Radicals in the reaction between peroxynitrite and uric acid identified by electron spin resonance spectroscopy and liquid chromatography mass spectrometry

Article abstract of DOI:10.1016/j.freeradbiomed.2010.04.010

Peroxynitrite is a reactive oxidant produced in vivo in response to oxidative and other stress by the diffusion-limited reaction of nitric oxide and superoxide. This article is focused on the identification of free radical intermediates of uric acid forme

Full text of DOI:10.1016/j.freeradbiomed.2010.04.010

Free Radical Biology & Medicine 49 (2010) 275–281
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Free Radical Biology & Medicine
journal homepage: www.elsevier.com/locate/freeradbiomed
Original Contribution
Radicals in the reaction between peroxynitrite and uric acid identified by electron
spin resonance spectroscopy and liquid chromatography mass spectrometry
b
b
b,c
Witcha Imaram a, , Christine Gersch , Kyung Mee Kim , Richard J. Johnson
,
⁎
George N. Henderson b, Alexander Angerhofer a
Department of Chemistry, University of Florida, Gainesville, FL 32611, USA
Division of Nephrology and Hypertension, Department of Medicine, University of Florida, Gainesville, FL 32610, USA
Division of Renal Diseases and Hypertension, University of Colorado Health Science Center, Denver, CO 80262, USA
a
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Peroxynitrite is a reactive oxidant produced in vivo in response to oxidative and other stress by the diffusion-
limited reaction of nitric oxide and superoxide. This article is focused on the identification of free radical
intermediates of uric acid formed during its reaction with peroxynitrite. The experimental approach included
the ESR spin trapping of the radical generated from the reaction between uric acid and peroxynitrite at pH
7.4 and mass spectrometry studies of the trapped radicals. Using PBN (N-tert-butyl-α-phenylnitrone) as the
spin trapping agent, a six-line ESR spectrum was obtained and its hyperfine coupling constants, aN =15.6 G
and aH =4.4 G, revealed the presence of carbon-based radicals. Further structural identification of the PBN–
radical adducts was carried out using liquid chromatography–mass spectrometry. After comparison with the
control reactions, two species were identified that correspond to the protonated molecules (M+1) at m/z
352 and 223, respectively. The ions of m/z 352 were characterized as the PBN–triuretcarbonyl radical adduct
and the m/z 223 ion was identified as the PBN–aminocarbonyl radical adduct. Their mechanism of formation
is discussed.
Received 7 December 2009
Revised 22 March 2010
Accepted 13 April 2010
Available online 18 April 2010
Keywords:
Uric acid
Peroxynitrite
ESR
Electron spin resonance
Mass spectrometry
LC–MS
Free radicals
Spin trap
© 2010 Elsevier Inc. All rights reserved.
PBN
During primate evolution, about 5–20 million years ago, two parallel
but distinct mutations occurred in early humanoids that rendered the
uricase gene nonfunctional [1]. As a result, humans and the great apes
have higher uric acid (1, Fig. 1) levels (in the range between 3 and
14 mg/dl) compared with most mammals (1 mg/dl) [2]. Thus, uric acid,
a metabolic product of purine, has been assumed to replace vitamin C as
the major antioxidant in humans [3]. Many studies have investigated
the role of uric acid and its ability to scavenge reactive oxygen and
nitrogen species. Among these reactive species, peroxynitrite, formed
by the fast reaction (k=6.7–19×109 M−1 s−1) between nitric oxide
and superoxide [4,5], has received great interest in toxicology as a
potent oxidant in vivo. Moreover, uric acid was found to scavenge
peroxynitrite very effectively in vivo [6]. Urate (1−, Fig. 10), the
conjugate base of uric acid, has been observed to inhibit tyrosine
nitration [7] by scavenging the radical intermediates generated in the
decomposition of peroxynitrite [8], which are responsible for tyrosine
nitration.
Despite its role as a major antioxidant in plasma, there is growing
evidence of uric acid being a true risk factor for the development of
obesity, hypertension, and cardiovascular disease, all conditions
associated with oxidative stress [2,9]. Uric acid can form free radicals
in various reactions [10], including the interaction with peroxynitrite
[8,11]. Reacting with peroxynitrite, urate decomposes to form
radicals, which are responsible for the amplification of lipid oxidation
products found in liposomes and low-density lipoprotein [12]. In fact,
one such radical was identified as the aminocarbonyl radical [12].
Thus the reaction between uric acid and oxidants such as peroxyni-
trite may initiate and propagate a radical chain reaction and damage
the cells [13].
The reactivity of peroxynitrite is strongly pH dependent [14]. Once
protonated peroxynitrous acid (ONOOH; pKa =6.8) is formed
(Eq. (1)). It can rapidly undergo homolysis to yield nitrogen dioxide
(•NO2) and hydroxyl (•OH) radicals (Eq. (2)) [14,15]. Furthermore,
peroxynitrite can react with carbon dioxide, leading to the formation
of the carbonate radical anion (CO•3−) and nitrogen dioxide radical
Abbreviations: ESR, electron spin resonance; PBN, N-tert-butyl-α-phenylnitrone;
LC–MS, liquid chromatography–mass spectrometry; DTPA, diethylenetriaminepentaacetic
acid.
⁎
Corresponding author. Fax: +1 352 392 0872.
E-mail address: wimaram@chem.ufl.edu (W. Imaram).
0891-5849/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.freeradbiomed.2010.04.010

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W. Imaram et al. / Free Radical Biology & Medicine 49 (2010) 275–281
LC–MS analysis
The LC–MS analyses were carried out with an Agilent 1100 liquid
chromatography system (Agilent Technologies, Palo Alto, CA, USA) and
a TSQ 7000 triple-quadrupole mass spectrometer (ThermoFinnigan, San
Jose, CA, USA) equipped with an APCI interface and operated in positive-
ion mode detection. In the TSQ 7000 instrument, nitrogen was used as
both the sheath and the auxiliary gas. The second quadrupole was used
as a collision chamber, with argon as a collision gas, at a pressure in the
vicinity of 2.5×10−3 Torr. Theoperation of theLC–MSand data analyses
was performed using the ThermoFinnigan Xcalibur 1.4 software.
Fig. 1. Structures of uric acid (1) and N-tert-butyl-α-phenylnitrone (PBN) (2).
(•NO2) (Eq. (3)) [14,15]. These peroxynitrite-derived radicals can
initiate one-electron oxidation:
Full-scan LC–MS
ONOO− þ Hþ⇆ONOOH
ð1Þ
Liquid chromatography analyses were performed in a gradient
elution mode using
a Phenomenex Luna 5 µ C18(2) 100 Å
•
ONOOH→•NO2 þ OH
ð2Þ
ð3Þ
(150×4.6 mm) column (Phenomenex, Torrance, CA, USA) coupled
with a Phenomenex Luna C18(2), 5-µm particle size guard column.
The mobile phase used included 5 mM ammonium acetate/0.1% acetic
acid (A) and methanol (B) as the gradient. The mobile phase flow was
0.6 ml min−1, and the injection volume was 20 μl. The gradient began
at 90% A. Composition was linearly ramped to 95% B over the next
10 min, remained constant for 3 min, and then reversed to the original
composition of 90% A over 1 min, after which it was kept constant for
1 min to reequilibrate the column. The extracted reaction products,
control samples and standard samples, were analyzed in the full-scan
mode at a mass range of m/z 90–450.
ONOO− þ CO2→ONOOCO−2 →•NO2 þ CO•3−
Because of the complexity of the peroxynitrite-mediated oxidation
of uric acid, its mechanism has not yet been established. During our
investigation to unfold the chemistry of uric acid when treated with
peroxynitrite at pH 7.4, we discovered a novel urate-derived radical—
triuretcarbonyl—which could be an intermediate for the production of
the aminocarbonyl radical. The radicals were studied by electron spin
resonance spectroscopy using the spin trapping method; N-tert-butyl-
α-phenylnitrone (PBN)1 (2, Fig. 1) was used as a spin trapping agent,
and the structures of the PBN–radical adducts were characterized by
liquid chromatography–mass spectrometry (LC–MS).
LC–MS tandem mass spectrometry analysis
LC analysis was performed as outlined above. The tandem mass
spectrometry (MS/MS) analysis was performed for the M+1 ions
with m/z of 223 and 352 in the positive mode at a collision energy of
20 V, with a mass scan range of m/z of 40–230 for the ion at 223 and a
mass scan range of m/z of 45–360 for the 352 ion.
Materials and methods
Chemicals
Uric acid was purchased from Sigma. Diethylenetriaminepentaa-
cetic acid (DTPA) was from Fluka. PBN was obtained from Alexis
Biochemicals. Peroxynitrite was synthesized following the method
reported by Uppu and Pryor [16]. The peroxynitrite concentration was
measured spectrophotometrically at 302 nm (ε=1670 M−1 cm−1).
Results
Electron spin resonance spin trapping
To probe the generation of the PBN–radical adducts at pH 7.4, the
reaction between urate with peroxynitrite was monitored by ESR
using the spin trapping method. The reaction between urate and
peroxynitrite resulted in a six-line ESR spectrum when the modula-
tion amplitude was set to 1 G (Fig. 2A). The trapped radical adducts
displayed average hyperfine coupling constants of aN =15.6 G and
aH =4.4 G. No trapped radicals were observed when the reactions
were conducted without urate or peroxynitrite. PBN alone, or mixed
with urate or peroxynitrite, did not yield any ESR signal (Fig. 2B).
Furthermore, the ESR intensities increased when the urate
concentration was increased (Fig. 3). These experiments confirmed
that the observed radicals were derived from urate, not artifacts.
Moreover, when the experiment was performed at higher resolution
(lower modulation amplitude), we observed additional line splitting
(data not shown) indicating that PBN might trap more than one
different carbon-based radical. The radical formation increased with
the concentration of peroxynitrite, but yielded a maximum at a
fourfold molar excess of peroxynitrite over urate (Fig. 4).
ESR experiments
Stock solutions of urate (100 mM) were prepared in 0.3 M
potassium hydroxide. The reactions, typically conducted in 0.3–0.5 M
potassium phosphate buffer at pH 7.4, contained a final concentration of
3 mM urate, 30 mM PBN, 0.1 mM DTPA, and 9 mM peroxynitrite. A
small aliquot of the reaction mixture was transferred into a quartz
capillary of approximately 1×3 mm i.d.×o.d. for ESR measurement.
After 2 min, the ESR spectrum was recorded at room temperature, using
a commercial Bruker Elexsys E580 spectrometer, employing the high-Q
cavity (ER 4123SHQE). Instrumental parameters were typically 100 kHz
modulation frequency, 1 or 0.5 G modulation amplitude, 20 mW
microwave power, 9.87 GHz microwave frequency, 82 ms time con-
stant, and 164 ms conversion time/point.
Sample preparation for LC–MS
Depending on pH, both uric acid and peroxynitrite will exist in
either their neutral or their anionic forms. Therefore, the radical
formation should be affected by pH as well. Indeed, we observed that
the urate–peroxynitrite reaction yielded more trapped radicals when
the pH increased (Fig. 5). This indicates that the radical production
depended on the availability of peroxynitrite in one of its anionic
forms. The simulated titration curve shows an inflection point at
pH 8.1. Our value is different from that reported previously [12];
The reaction mixtures were prepared at room temperature in
0.3 M potassium phosphate buffer, pH 7.4, and contained a final
concentration of 10 mM urate, 30 mM PBN, 0.1 mM DTPA, and 30 mM
peroxynitrite in a final volume of 10 ml. The reaction mixtures were
subsequently extracted by 2×20 ml of CH2Cl2, dried under nitrogen
gas, and resuspended in 1 ml CH3CN.

W. Imaram et al. / Free Radical Biology & Medicine 49 (2010) 275–281
277
Fig. 2. ESR spectra of PBN–radical adducts obtained from the incubation of 3 mM urate,
30 mM N-tert-butyl-α-phenylnitrone (PBN), 0.1 mM DTPA, and 9 mM peroxynitrite in
phosphate buffer, pH 7.4. At room temperature, the ESR spectrum was recorded 2 min
after addition of peroxynitrite. (A) The ESR spectrum of the PBN–radical adducts using
the instrumental parameters of 100 kHz modulation frequency, 1 G modulation
amplitude, 20 mW microwave power, 9.87 GHz microwave frequency, 82 ms time
constant, and 164 ms conversion time/point. (B) The control reaction conducted under
the same conditions as described for (A) but without urate.
Fig. 4. Effect of peroxynitrite concentration on the production yield of the PBN–radical
adduct derived from urate obtained from the oxidation of urate by peroxynitrite. The
ESR spectra were recorded after 2 min of incubation of 3 mM urate, 30 mM PBN,
0.1 mM DTPA, and various concentrations of peroxynitrite in 0.5 M phosphate buffer,
pH 7.4. The instrumental parameters were 100 kHz modulation frequency, 1 G
modulation amplitude, 20 mW microwave power, 9.87 GHz microwave frequency,
20 ms time constant, and 82 ms conversion time/point.
however, it is still in the range of the pKa of peroxynitrous acid, which
varies from 6.5 to 8.6 depending on the composition of the solution,
determined previously by Kissner et al. [4]. We suspected that the use
of a higher concentration in our reaction mixture may cause the shift
of the pH profiles as well as the pKa of peroxynitrous acid. In addition
to the pH effect, we have investigated the effect of CO2 concentration
on the production of PBN–radical adducts. Increased [CO2] decreased
the observed ESR signal intensity (Fig. 6).
(Fig. 7B)—the reaction without urate—the full-scan LC–MS showed
two products, at the retention times of 10:56 and 13:13 min. These
peaks correspond to the ions of m/z 223 and m/z 352, respectively
(Fig. 7A). As displayed in Figs. 7A and B, the large area under the total
ion current chromatography starting at a retention time of 11:12 min
up to 15:00 min arises from ions derived from PBN (M+1=178), and
the small peak at 9 min (m/z 122), which was observed in the control
reaction as well, could be a degradation product of PBN, which is
tentatively identified as benzaldehyde oxime.
Product identification of the radical adducts by LC–MS analysis
In addition to the full scan, the ions at m/z 223 and 352 were selected
and analyzed by MS/MS. At m/z 223, the following fragment ions were
identified: m/z (% intensity) 223 (4), 167 (30), 149 (29), 134 (12), 132
From the reaction between urate and peroxynitrite in phosphate
buffer, pH 7.4, the extracted radical adducts were separated and
characterized by LC–MS. After comparison with the control reaction
Fig. 5. Effect of pH on the yield of the PBN–radical adduct derived from urate obtained
from the oxidation of urate by peroxynitrite. The ESR spectra were recorded after 2 min
of incubation of 3 mM urate, 30 mM PBN, 0.1 mM DTPA, and 9 mM peroxynitrite in
0.5 M phosphate buffer at various pH's. The instrumental parameters were 100 kHz
modulation frequency, 1 G modulation amplitude, 20 mW microwave power, 9.87 GHz
microwave frequency, 20 ms time constant, and 82 ms conversion time/point. The
measured ESR intensities corresponded to the low-field PBN–radical adduct peak-to-
peak heights.
Fig. 3. Effect of urate concentration on the production yield of the PBN–radical adduct
derived from urate obtained from the oxidation of urate by peroxynitrite. The ESR
spectra were recorded after 2 min of incubation with various urate concentrations,
19 mM PBN, 0.1 mM DTPA, and 23 mM peroxynitrite in 0.3 M phosphate buffer, pH 7.4.
The instrumental parameters were 100 kHz modulation frequency, 0.5 G modulation
amplitude, 20 mW microwave power, 9.87 GHz microwave frequency, 82 ms time
constant, and 164 ms conversion time/point.

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W. Imaram et al. / Free Radical Biology & Medicine 49 (2010) 275–281
Fig. 8. MS/MS spectrum of the m/z 223 (M+1) ion identified as the PBN–
aminocarbonyl radical adduct.
Fig. 6. Effect of [CO2] on the yield of the PBN–radical adduct derived from urate obtained
from the oxidation of urate by peroxynitrite. The ESR spectra were recorded after 2 min of
incubation of 3 mM urate, 30 mM PBN, 0.1 mM DTPA, and 9 mM peroxynitrite in 0.5 M
phosphate buffer at pH 7.4 in the presence of various concentrations of bicarbonate
(HCO−3 ). The instrumental parameters were 100 kHz modulation frequency, 1 G modula-
tion amplitude, 20 mW microwave power, 9.87 GHz microwave frequency, 82 ms time
constant, and 164 ms conversion time/point.
Discussion
Identification of the free radicals
(5), 122 (24), and 104 (100) (see Fig. 8). The fragments of m/z 352
exhibited the following m/z ratios (% intensity): 352 (2), 335 (13), 296
(33), 279 (25), 263 (100), 246 (83), 218 (7), 193 (12), 177 (24), 175
(22), 167 (9), 147 (24), 122 (8), 118 (6), 104 (9), and 61 (1) (see Fig. 9).
Spin trapping allows the trapping of short-lived radicals to form a
more stable radical adduct amenable to ESR analysis [17]. However, it
is usually not possible to obtain detailed structural information about
the trapped radical from ESR alone. The identification of the radical
adducts therefore relies on the interpretation of the LC–MS experi-
ments. Their proposed formation mechanism in the peroxynitrite–
urate reaction will be discussed in the following paragraphs.
The first spin trapping studies on urate-derived radicals were
reported by Santos et al. on the peroxynitrite–urate system [12]. More
than one DMPO–radical adduct were observed from the ESR
spectrum. However, only one radical was identified as the amino-
carbonyl radical by MS (13, Fig. 10). Our experiments also showed
that PBN could trap at least two carbon-based radicals. The
assignment to carbon-based radicals was made by comparison with
the literature [18]. The full-scan LC–MS study showed two distinctive
products with m/z 223 (M+1) and 352 (M+1), which were absent
in the control reaction (Fig. 7).
The analysis of the LC–MS/MS spectra (Figs. 8 and 9) revealed that
both ions showed the same initial fragment loss of a neutral compound
with a molecular weight (MW) of 56, corresponding to the loss of 2-
methylpropene ([M+H–C4H8]+). This fragment loss can be explained
by the cleavage of the tert-butyl group from the PBN moiety. Moreover,
Fig. 7. LC–MS study of the reaction between urate and peroxynitrite in phosphate
buffer, pH 7.4. Before being subjected to LC, the reaction products were extracted by
methylene chloride (CH2Cl2), dried under nitrogen gas, and resuspended in acetonitrile.
(A) LC chromatogram of 10 mM urate treated with 30 mM peroxynitrite in the
presence of 30 mM PBN and 0.1 mM DTPA. (B) LC chromatogram of the control reaction
in which every reagent had the same concentration as described in (A) but without
urate.
Fig. 9. MS/MS spectrum of the m/z 352 (M+1) ion identified as the PBN–
triuretcarbonyl radical adduct.

W. Imaram et al. / Free Radical Biology & Medicine 49 (2010) 275–281
279
Fig. 10. Proposed radical formation mechanism of the reaction between urate and peroxynitrite.
the fragmentation pattern of both parent ions contained the same
fragment daughter ion at m/z 122. This ion was identified as protonated
benzaldehyde oxime (Supplementary Figs. 1 and 2), which was derived
from PBN as well. These results indicated that both parent ions
contained PBN in their structure. Furthermore, the loss of formamide
(MW 45), from both 223 and 352 ions, and isocyanic acid (HNCO; MW
43), in the case of the 352 ion, indicated that at least one amide group
was present in both compounds. This assignment is further supported
by the loss of ammonia (MW 17) from both 223 and 352 ions, which
reflects the fact that the parent compounds have a functional group that
containsa primary aminesuch as an amide. Based on theseobservations,
the MS/MS fragmentation analysis confirmed that the structure of the
product corresponding to m/z 223 was consistent with the hydroxyl-
amine form of the PBN–aminocarbonyl radical adduct 14 (Fig. 10), and
the structure corresponding to m/z 352 is proposed to be the
hydroxylamine form of the PBN–triuretcarbonyl radical adduct 15
(Fig. 10). The existence of the triuret moiety in compound 15 is
further confirmed by the fragment ions at m/z 147, 104, and 61
(Supplementary Fig. 2), which is a signature fragmentation pattern
of triuret (M+1 = 147) [19].
possible pathways have been proposed for its generation. Santos
et al. suggested that the aminocarbonyl radical could be produced
from one of the products of the urate–peroxynitrite reaction, such
as alloxan, parabanic acid, or allantoin, even though they did not
observe any trapped radical when these compounds were incubated
with peroxynitrite [12]. Robinson et al. later suggested that triuret
(16, Fig. 10), a novel product from peroxynitrite-induced oxidation
of urate, could account for the detection of the aminocarbonyl
radical [19]. We have tested the reaction of the proposed
intermediates, alloxan, parabanic acid, allantoin, and triuret, with
peroxynitrite under the same conditions as the oxidation of urate,
but did not observe any radical adduct formation. We therefore
conclude that the aminocarbonyl radical was not derived from these
known urate-derived products. In our study, the trapping of radical
adduct 15 led us to postulate that the triuretcarbonyl radical 12 is an
intermediate in the production of the aminocarbonyl radical 13, as
proposed in Fig. 10.
How is the oxidation of urate initiated? It is not clear whether
the oxidant is peroxynitrite itself or one of its possible derivatives.
Depending on the pH of the reactions, a variety of peroxynitrite-
derived compounds can coexist. At pH 7.4, a mixture of the
peroxynitrite anion (ONOO−) and ONOOH or its decomposition
products is present [14,15,19]. It is highly unlikely that the
negatively charged peroxynitrite anion reacts with the negatively
charged urate anion. Rather, at pH 7.4, it is more feasible for urate to
Mechanism of the radical formation
Although the aminocarbonyl radical has been previously char-
acterized, little is known about its formation mechanism. Two

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W. Imaram et al. / Free Radical Biology & Medicine 49 (2010) 275–281
be oxidized by neutral uncharged oxidants, for example, ONOOH or
its decomposition radicals, •NO2 and •OH [14,15]. These oxidants can
initiate a one-electron oxidation [14,15,20,21]. Moreover, the initial
step of the oxidation of many purine-based compounds is
considered to be a one-electron oxidation as well [10,22,23]. Thus,
it is likely that urate is initially oxidized via one-electron transfer,
forming the urate radical 3 (Eq. (4)).
radical formation was at a maximum when a fourfold excess of
peroxynitrite over urate was used (Fig. 4). By analogy, similar to the
degradation of 5, the triuretcarbonyl radical 12 is obtained. The β-
cleavage of triuretcarbonyl radical 12 yields isocyanic acids and the
aminocarbonyl radical 13, which is subsequently trapped by PBN
(Fig. 10).
Aside from being the precursor of the aminocarbonyl radical, the
triuretcarbonyl radical could also be an intermediate candidate for the
generation of triuret 16, which could be achieved by eliminating
carbon monoxide or carbon dioxide by reaction with oxygen (Fig. 10).
Given the high concentrations of both urate and peroxynitrite used in
this study, which were necessitated by the limited sensitivity of the
ESR spin trap method, one can question the relevance of the proposed
mechanism to the intracellular fate of urate. However, we point out
that triuret is a unique product of the reaction between urate and
peroxynitrite that not only was observed in vitro [19] but also has
been detected in urine samples of hypertensive patients [31].
ð4Þ
When the pH of the reaction is increased, the chemistry of
peroxynitrite is changed. At high pH, both peroxynitrite and urate are
present in their anion or dianion forms, and their direct reaction is
unfavorable because of the charge repulsion. The only oxidant that
Conclusion
The observation of a novel intermediate, triuretcarbonyl radical,
sheds light on the formation mechanism of triuret [19] and the
aminocarbonyl radical [12] in the reaction between urate and
peroxynitrite. In contrast to other known oxidants that can react
with uric acid, triuret is observed only in the peroxynitrite–urate
reaction [32]. This mechanism rationalizes the uniqueness of
peroxynitrite-mediated oxidation of uric acid to produce triuret as a
major product.
•
reacts with urate under basic conditions should be NO2 generated
from the reaction between peroxynitrite and carbon dioxide [14,15].
In this case the diimine 4, an oxidative intermediate of uric acid
described in many urate oxidation studies [12,24], can be produced by
one-electron and one-proton transfer from the urate radical 3 to
another peroxynitrous acid or its derived radicals as described above.
The diimine 4 is susceptible to nucleophilic addition and can rapidly
react with nucleophiles such as water or ammonia [24] and, in our
case, the peroxynitrite anion. The peroxynitrite anion has been
reported to undergo nucleophilic addition in many reactions [25–29].
Moreover, both the observed pH dependence and the dependence on
CO2 concentration suggest one important conclusion, which is that
ONOO− is necessary for the production of radicals. Our observation
confirms the results reported by Santos et al. in which urate-derived
radical formation was dependent on peroxynitrite anion [12]. At high
pH, peroxynitrite is monoanionic and we observed higher yields of
radical production (Fig. 5). In the presence of CO2, which can rapidly
react with ONOO− and reduce its concentration [30], the radical
formation was decreased (Fig. 6). These observations suggest that
peroxynitrite anion can act as a nucleophile and react with the
diimine 4 to produce the peroxo adduct 5.
Acknowledgments
This work was financially supported by the National Science
Foundation (CHE-0809725), in part by NIH Grant HL-68607 and
generous funds from Gatorade. We also acknowledge helpful
discussions with Dr. William Dolbier, Jr.
Appendix A. Supplementary material
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.freeradbiomed.2010.04.010.
References
After undergoing homolysis of the weak peroxo bond (O–O bond)
[22,25], the peroxo adduct 5 decomposes to yield •NO2 and the radical
intermediate 6, which then rearranges to form a carbonyl radical, 7.
The carbonyl radical 7 can be converted to radical 8 by either losing
carbon monoxide (CO) or reacting with oxygen (O2) and releasing
carbon dioxide (CO2). We have performed the reactions under
anaerobic conditions with the ESR spin trapping experiments (data
not shown). Interestingly, we found that the yield of the trapped
radicals under anaerobic conditions was higher than under aerobic
conditions. On the other hand, when reactions were carried out under
O2, we observed less or no ESR signal from the reaction. But the work
by Santos et al. has shown that the urate–peroxynitrite reaction
consumed oxygen [12]. We rationalized that oxygen may either
quench the radical adducts or quickly react with radical 12 to form
triuret, which will diminish the probability of trapping the radicals.
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oxidation of 8-oxoguanine [22]. Another molecule of peroxynitrite
anion may react with intermediate 9, which is produced by the
reduction of compound 8, to generate the allantoin–peroxo adduct 10.
The multimolar equivalent consumption of peroxynitrite leading to
radical formation was supported by our observation that the yield of
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