Chem. Res. Toxicol. 1995,8, 859-864
859
Reaction between Peroxynitrite and Hydrogen Peroxide: Formation of Oxygen and Slowing of Peroxynitrite Decomposition Beatriz Alvarez,+ Ana Denicola,t and Rafael Radi*>' Department of Biochemistry, Facultad de Medicina, and Department of Physical Biochemistry, Facultad de Ciencias, Universidad de la Republica, 11800 Montevideo, Uruguay Received February 28, 1995@ Peroxynitrite, the reaction product of nitric oxide and superoxide, is a potent and versatile oxidant that can attack a wide range of targets. I n this work, we studied the oxidation of hydrogen peroxide by peroxynitrite, which led to oxygen evolution. Oxygen yields increased at alkaline pH with a n apparent pKa of 7.05 f 0.04. The maximum yields were 16% and 32% of added peroxynitrite at pH 5.9 and 7.4, respectively, assuming t h a t two molecules of peroxynitrite are needed to produce one of oxygen. Hydroxyl radical scavengers (dimethyl sulfoxide, mannitol, ethanol, formate, and acetate) inhibited oxygen evolution to a similar extent to that predicted from their rate constants with hydroxyl radical. The apparent rate constant of peroxynitrite decomposition was zero-order in hydrogen peroxide at acidic pH. At neutral and alkaline pH, the rate of peroxynitrite disappearance decreased in the presence of millimolar concentrations of hydrogen peroxide by up to 50%. The apparent activation enthalpy and entropy for peroxynitrite decomposition increased by 1.7 kcal mol-' and 4.7 cal mol-l K-l, respectively, in the presence of hydrogen peroxide. We propose that a n activated intermediate of peroxynitrous acid is responsible for hydrogen peroxide oxidation at acidic pH, while a t more alkaline pH the formation of a stabilizing complex between hydrogen peroxide and trunsperoxynitrite anion is involved.
Introduction Nitric oxide ('NO) reacts with superoxide ( 0 2 ' - ) a t a nearly diffusion-controlled rate (k = 6.7 x lo9 M-' s-' 1 ( I ) to produce peroxynitrite anion (ONOO-), which can rapidly protonate (pK, = 6.8) (2) to its conjugate acid, peroxynitrous acid (ONOOH). Peroxynitritel is a strong and relatively long-lived oxidant capable of oxidizing deoxyribose ( 3 ) ,thiols (2), lipids ( 4 ) , methionine (5), and ascorbic acid (6). In addition, it can nitrate tyrosine and other aromatics (7, 8). The biological formation of peroxynitrite has been recently established (9, l o ) , and peroxynitrite is postulated to constitute a key biomolecule in mediating toxic effects of superoxide and nitric oxide (3,2, 11 -14). The reactivity of peroxynitrite is strongly pH-dependent (2, 3, 15). A vibrationally activated intermediate derived from trans-peroxynitrous acid, with a reactivity similar to that of hydroxyl radical ('OH) in 20-30% yield, has been proposed (16)in the pathway leading to peroxynitrite isomerization to nitrate. Nevertheless, the exact nature of the oxidizing intermediate remains controversial (5,17),and experimental data from which the mechanisms of peroxynitrite reactivity can be discerned are still scanty. The reaction between peroxynitrite and excess hydrogen peroxide elicits the evolution of oxygen. In an early paper, Mahoney (18)reported oxygen evolution in acidic * To whom correspondence shbuld be addressed, at the Departamento de Bioquimica, Facultad de Medicina, Universidad de la Republica. Av. Gral. Flores 2125, 11800 Montevideo, Uruguay. Fax: (59821949563. E-mail: RRADIC2BQRAD.EDU.W. Department of Biochemistry, Facultad de Medicina. Department of Physical Biochemistry, Facultad de Ciencias. Abstract published in AdLiance ACS Abstracts, July 15, 1995. Note: The term peroxynitrite is used to refer to both ONOO- and @
ONOOH.
solutions of nitrite and hydrogen peroxide and attributed the oxidation of hydrogen peroxide to free hydroxyl radicals arising from the homolytic decomposition of peroxynitrite. In light of the complex oxidative chemistry of peroxynitrite and the recently proposed models of peroxynitrite reactivity (5,251,we studied the reaction of peroxynitrite with hydrogen peroxide over a broad pH range, focusing on the kinetics of peroxynitrite decomposition, the yields of oxygen evolution, and the role of hydroxyl radical scavengers:
Materials and Methods Chemicals. Peroxynitrite was prepared from sodium nitrite and hydrogen peroxide in a quenched-flow reactor a s previously described (2, 3). Peroxynitrite was treated with manganese dioxide in order to eliminate excess hydrogen peroxide, and its concentration was determined spectrophotometrically a t 302 nm in 1M sodium hydroxide ( E = 1670 M-l cm-') (19). The absence of hydrogen peroxide in peroxynitrite solutions was further confirmed by the lack of oxygen release after catalase addition to decomposed peroxynitrite. The concentration of hydrogen peroxide solutions was measured at 240 nm ( E = 43.6 M-l cm-l) (20). Diethylenetriaminepentaacetic acid (DTPAY (0.1 mM) was added to hydrogen peroxide solutions and potassium phosphate and pyrophosphate buffers to avoid metal interference. Hydrogen peroxide, sodium nitrite, DTPA, mannitol, formate, acetate, and dimethyl sulfoxide (DMSO) were purchased from Sigma Chemical Co. (St. Louis, MO). Desferrioxamine was a kind gift of Ciba-Geigy (Basel, Switzerland). All other reagents were of analytical grade. Oxygen Measurements. Oxygen evolution from peroxynitrite and hydrogen peroxide was determined with a Clark-type
* Abbreviations: DTPA, diethylenetriaminepentaaceticacid; DMSO. dimethyl sulfoxide; ABTS, 2,2'-azinobis(3-ethyl-l,Z-dihydrobenzothiazoline 6-sulfonate).
0893-228x/95/2708-0859$09.00/0 0 1995 American Chemical Society
860 Chem. Res. Toxicol., Vol. 8, No. 6, 1995 oxygen electrode (YSI Model 5300) in a water-jacketed chamber a t a temperature of 25 "C unless otherwise specified. The effect of different scavengers known to react with hydroxyl radical such as mannitol, formate, acetate, ethanol, and DMSO on peroxynitrite-mediated oxygen evolution was studied. Desferrioxamine was used as a scavenger of peroxynitrous acid (21). Control experiments were carried out to rule out changes in oxygen concentration due to peroxynitrite reactions with the scavengers. Kinetics. The decomposition of peroxynitrite in the presence and absence of hydrogen peroxide was followed at 302 nm in a stopped-flow spectrophotometer (Applied Photophysics SF.17MV) with a mixing time of less than 2 ms. The rate of peroxynitrite disappearance in the presence of hydrogen peroxide was studied under pseudo-first-order conditions using a 2 10-fold excess of hydrogen peroxide unless otherwise specified. At 302 nm, no interference from hydrogen peroxide nor DTPA absorbance was detected. Apparent first-order rate constants for peroxynitrite decay were determined by nonlinear least-squares fitting of stopped-flow data, and the values reported are the average of a t least 7 separate determinations. The temperature was maintained to within 0.1 "C, and the pH was measured a t the outlet to detect changes caused by the addition of alkaline peroxynitrite. Data Analysis. To estimate the maximum yields of hydrogen peroxide oxidation by peroxynitrite, a Scatchard-like plot was used ( 3 ) . If the apparent rate constant of the reaction between peroxynitrite or a n oxidizing intermediate derived from it (OX) and hydrogen peroxide resulting in oxygen evolution is h, and the pooled rate constant of OX undergoing all other reactions is h,, then the molar concentration of oxygen formed in a n assay for a given hydrogen peroxide concentration may be written as:
This equation can be linearized to give the equivalent form of a Scatchard plot in which
In order to express the results independently of peroxynitrite concentration, both terms of eq 2 can be divided by the amount of peroxynitrite used:
Thus, in a plot of ([O~]/[ON00-1)/[H~0~1 versus [0~1/[ON00-] for a range of hydrogen peroxide concentrations, the x axis intercept represents the concentration of oxidant per added peroxynitrite t h a t can be trapped a t infinite hydrogen peroxide concentrations. The inhibition of oxygen production by different scavengers was analyzed according to Winterbourn (22). If the oxidizing species derived from peroxynitrite that reacts with hydrogen peroxide were hydroxyl radical or a species with similar reactivity, then simple competitive kinetics predict
where F, is the fraction of inhibition from scavenger i at concentration [SJ, h, is the rate constant for the reaction of hydroxyl radical with scavenger i, and h, is the rate constant for the reaction of hydroxyl radical with hydrogen peroxide, which has been estimated a t 2.7 x lo7 (23). Plots of F,[H20& (1 - FJS,] against the known rate constants of hydroxyl radical with the different scavengers as taken from the literature (23) will yield a linear relationship of slope llh, as long as the reactive species derived from peroxynitrite were hydroxyl radical or a species with hydroxyl radical-like reactivity (3,17).
Results Oxygen Evolution. The addition of peroxynitrite to hydrogen peroxide-containing buffer solutions resulted
Alvarez et al.
15~1
I" // 100
h
5, Y
T
/
I
50
0 0.0
0.4
0.8
1.2
[ONOO.](mM)
Figure 1. Yield of oxygen from hydrogen peroxide as a function of added peroxynitrite. Reactions were started by the addition of peroxynitrite to 40 mM hydrogen peroxide in 50 mM potassium phosphate a t 37 "C, final pH 6.8. Data are the mean Z! SD.
in significant oxygen evolution. The amount of oxygen formed under excess hydrogen peroxide increased linearly as a function of the initial peroxynitrite concentration (Figure 1). At pH 7.36 f 0.03, oxygen evolution was complete in less than 20 s. This period corresponds to the time needed for > 99% decomposition of peroxynitrite, since the observed rate constant for peroxynitrite disappearance in the presence of 100 mM hydrogen peroxide (see, for example, Figure 5), a t this pH is 0.21 corresponding to a half-life of 3.3 s. Oxygen formation from the spontaneous decomposition of hydrogen peroxide alone was negligible, and that from peroxynitrite alone added to the buffer was representatively less than 4% relative to the total amount of peroxynitrite added. Oxygen release from peroxynitrite alone, presumably due to nitrosodioxyl radical (ONOO) formation and decomposition, has been reported before ( 4 ) and increased a t a n alkaline pH (Figure 2). The oxygen yield from peroxynitrite reacting with hydrogen peroxide increased hyperbolically with the concentration of hydrogen peroxide added (Figure 2). Based upon the estimates from Scatchard-like plots for each of the different pHs tested, the maximum yields of oxygen release relative to the amount of peroxynitrite added increased with pH from 15.6%a t pH 5.92 f 0.03 to 18.2%a t pH 6.56 f 0.02 and 32.2%a t pH 7.36 f 0.03, assuming a two-to-one reaction stoichiometry so that two molecules of peroxynitrite are needed to produce one of oxygen. Thus, for a given concentration of hydrogen peroxide and peroxynitrite, oxygen yields were greater a t alkaline pH values (Figure 3) and the pH dependence could be described by the Henderson-Hasselbalch equation, with the apparent pKa being 7.05 f: 0.04. At pH 5.88 f 0.05, where most peroxynitrite is in the protonated form, hydroxyl radical scavengers such as mannitol, acetate, formate, ethanol, and DMSO resulted in a concentration-dependent inhibition of oxygen yield. In addition, as seen on the Winterbourn plot in Figure 4, the k$kp values obtained considering that peroxynitrous acid reacted with the targets a t the rates of hydroxyl radical were similar to the theoretical values expected. Desferrioxamine, postulated to react with the
Chem. Res. Toxicol., Vol. 8, No. 6, 1995 861
Peroxynitrite Reaction with Hydrogen Peroxide 0.04
0.25
7
800
1
I I
1
I
/
0 2
0
I
I
4
6
k,
I
I
I
I
8 1 0 1 2 1 4
(b4.ls.l)(
~10~)
Figure 4. Inhibition of oxygen evolution by 'OH scavengers. Figure 2. Yield of oxygen per peroxynitrite added as a function of hydrogen peroxide concentration. Peroxynitrite was added to various concentrations of hydrogen peroxide in 50 mM potassium phosphate, final pH 5.92 f 0.03 (m), 6.56 f 0.02 (A),and 7.36 f 0.03 ( 0 ) .(Inset) Data are transformed to a Scatchardlike plot, eq 3, after substracting the oxygen formed in the absence of hydrogen peroxide.
Peroxynitrite (500 pM) was mixed with potassium phosphate (100 mM), pH 5.88 & 0.05, hydrogen peroxide (100 mM), and the scavenger in concentrations optimized to span the range giving 50%inhibition. The scavengers were acetate (VI, formate (+), ethanol (m), mannitol (+I, DMSO (A),and desferrioxamine ( 0 ) .Points represent the mean & SD of different scavenger concentrations tested. The open symbols represent the theoretical points expected if the reactant was free hydroxyl radical.
21 100-
-T
e*
20! 0
4
5
6
7
8
9
1
8 0
PH
Figure 3. Oxygen evolution as a function of pH. Peroxynitrite (250 pM) was added to hydrogen peroxide (133 mM) a t 37 "C. Potassium phosphate (50 mM) was used over the pH range of 4.0-8.0, while potassium pyrophosphate was used over the range of 8.0-9.0. The solid line was fitted by nonlinear regression using the Henderson-Hasselbalch equation. Data are the mean i SD.
ground state of trans-peroxynitrous acid (211, inhibited oxygen evolution to a somewhat larger extent than predicted according to its rate constant with hydroxyl radical. Stopped-Flow Experiments. As previously described (16),peroxynitrite decomposition in phosphate and pyrophosphate buffers followed first-order kinetics in the range of temperatures tested (15-45 "C). In the presence of hydrogen peroxide, the decomposition of peroxynitrite also followed pseudo-first-order kinetics. Surprisingly, the apparent rate did not increase but actually decreased. Figure 5 shows the pH dependence of peroxynitrite decomposition in the presence and absence of hydrogen peroxide. The first-order rate constant of peroxynitrite
0.01
'
I
I
3
4
5
I
I
I
6
7
8
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PH
Figure 5. Peroxynitrite decomposition rate as a function of pH. Hydrogen peroxide was 100 mM, and the buffer was either potassium phosphate (80 mM) or pyrophosphate (180 mM), beyond pH 7.8. Open symbols are used in the absence of hydrogen peroxide. Data are the mean of a t least 7 experiments, and standard deviations are smaller than symbols. The solid line was fitted by nonlinear regression to eq 5.
decomposition in the absence of hydrogen peroxide as a function of pH can be described by the equation first proposed by Keith and Powell (24)
where k is the apparent rate constant of peroxynitrite decomposition a t a given pH, km is the first-order rate constant for peroxynitrite decomposition, and K, is its acid dissociation constant. The pKa measured in our experiments was 6.9 f 0.1, and km was 1.13 f 0.01, in good agreement with the literature (2,16). The addition of 100 mM hydrogen peroxide caused a shift in the apparent pKa of peroxynitrite decomposition to the value of 6.63 f 0.06. At neutral to alkaline pH, the apparent
Aluarez et al.
862 Chem. Res. Toxicol., Vol. 8, No. 6, 1995
peroxide was calculated to be 17.4 f 0.1 kcal mol-'. This agrees well with the value of 18 f 1 kcal mol-' previously found a t pH 5 (161,confirming that the standard enthalpy of dissociation of peroxynitrous acid is close to 0 kcaU mol (16). The apparent activation entropy was -1.8 & 0.2 cal mol-' K-'. When, instead of plotting the observed rate constants directly, corrections were made for pH effects according to eq 5, the activation entropy calculated was 1.3 f 0.2, in agreement with the value reported by Koppenol et al. (16). When 30 mM hydrogen peroxide was added, the apparent activation enthalpy and entropy were 19.1 f 0.1 kcal mol-' and 2.9 f 0.4 cal mol-l K-I, respectively. Thus, the activation enthalpy increased by 1.7 kcal mol-l and the activation entropy by 4.7 cal mol-' K-l in the presence of hydrogen peroxide. These shifts were consistent within different experiments.
1.41
a
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.
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2
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0.41 0.34
1
0.0 0.00
0.02
0.04
0.06
0.08
(M) Figure 6. Peroxynitrite decomposition in the presence of hydrogen peroxide. Peroxynitrite (375 ,uM) was mixed with varying amounts of hydrogen peroxide and potassium phosphate (70 mM), pH 5.75 0.02 (A)or pH 7.54 & 0.02 (0).The solid lines are results of nonlinear regression to eq 6. Data are mean
Discussion
IHZOZl
*
* SD.
-65
-
0.0030
0.0032
1/T
0.0034
0.0036
(K.')
Figure 7. Eyring plot of the first-order peroxynitrite decomposition. The pH was 7.35 0.02, and the temperature was varied from 15 to 45 "C. Peroxynitrite was 400 pM with no (0)
*
or 30 mM hydrogen peroxide ( 0 ) .
rate constant in the presence of excess hydrogen peroxide was near 50% smaller than in its absence, whereas a t acid pH there was no significant difference in the rate constants, with hydrogen peroxide causing no detectable slowing of peroxynitrite decomposition. The apparent rate constant for peroxynitrite decomposition decreased as hydrogen peroxide increased (Figure 61, following a n hyperbolic function according to:
k = c + l/(a[H,O,l
+ b)
(6)
where k is the observed rate constant and a , b, and c are empirical parameters that differ for the pHs tested. The temperature dependence in the range of 15-45 "C of the rate constant of peroxynitrite disappearance in the presence and absence of hydrogen peroxide a t pH 7.35 & 0.02 is shown in Figure 7. For the representative experiment shown, the apparent activation enthalpy for peroxynitrite decomposition in the absence of hydrogen
A rationalization of the reaction between peroxynitrite and hydrogen peroxide should take into account, on one hand, that oxygen yields were the smallest at acidic pH and that hydroxyl radical scavengers inhibited oxygen evolution. On the other hand, hydrogen peroxide decreased the apparent first-order rate of peroxynitrite disappearance a t neutral to alkaline pH. Oxidation reactions of peroxynitrite can be explained as involving two-electron processes, such as S Ndisplace~ ments, or a one-electron transfer reaction in which the substrate is oxidized by one electron and peroxynitrite is reduced (5). From a thermodynamic perspective, the oxidation of hydrogen peroxide by the ground state of peroxynitrite could take place by a one- or two-electron mechanism, since both reduction potentials, E"'(ONOO-,2Hf/"0~,H~O) 1.4 and 0.99 V, respecand E"'(ONOO-,2Hf/N0~-,H~O), tively (16, 61, are higher than Eo'(02'-,2H+/H202), 0.94 V, and E"'(02,2Hf/H202), 0.3 V (25). However, the most thermodynamically favored reaction appears to be the one-electron oxidation of hydrogen peroxide by the highenergy intermediate derived from trans-peroxynitrous acid, ONOOH*, which has a n estimated redox potential 0.7 V higher than that of the ground state (161, E"'(ONOOH*,H+/"O~,H~O)of 2.1 V. A two-electron S N displacement ~ would imply the reaction of hydrogen peroxide with peroxynitrous acid to yield oxygen and nitrite as follows: ONOOH
+ H,O, - NO,- + 0, + H+ + H,O
(7)
Since peroxynitrous acid is in rapid equilibrium with peroxynitrite anion, first-order kinetics with respect to hydrogen peroxide would be expected if this mechanism was operative. Yet, hydrogen peroxide did not increase the rate of peroxynitrite disappearance. Hence, a oneelectron mechanism is more likely. In a one-electron mechanism, hydrogen peroxide would be oxidized to superoxide, which would dismutate spontaneously ( k lo5 M-' 5-l) (26) to yield oxygen and hydrogen peroxide as shown: ^v
+ H,O, - 02*-+ H,O + 'NO, + H+ ( 8 ) HO,' + 0,'- + H,O H,O, + 0, + OH- (9)
ONOOH
-
so that two peroxynitrite molecules would be needed to produce one of oxygen, as proposed originally by Mahoney (18).
Peroxynitrite Reaction with Hydrogen Peroxide
Chem. Res. Toxicol., Vol. 8, No. 6, 1995 863
alkaline pH has already been shown for sulfhydryl oxidation by peroxynitrite (2). In contrast with the .T reaction with hydrogen peroxide, the rate constant for [ONOO -...H 2 0 2 ]% [ONOO' ... H 2 0 2 ] *+ NO,' this reaction is first order in sulfhydryls and the oxidative yield a t alkaline pH reaches 100% relative to peroxyniH202 trite. i The pH dependence of hydrogen peroxide oxidation Pans-ONOO[ONOO-1' NO,' suggests that a n anionic form of peroxynitrite may be involved. It has been postulated that isomerization of cis- to trans-peroxynitrous acid represents a slow step c/s-ONOOH h rrans-ONOOH [ONOOH]. HNO, in the acid-catalyzed conversion of peroxynitrite to nitrate (15,211 and that the loss of hydroxyl radical-like reactivH202 ity of peroxynitrite a t alkaline pH depends on the direct cis-0 NOOisomerization of an activated state of trans-peroxynitrite 0 2 anion, ONOO-*, to nitrate (15). ONOO-* is unlikely to Figure 8. Proposed mechanism for the reaction of peroxynitrite directly oxidize other substrates, because this would with hydrogen peroxide. Protonation of cis-peroxynitrite anion result in placing a further negative charge on the promotes isomerization to trans-peroxynitrous acid, which can ionize to yield trans-peroxynitrite anion. trans-Peroxynitrous molecule. However, hydrogen peroxide could have a role acid can undergo transition to an activated form which can in stabilizing trans-peroxynitrite anion, partially diverteither rearrange to nitric acid or oxidize hydrogen peroxide. ing the route of decay from isomerization to reduction trans-Peroxynitrite anion can also rearrange to nitrate via a n by means of hydrogen peroxide oxidation. Indeed, the energized intermediate or form a stabilizing transient complex increase in the activation enthalpy and entropy in the with hydrogen peroxide that can undergo activation and decay to superoxide and eventually molecular oxygen. Adapted from presence of hydrogen peroxide is consistent with the Crow et al. (15). existence of a relatively more stabilized and ordered ground state. Formation of this complex would result Alternatively, superoxide could be further oxidized by in apparent slowing of the rate of peroxynitrite decay if nitrogen dioxide ('Nod ( K lo8 M-l s-l) (23) to yield more energy is needed to reach the vibrationally excited oxygen. species, from a stabilized ground-state trans-peroxynitrite anion (see Figure 8). 'NO, 0;NO,0, (10) The importance of water interactions and hydrogen bonding for peroxynitrite stabilization has been reported In order to explain the reactivity of peroxynitrite (27,281. Perhaps hydrogen peroxide, whose small barrier toward hydrogen peroxide a t acidic pH, a mechanism for rotation around the 0-0 bond allows for flexibility involving the high-energy form of trans-peroxynitrous of hydrogen bonding (291, could form a two-hydrogen acid, ONOOH*, can be suggested in agreement with bonded complex with peroxynitrite anion, although this previous data (5, 15, 16) (see Figure 8). Indeed, the would result in a decrease of rotational freedom. In apparent upper limit of 15.6% yield of oxygen from principle, water could also hydrogen bond with peroxperoxynitrite even a t infinite hydrogen peroxide concenynitrite, but this would not be as favorable, since two trations, and the inhibition by hydroxyl radical scavenwater molecules would be needed compared with one of gers to an extent similar to that predicted according to hydrogen peroxide, resulting in a loss of translational their published rate constants, are consistent with the entropy. Hydrogen peroxide would also stabilize cisoxidation via the hydroxyl radical-like intermediate, a peroxynitrite anion, since the charge distribution is not process that competes with the direct rearrangement of likely to be much different than for the trans-rotamer. ONOOH* to nitric acid. Additionally, in this mechanism The results presented herein contribute to the underthe formation of ONOOH* previous to target molecule standing of the reaction chemistry of peroxynitrite. In reactions is the rate-limiting step, and the decomposition addition, the significance of contaminating hydrogen of peroxyninitrite is zero-order in hydrogen peroxide a t peroxide in stock solutions is highlighted. Nevertheless, acidic pH. the biological relevance of the findings, in terms of Controversy exists as to whether the oxidant is potential reactions between peroxynitrite and hydrogen ONOOH* or 'OH and 'NO2 radicals derived from the peroxide, requires further investigation. Even though dissociation of peroxynitrous acid in a cage of water both molecules will coexist in cells and tissues producing molecules. For further discussion, see Pryor (5). nitric oxide and superoxide, other intracellular reactions According to previous papers (3,4,15,21), the yield of of peroxynitrite such as thiol oxidation and nitration of expected hydroxyl radical products from deoxyribose, aromatics would be significantly faster. However, it is DMSO, ABTS (2,2'-azinobis(3-ethyl-1,2-dihydrobenzothi- possible that in extracellular compartments, where direct azoline-6-sulfonate)), desferrioxamine, and polyunsatutargets for peroxynitrite are a t significantly lower conrated fatty acids by peroxynitrite decreases a t alkaline centrations, the reaction between peroxynitrite and pH. In contrast, oxygen yields from hydrogen peroxide hydrogen peroxide could occur to some extent. Finally, increased a t alkaline pH. Furthermore, a t infinite while this reaction may allow for the annihilation of two concentrations of hydrogen peroxide the maximum oxypotent oxidants, the formation of secondary oxidizing gen yield was 32.2% a t pH 7.36,larger than a t acid pH intermediates such as superoxide (eq 81, nitrogen dioxide but not reaching 100%. The pH dependence of the (eq lo), and singlet oxygen (30)may ultimately result in reaction between peroxynitrite and hydrogen peroxide a redirection of peroxynitrite and hydrogen peroxidecan be only attributable to the ionization of peroxynitrite, mediated toxicity. since the first pKa of hydrogen peroxide is 11.7 a t 25 "C (25) so that its dissociation does not significantly occur Acknowledgment. We thank Drs. W. A. Pryor, G. a t the pHs tested. An increase in oxidative yield a t L. Squadrito, J. Hayes, J. S. Beckman, and W. H. 0 2
1
---j
I
D __
+
-
1
+
Alvarez et al.
864 Chem. Res. Toxicol., Vol. 8, No. 6, 1995
Koppenol for sharing unpublished data and helpful discussions. This work was supported by a grant from CONICYT, Uruguay, to R.R. B.A. was partially supported by PEDECIBA and CONICYT.
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