicarbonate and phosphate buffers. There was no effect of intensity by 5 s at pH 10.5 and was followed by an exponential bicarbonate concentration (50-400 mM) on oxidation yield decay (Figure 2). The emission decay followed first-order kinetics (results not shown). 3 21 cn ao 0, 4) um 0 x 10 1 0 500 80 [HCO3-1 (mM) Time (s) Figure 4 kd as a function of bicarbonate concentration (a) Assay conditions were: 100 ,uM ONOO-, 400 ,uM luminol and 50-400 mM bicarbonate at pH 10.5 (A) or 9.5 (0) at 25 °C. kd was determined as the tirst-order constant of exponential decay for each buffer concentration. (b) Chemiluminescence records were obtained in 50 mM (0) and 300 mM (A\) bicarbonate, pH 10.5. Results are means+S.E.M. 54 R. Radi and others Table 1 Luminol oxidation as a function of ONOO- concentration Table 2 Effect of antioxidants on luminol oxidation Peroxynitrite (1 00-400 ,uM) was added to 400 ,uM luminol in 50 mM bicarbonate, pH 10.5 Assay conditions were as described in Figure 6. Incubations were performed for 10 min at or 7.5, at 25 OC and incubated for 10 min. Data represent means+ S.D. (n = 4). 25 °C. Data represent means+ S.D. (n = 3). Superscripts denote signiticance of difference from control (a) and ONOO- (b)-treated (no addition) condition after ANOVA and analysis by Luminol (uM) Duncan's multiple-range test (P < 0.05). [ONOO-] (,uM) pH 10.5 pH 7.5 Condition Luminol (IuM) 0 400 + 5 400 + 5 Control 396 + 2 100 366 + 8 379 + 6 + ONOO- 250 340 + 8 364 + 5 No addition 371 + 2" 500 307 + 7 333 +12 +100 mM mannnitol 372 + 3a 750 274 + 5 305 + 20 +100 mM DMSO 372 + 3a 1000 243 + 4 274 + 20 +3 mM cystine 371 + 4 +3 mM cysteine 395 +3 +1 mM urate 397 + 4 + 2.3 units/ml SOD 378 + 4a + 23.0 units/ml SOD 386 + 3a 20 15 10 20 15 - x5 15- Co 0 25 50 75 100 o10 Time (s) 0)~~~ x C10 1 x o 0 5 5 0 0 0 A A~U AU A 0 25 50 75 100 0 25 50 75 100 Time (s) Time (s) Figure 5 Effect of Cu/Zn SOD and oxygen on luminol chemiluminescence Figure 6 Effect of antioxidants on luminol chemiluminescence Assay conditions were: 100,uM ONOO-, 400 uM luminol with no SOD (O), 0.23 units/ml Assay conditions were: 100 ,tM ONOO-, 400 ,#M luminol with no additions (M), 100 mM native SOD, (A, 3.75 nM), 2.3 units/ml native SOD (+, 37.5 nM), 0.27 units/ml inactivated DMSO (0), 100 mM mannitol (C]), 3 mM cysteine (0), 1 mM urate (A) and 0.375 PuM SOD SOD (0, protein equivalent 37.5 nM) and 2.7 units/ml inactivated SOD (El, protein equivalent in 50 mM bicarbonate, pH 10.5, at 25 °C. Where indicated, results are means+ S.E.M. 2.7 units/ml) in 50 mM bicarbonate, pH 10.5, at 25 OC. The inset shows the effect of oxygen on chemiluminescence. Assay conditions were as above with no SOD present. Reactions were saturated with air (@), nitrogen (V) or oxygen (O). duration by about 10-20 %, whereas nitrogen saturation only slightly inhibited chemiluminescence (Figure 5, inset). Similar extents of SOD inhibition of ONOO--induced luminol chemi- Effect of Cu/Zn SOD luminescence occurred in oxygen, air or nitrogen-saturated conditions. SOD caused a dose-dependent inhibition of luminol chemi- luminescence and a small increase in kd (Figure 5). When H202- inactivated SOD replaced native SOD, the inhibition of light Effect of antioxidants emission corresponded to that expected for the residual SOD Uric acid (1 mM) and cysteine (3.0 mM) completely inhibited activity remaining in the inactivated enzyme preparation (Figure both luminol chemiluminescence and luminol oxidation (Figure 5). A significantly greater SOD activity than that used to inhibit 6, Table 2). The OH scavengers mannitol (100 mM) and DMSO chemiluminescence only partially inhibited luminol oxidation (100 mM), and the disulphide cystine (3.0 mM), did not affect (Table 2). With the experimental conditions of Figure 5, cupric luminol chemiluminescence and luminol oxidation (Table 2). sulphate (1 and 20 1tM) inhibited luminol chemiluminescence by 70% and 95 % respectively. The copper chelate Cu/EDTA (1: 1.1, mol/mol; up to 200 ,#M) did not inhibit light emission. Secondary products from ONOO-ireactlon with luminol Reactions conducted in 100 % oxygen-saturated bicarbonate After luminol reacted with ONOO-, a pronounced 425 nm yellow buffer, pH 10.5, increased both chemiluminescence yield and absorbance was found in samples which progressively disap- Peroxynitrite and chemiluminescence 55 there was no direct source of H202 in our reaction mixtures. Exogenous addition of H202 had no significant effect on light yield as well. Formation of a nitrated luminol endoperoxide by ONOO- adduct formation with the diazaquinone, followed by internal rearrangement, also does not account for light emission because nitrophthalate is not chemiluminescent (G. Merenyi and J. Lind, personal communication). The inhibitory effects of SOD and free Cu2+ on chemilumi- A2 nescence imply the participation of 02- in chemiexcitation. This concept is reinforced by the observation that Cu/EDTA, which lacks SOD activity , did not inhibit chemiluminescence. Different mechanisms could operate to generate 02O- during ONOO- reaction with luminol. 02- could have been produced by a luminol radical-dependent univalent reduction of molecular oxygen . This mechanism was probably not the main source of 02@- since saturation with oxygen or nitrogen had a marginal 380 420 effect on chemiluminescence yield (Figure 5, inset). Moreover, Wavelength (nm) SOD inhibited light emission in both aerobic and anaerobic Figure 7 Absorption spectra of luminol after oxidation by ONOO- conditions. The direct formation of 02-- (plus 'NO) from ONOO- did not occur for thermodynamic and kinetic Luminol (400 uM) reacted with 100 ,uM ONOO- in 50 mM bicarbonate, pH 10.5 at 25 OC. considerations. The AGO' for formation of ONOO- from O2.- Spectra were recorded immediately before (----) and 3 min after ( ) ONOO- addition. and 'NO is about -92 kJ/mol (-22 kcal/mol), thus Keq = A decrease in A350 and the appearance of a peak in the 425 nm region was observed. Whereas A350 did not change with time, the 425 nm absorbance was transient and decayed (inset). 5 x 101' M-l at 25 °C . With the k2 for formation of ONOO- -being at least 3.4 x 107 M-1 * s-' , it can be estimated that the reverse re.action (formation of O2 - from ONOO-) will proceed slowly at 10-8 s-1. Thus O2- must be generated after direct peared over 15-25 min at both pH 10.5 and 7.5 in either 50 mM reaction of ONOO- with luminol and by a bicarbonate- bicarbonate or phosphate buffers (Figure 7 and inset). This stimulated mechanism (Figures 1 and 4). When bicarbonate was 425 nm absorbance was pH insensitive. Addition of uric acid absent, luminol could still be oxidized by ONOO- (Table 1), but (1 mM) and cysteine (3 mM) but not DMSO (100 mM) or the presence of bicarbonate led the oxidation process to a light- mannitol (100 mM) to ONOO--luminol reactions inhibited the emitting pathway, presumably by favouring O2- production, appearance of the 425 nm absorbance. Luminol reaction with thus supporting a greater chemiluminescence quantum yield. In tetranitromethane (100 ,uM) or much higher concentrations of the presence of high bicarbonate concentrations, the rate of nitronium tetrafluoroborate (5 mM) resulted in a stable yellow light decay was independent of both ONOO- and luminol product with a broad absorbance at 400-500 nm rather than the concentration and was pseudo-first-order with respect to sharper 425 nm peak after luminol reaction with ONOO-. bicarbonate. Thus, the formation of the key oxidant might Oxidation of luminol with horseradish peroxidase plus H202 did precede the reaction of ONOO- with luminol. A mechanism not generate this 425 nm-absorbing species (results not shown). consistent with these observations can involve a peroxynitrite- bicarbonate intermediate: DISCUSSION ONOO- + HC03- + H+ -÷ ONOOC(O)0- + H20 (4) Peroxynitrite-induced luminol chemiluminescence was greatly ONOOC()0-+ LH- - L- +02--+ NO- + CO2 + H+ (5) enhanced by HCO3- (Figure 1). Spectral analysis of luminol chemiluminescence (Figure 1) indicated that excited amino- L--+ 02 -- light + aminophthalate (6) phthalate was the emitting species . Since formation of With phosphate-buffered systems, a similar but minor reaction aminophthalate depends on decomposition of luminol endo- could be responsible for some light generation, with the principal peroxide, ONOO- reaction with luminol must yield this unstable light emission being due to the reduction of ONOO- to NO2 and intermediate. H20 by luminol. The hallmark of eqns. (4)8(6) is that the Peroxynitrite is an effective one-electron oxidant (E'ONOO/-NO2) = reduction of ONOO- can yield 02n-' with the efficiency of this + 1.4 V ), thus oxidation of luminol by ONOO- is thermo- reaction increasing after ONOO- reaction with bicarbonate. dynamically feasible. The first oxidation by ONOO- or its Other mechanisms might also participate in ONOO--depen- conjugate acid would lead to the formation of luminol radical dent bicarbonate-mediated luminol-oxidation reactions. For (L*-) as follows: example, formation of a bicarbonate radical from ONCO- reaction may occur, with the EL'(HC03/HC03-) being + 1.5 V . ONOO- + LH- -*'NO2 + L- + OH- (2) ONOO-/'NO2 and HCO3*/HCO3- couples have similar one- If NO2 is in close proximity to luminol radical, an immediate electron standard redox potentials. Thus, under conditions second oxidation will take place to yield a diazaquinone (L), with where ONOO- reacts with excess HCO3-, one-electron oxidation the overall process a two-electron oxidation according to: of bicarbonate by ONOO- is thermodynamically favourable ONOO-+ LH- -- NO2-+OH-+L (3) according to: Alternatively, luminol radical can be oxidized to the diazaquinone ONOO-+HCO3-+ H+-HCO3 NO2 + OH- + (7) by direct reaction with a second molecule of ONOO-. If the diazaquinone reacts with H202, the luminol endoperoxide in- Bicarbonate radical is known to oxidize luminol  and other termediate is formed . A diazaquinone-H202 reaction was aromatic and heterocyclic molecules with second-order rate not responsible for ONOO--induced chemiluminescence, since constants ranging from 5 x 105 to 5 x I07 M-1 s-s . 56 R. Radi and others Alternatively, ONOO- may peroxidize bicarbonate to potassium superoxide and 200 ,uM 'NO. This concurs with peroxybicarbonate , another strong oxidizing species. Thus, previous observations of poor reactivity between O2-- and there are several potential mechanisms by which bicarbonate luminol . When 02'- participates in luminol chemilumi- could influence the overall reactivity of ONOO- in both model nescence reactions, it does so by reacting with luminol radical and biological conditions. Interestingly, bicarbonate is expected rather than luminol. Luminol radical can be formed through a to accumulate in tissues undergoing ischaemia-reperfusion, a one-electron oxidation of luminol by 'OH, ferryl ion (FeO2+) or condition for which peroxynitrite is proposed to contribute by other oxidants of similar reactivity [18,20,39]. When 02'- is significantly to net oxidant stress [11,14,15,28]. formed after reaction of ONOO- with HCO3-, it is possible that Smaller yields of ONOO--induced luminol chemiluminescence H202 (formed after dismutation of 02'-) can contribute to were found at lower pH (Figure 3). This could be due to the fact chemiluminescence by reacting with the diazaquinone formed that only the decomposition of luminol monoanion (apparent after reaction with a second molecule of ONOO- (eqns. (2) and pK. = 8.2) leads to the light-emitting route . In addition, (3)]. proton-catalysed decomposition of ONOO- becomes a more Peroxynitrite is, in addition to being an oxidant, a potent efficient competing reaction by decreasing pH . Consistent nitrating species [16,40]. Luminol nitration occurring con- with this concept, peroxynitrite oxidized luminol more efficiently comitantly with oxidation by ONOO- is suggested by the at pH 10.5 than at 7.5 (Table 2) and there was a shorter half-life observed transient 425 nm absorbance. This by-product was not of luminol chemiluminescence at lower pH (Figure 3). Luminol- formed when other oxidants such as H202 attacked luminol. oxidation studies were conducted at alkaline pH in order to The mechanism of luminol nitration by ONOO- is expected to define mechanisms of luminol reaction with ONOO- and limit be different from other nitrating agents such as tetranitro- the proton-catalysed decomposition of ONOO-, so that in- methane and nitronium tetrafluoroborate. Nitration of aromatic formation could be more precisely obtained about the kinetics molecules by ONOO- occurs in concert with the 'OH-like and mechanism of luminol oxidation. There were no differences reaction of ONOO- with double bonds . This mechanism in the mechanisms of luminol oxidation at physiological versus requires the formation of a complex with ONOOH, and attack alkaline pH, thus all results relate to biological events. of the peroxynitrous acid hydroxyl with the aromatic molecule to Cysteine and uric acid inhibited luminol chemiluminescence. form an aromatic radical species which then combines with the Cysteine reacts with ONOO- with an apparent second-order rate remaining NO2 of peroxynitrous acid to yield the nitrated product constant of 30-40 M-1 * s-1 at pH 10.5 . Cysteine can therefore . In agreement with the concept of a concerted nitration- inhibit luminol chemiluminescence by both direct reaction with oxidation reaction, we observed that uric acid and cysteine ONOO- and by reacting with bicarbonate-derived oxidizing inhibited both luminol oxidation and the appearance of the species . Cystine, which does not react at significant rates 425 nm absorbance peak. The final nitrated adduct was probably with ONOO- , did not have any effect on luminol chemi- a derivative of luminol oxidation products, since exposure of luminescence or oxidation. Uric acid inhibits xanthine/xanthine luminol to nitrating agents did not result in the characteristic oxidase and H202/cytochrome c-induced luminol chemilumi- 425 nm peak. The transient nature of the nitrated adduct was not nescence by scavenging OH and iron complexes having 'OH-like related to chemiluminescence, since the 425 nm absorbance was reactivity [20,24]. However, the OH radical scavengers mannitol longer lived than light emission (Figure 7). and DMSO did not affect light emission, ruling out a primary Superoxide is frequently invoked as being responsible for role for 'OH in chemiexcitation. We propose that uric acid biologically produced luminol chemiluminescence due to the reacted directly with ONOO- and/or bicarbonate-derived oxidi- inhibitory effects of SOD.. Still, oxidants stronger than 02' and zing species, for which we have supporting preliminary h.p.l.c.- even H202 may be needed to explain the observed light yields based product analyses. Uric acid and cysteine might also directly . Since macrophages and neutrophils will simultaneously reduce luminol radical to its ground state, preventing chemi- produce 'NO and 02'-  and we have recently observed the excitation . Scavenging of reactive species rather than formation of ONOO- by macrophages , it will be important quenching of excited species (resulting in non-radiative pathways to assess what proportion of luminol chemiluminescence induced of relaxation) was the mechanism of cysteine and urate inhibition by activated macrophages and neutrophils depends on ONOO-. of luminol chemiluminescence, because luminol oxidation was For example, 'NO- and 0 ;--derived products from macrophage- prevented by these compounds (Table 2). like liver Kupffer cells synergistically contribute to luminol Whereas urate and cysteine inhibited both luminol chemiluminescence. This observation strongly supports the role chemiexcitation and oxidation to similar extents, SOD of ONOO- as a source of hepatic oxidant stress . If ONOO- significantly inhibited only chemiluminescence. SOD activity, ten is a critical mediator of cell-derived luminol clhemiluminescence, times greater (23.0 units/ml) than that required to almost the inhibitory effects of SOD would depend on at least three completely inhibit light emission (2.3 units/ml, Figure 5), only different mechanisms: (1) inhibition of ONOO- formation, partially inhibited luminol oxidation (Table 2). SOD effects on preventing the reaction of 02-- with 'NO, (2) inhibition of 02'-- light yields are explained by the scavenging of O2e, whereas the mediated oxidation of luminol radical [eqn. (6)] and (3) catalysis influence on oxidative yields occurring with higher SOD activities of the direct decomposition of ONOO-. Mechanisms (2) and (3) were probably due to a direct reaction between ONOO- and the would complement mechanism (1) because SOD is unable to enzyme. Indeed, Cu/Zn SOD catalyses decomposition of totally inhibit ONOO- production by activated macropLages, ONOO- to a species with a reactivity similar to nitronium ion since a fraction of ONOO- was apparently formed in sites where . Thus SOD can directly inhibit ONOO--mediated luminol- SOD does not have access . oxidation reactions by also scavenging ONOO-. Since nitronium Cell and tissue toxicity from excess production of peroxynitrite ion could then attack luminol, we added nitronium may occur because of its strong oxidizing properties. In addition, tetrafluoroborate to luminol and saw that it did not yield our data suggest that ONOO- will react with bicarbonate, chemiluminescence. forming secondary bicarbonate-derived oxidizing species. Unlike ONOO-, its biological precursors 02- and NO did not Extracellular and intravascular compartments ean have up to stimulate chemiexcitation of luminol. We observed no direct 25 mM bicarbonate, inferring that products of the reaction of oxidation of luminol or light emission with up to 2.5 mM ONOO- with bicarbonate will be an important potential mech- Peroxynitrite and chemiluminescence 57 anism of oxidant tissue injury. The formation of bicarbonate 13 Ischiropoulos, H., Zhu, L. and Beckman, J. S. (1992) Arch. Biochem. Biophys. 298, radicals greatly increases 02--induced lysis of erythrocytes , 446-451 stimulates enzyme inactivation and enhances luminol chemi- 14 Radi, R., Beckman, J. S., Bush, K. and Freeman, B. A. (1991) J. Biol. Chem. 266, 4244-4250 luminescence . We envisage that, after ONOO- production 15 Radi, R., Beckman, J. S., Bush, K. and Freeman, B. A. (1991) Arch. Biochem. in vivo, several competing pathways will contribute to ONOO- Biophys. 288, 481-487 reactions and decomposition. Some of these pathways will include 16 Ischiropoulos, H., Zhu, L., Chen, J., Tsai, H. M., Martin, J. C., Smith, C. D. and (a) proton-catalysed ONOO- decomposition to potent secondary Beckman, J. S. (1992) Arch. Biochem. Biophys. 298, 431-437 oxidants, (b) direct reaction of ONOO- with biomolecules such 17 Radi, R., Rubbo, H. and Prodanov, E. (1989) Biochim. Biophys. Acta 994, 89-93 as methionine  or thiols and (c) reaction of ONOO- with 18 Allen, R. C. (1986) Methods Enzymol. 133, 449-493 19 Archer, S. L., Nelson, D. P. and Weir, K. E. (1989) J. Appl. Physiol. 67, 1903-1911 bicarbonate to form toxic secondary reactive species. 20 Radi, R., Rubbo, H., Thomson, L. and Prodanov, E. (1990) J. Free Rad. Biol. Med. 8, 121-126 This work was supported by NIH grants NS24275, HL48676 and HL40458 (B.A.F.) 21 Merenyi, G., Lind, J. and Eriksen, T. E. (1990) J. Biolum. Chemilum. 5, 53-56 and HL46407 (J.S.B.). J.S.B. is also an Established Investigator of the American 22 Hodgson, E. K. and Fridovich, I. (1973) Photochem. Photobiol. 18, 451-455 Heart Association. We thank Dr. Gabor Merenyi and Dr. Johan Lind from the Royal 23 Miller, E. K. and Fridovich, I. (1986) J. Free Rad. Biol. Med. 2,107-110 Institute of Technology in Stockholm, Sweden, for suggestions regarding the 24 Radi, R., Thomson, L., Rubbo, H. and Prodanov, E. (1991) Arch. Biochem. Biophys. mechanism of luminol chemiexcitation by peroxynitrite. We also thank Dr. Harry 288, 112-117 Ischiropoulos for discussions throughout the course of this work and Yvonne Lambott 25 Merenyi, G., Lind, J. and Eriksen, T. E. (1985) Photochem. Photobiol. 41, 203-208 for manuscript preparation. 26 Mulligan, M. S., Hevel, J. M., Marletta, M. A. and Ward, P. A. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 6338-6342 REFERENCES 27 Mulligan, M. S., Warren, J. S., Smith, C. W., Anderson, D. C., Yeh, C. G., Rudolph, A. R. and Ward, P. A. (1992) J. Immunol. 148, 3086-3092 1 Rosen, G. M. and Freeman, B. A. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 28 Matheis, G., Sherman, M. P., Buckberg, G. D., Haybron, D. M., Young, H. H. and 7268-7273 Ignarro, L. J. (1992) Am. J. Physiol. 262, H616-620 2 Marletta, M. A. (1989) Trends Biochem. Sci. 14, 488-492 29 Valentine, J. S., Miksztal, A. R. and Sawyer, D. (1984) Methods Enzymol. 105, 3 Ding, A. H., Nathan, C. F. and Stuehr, D. J. (1988) J. Immunol. 141, 2407-2412 71-81 4 Matsubara, T. and Ziff, M. (1986) J. Cell. Physiol. 127, 207-210 30 Hodgson, E. K. and Fridovich, I. (1975) Biochemistry 14, 5294-5299 5 Stuehr, D. and Marletta, M. T. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 7738-7742 31 Flohe, L. and Oiting, F. (1984) Methods Enzymol. 105, 93-104 6 Wagner, D. A., Young, V. R. and Tannenbaum, S. R. (1983) Proc. Nati. Acad. Sci. 32 Rauhut, M. M., Semsel, A. M. and Roberts, B. G. (1966) J. Org. Chem. 31, U.S.A. 80, 4518-4521 2431-2436 7 Wang, J. F., Komarov, P., Sies, H. and de Groot, H. (1991) Biochem. J. 279, 33 Thorpe, G. H. G. and Kricka, L. J. (1986) Methods Enzymol. 133, 331-353 311-314 34 Koppenol, W. H., Moreno, J. J., Pryor, W. A., Ischiropoulos, H. and Beckman, J. S. 8 Saran, M., Michel, C. and Bors, W. (1990) Free Radical Res. Commun. 10, 221-226 (1992) Chem. Res. Toxicol. 5, 834-842 9 Gryglewski, R. J., Palmer, R. M. J. and Moncada, S. (1986) Nature (London) 320, 35 Beyer, W. F. Jr. and Fridovich, I. (1988) Anal. Biochem. 173, 160-165 454-456 36 Koppenol, W. and Rush, J. D. (1987) J. Phys. Chem. 91, 4430-4431 10 Albina, J. E., Mills, C. D., Henry, W. L. and Caldwell, M. D. (1989) J. Immunol. 143, 37 Michelson, A. M. and Maral, J. (1983) Biochimie 65, 95-104 3641-3646 38 Chen, S. N. and Hoffman, M. Z. (1973) Radiat. Res. 56, 40-47 11 Beckman, J. S., Beckman, T. W., Chen, J., Marshall, P. A. and Freeman, B. A. (1990) 39 Keith, W. G. and Powell, R. E. (1969) J. Chem. Soc. (A), 90 Proc. Natl. Acad. Sci. U.S.A. 87, 1620-1624 40 Halfpenny, E. and Robinson, P. L. (1952) J. Chem. Soc., 939-946 12 Hogg, N., Darley-Usmar, V. M., Wilson, M. T. and Moncada, S. (1992) Biochem. J. 41 Michelson, A. M. and Durosay, P. (1977) Photochem. Photobiol. 25, 55-63 28, 419-424 42 Moreno, J. J. and Pryor, W. A. (1992) Chem. Res. Toxicol. 5, 425-431 Received 3 August 1992; accepted 27 August 1992
"Peroxynitrite-induced luminol chemiluminescence"