Peroxynitrite is the major species formed from different flux ratios of co-generated nitric oxide and superoxide: Direct reaction with boronate-based fluorescent probe

Article abstract of DOI:10.1074/jbc.M110.110080

There is much interest in the nitration and oxidation reaction mechanisms initiated by superoxide radical anion (O₂^.- ) and nitric oxide (^.NO). It is well known that O₂^.- and ^.NOrapidly react to form a potent oxidant, peroxynitrite anion (ONOO^-). However, indirect measurements with the existing probes (e.g. dihydrorhodamine) previously revealed a bell-shaped response to co-generated ^.NO and O_2.^.- fluxes, with the maximal yield of the oxidation or nitration product occurring at a 1:1 ratio. These results raised doubts on the formation of ONOO^- per se at various fluxes of ^.NO and O_2.^.- Using a novel fluorogenic probe, coumarin-7-boronic acid, that reacts stoichiometrically and rapidly with ONOO^- (k = 1.1 x 10⁶ M^-1s ^-1), we report that ONOO^- formation increased linearly and began to plateau after reaching a 1:1 ratio of co-generated ^.NO and O₂^.- fluxes. We conclude that ONOO^- is formed as the primary intermediate during the reaction between ^.NO and O₂^.- co-generated at different fluxes.

Full text of DOI:10.1074/jbc.M110.110080

Signal Transduction:
Peroxynitrite Is the Major Species Formed
from Different Flux Ratios of Co-generated
Nitric Oxide and Superoxide: DIRECT
REACTION WITH BORONATE-BASED
FLUORESCENT PROBE
Jacek Zielonka, Adam Sikora, Joy Joseph and
Balaraman Kalyanaraman
J. Biol. Chem. 2010, 285:14210-14216.
doi: 10.1074/jbc.M110.110080 originally published online March 1, 2010
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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 19, pp. 14210–14216, May 7, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Peroxynitrite Is the Major Species Formed from Different Flux
Ratios of Co-generated Nitric Oxide and Superoxide
□
S
ꢀ
DIRECT REACTION WITH BORONATE-BASED FLUORESCENT PROBE*
Received for publication, February 2, 2010, and in revised form, March 1, 2010 Published, JBC Papers in Press, March 1, 2010, DOI 10.1074/jbc.M110.110080
Jacek Zielonka‡, Adam Sikora‡§, Joy Joseph‡, and Balaraman Kalyanaraman‡1
From the ‡Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
and the §Institute of Applied Radiation Chemistry, Technical University of Lodz, 90-924 Lodz, Poland
ꢁ
ꢁ
There is much interest in the nitration and oxidation reaction
hydroxyl radical ( OH) and nitrogen dioxide ( NO2) (Reaction
.
3, k3 ϭ 1.25 sϪ1) (2, 13).
mechanisms initiated by superoxide radical anion (O2) and
.
ꢁ
ꢁ
nitric oxide ( NO). It is well known that O2 and NO rapidly react
؊
ONOOϪ ϩ Hϩ ^ ONOOH
to form a potent oxidant, peroxynitrite anion (ONOO ). How-
REACTION 2
ever, indirect measurements with the existing probes (e.g. dihy-
drorhodamine) previously revealed a bell-shaped response to
ꢁ
ꢁ
ONOOH 3 HNO3͑ϳ70%͒; OH ϩ NO2͑ϳ30%͒
.
ꢁ
co-generated NO and O2 fluxes, with the maximal yield of the
REACTION 3
oxidation or nitration product occurring at a 1:1 ratio. These
؊
results raised doubts on the formation of ONOO per se at var-
In most biological systems, carbon dioxide is a likely scavenger
.
ꢁ
ious fluxes of NO and O2. Using a novel fluorogenic probe, cou-
Ϫ
of ONOO , yielding a short-lived nitrosoperoxycarbonate
marin-7-boronic acid, that reacts stoichiometrically and rapidly
Ϫ
anion (ONOOCO2 , Reaction 4, k4 ϭ 2.9 ϫ 104 Ϫ1sϪ1 (14)).
M
with ONOO (k ؍ 1.1 ؋ 106 ؊1s؊1), we report that ONOO
M
؊
؊
Ϫ
During the decomposition of ONOOCO2 , nitrate and carbon
dioxide are formed, as well as nitrogen dioxide radical and car-
bonate radical anion (Reaction 5) (2, 13, 15).
formation increased linearly and began to plateau after reaching
.
ꢁ
a 1:1 ratio of co-generated NO and O2 fluxes. We conclude that
؊
ONOO is formed as the primary intermediate during the reac-
.
ONOOϪ ϩ CO2 3 ONOOCOϪ2
ꢁ
tion between NO and O2 co-generated at different fluxes.
REACTION 4
Ϫ
Ϫ
.
Ϫ
Peroxynitrite (ONOO ) is an unstable intermediate formed
ꢁ
ONOOCO2 3 NO3 ϩ CO2͑ϳ65%͒; CO3 ϩ NO2͑ϳ35%͒
from the diffusion-controlled reaction between nitric oxide
REACTION 5
.
ꢁ
( NO) and superoxide radical anion (O2, Reaction 1, k1
ϭ
(0.38 Ϫ 1.6) ϫ 1010
M
Ϫ1sϪ1) (1–6).
Due to the occurrence of Reactions 4 and 5, as well as the scav-
enging by peroxiredoxins or oxyhemoglobin in specific subcel-
.
Ϫ
ꢁ
Ϫ
NO ϩ O2 3 ONOO
lular compartments, the lifetime of ONOO in biological sys-
REACTION 1
tems is limited to only a few milliseconds (2, 13). The current
Ϫ
methodologies for detection of ONOO are based on the
Ϫ
The early indication of the occurrence of this reaction in bio-
logical systems came from the report on the inhibitory effect of
detection of radical species formed from ONOO decomposi-
.
ꢁ
ꢁ
tion, i.e. NO2 and CO3 or OH, using tyrosine that forms nitro-
.
ꢁ
O2 on the activity of endothelium-derived relaxing factor (7).
tyrosine (TyrNO2) as a marker product of intracellular NO2
and dihydrorhodamine 123 (DHR)2 as a fluorogenic probe for
After endothelium-derived relaxing factor identity was estab-
.
.
ꢁ
ꢁ
ꢁ
ꢁ
lished as NO (8, 9), its scavenging by O2 was first proposed as a
oxidants ( NO2, OH, CO3). However, NO2 radical formed from
Ϫ
contributing factor to endothelial injury (10). Reaction 1 has
the ONOO -independent processes, e.g. via myeloperoxidase-
ꢁ
great physiological significance as both NO and hydrogen per-
catalyzed oxidation of nitrite by H2O2 (16), could make data inter-
pretation more tenuous (17, 18). Additional problems with this
indirect approach may arise from alternate mechanisms through
.
oxide (H2O2, the product of dismutation of O2) act as impor-
tant second messengers in redox cell signaling (11, 12).
Ϫ
ꢁ
In the absence of scavengers, ONOO decomposes at neu-
which TyrNO2 can be formed without the involvement of NO2
tral pH via protonation to peroxynitrous acid (pKa ϭ 6.7, Reac-
radicals (19). DHR can be oxidized to the fluorescent rhodamine
molecule by various one-electron oxidants, including compounds
I and II of peroxidases (20, 21).
Ϫ
tion 2) to yield nitrate (NO3 ) and free radical intermediates:
* Thisworkwassupported, inwholeorinpart, byNationalInstitutesofHealth
Grant HL063119 (to B. K.).
ꢀ This article was selected as a Paper of the Week.
2 The abbreviations used are: DHR, dihydrorhodamine 123; CBA, coumarin-7-
boronic acid; CBE, coumarin-7-boronic acid, pinacolate ester; COH, 7-hy-
droxycoumarin; PAPA-NONOate, (Z)-1-[N-(3-ammoniopropyl)-N-(n-pro-
pyl)amino]diazen-1-ium-1,2-diolate; X, xanthine; XO, xanthine oxidase;
SOD, superoxide dismutase; DTPA, diethylenetriaminepentaacetic acid;
HPLC, high pressure liquid chromatography.
□S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. S1–S8, Table S1, and references.
1 To whom correspondence should be addressed: Dept. of Biophysics, Medi-
cal College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226.
Tel.: 414-456-4035; Fax: 414-456-6512; E-mail: balarama@mcw.edu.
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.
؊
ꢁ
Quantitation of ONOO from the Reaction between NO and O2
Science, and PAPA-NONOate ((Z)-1-[N-(3-ammoniopropyl)-
N-(n-propyl)amino]diazen-1-ium-1,2-diolate) was from Cay-
man Co. DHR was from AnaSpec Inc. All other chemicals were
from Sigma-Aldrich and were of highest purity available. All
solutions were prepared using the deionized water (Millipore
Ϫ
Milli-Q system). ONOO was prepared by reacting nitrite with
H2O2, according to the published procedure (31). The concen-
Ϫ
tration of ONOO in alkaline aqueous solutions (pH Ͼ 12) was
determined by measuring the absorbance at 302 nm (⑀ ϭ 1670
M
Ϫ1cmϪ1). The pinacolate ester of coumarin boronic acid
(CBE) was synthesized following the procedure described else-
where (32). CBA was prepared by acidic hydrolysis of CBE.
UV-visible Absorption and Fluorescence Measurements—
The UV-visible absorption spectra were collected using an Agi-
lent 8453 spectrophotometer equipped with a diode array
detector and thermostated cell holder. Fluorescence spectra
were collected using the PerkinElmer Life Sciences LS 55 lumi-
nescence spectrometer. The kinetic absorption and fluores-
cence measurements were carried out at room temperature
FIGURE 1. Scheme showing the conversion of CBA and CBE into the fluo-
rescent product, COH.
Previous reports suggest that oxidative and nitrative modifi-
cations of tyrosine and DHR observed in the presence of co-
.
ꢁ
generated NO and O2 in cell-free and cellular systems dis-
played a characteristic bell-shaped response with maximal
.
ꢁ
response occurring at a 1:1 ratio of NO to O2 (22–26).
Ϫ
Although these findings could implicate that ONOO is
.
ꢁ
using the same instruments.
formed at the maximal yield only at the 1:1 ratio of NO/O2, one
.
ꢁ
ꢁ
Determination of O2 and NO Fluxes— NO fluxes were deter-
mined from the measured rate of the decomposition of PAPA-
NONOate by following the decrease of its characteristic
could also question the interpretations because of the con-
founding effects of radical-radical interactions from free radical
Ϫ
species derived from ONOO decomposition and probe-de-
absorbance at 250 nm (⑀ ϭ 8.1 ꢁ 103
M
Ϫ1cmϪ1). Under the con-
rived radicals (tyrosyl radical or rhodamine radical) (22, 27–29).
Clearly, there is an urgent need for developing direct probe(s)
ꢁ
ditions used, the NO donor decomposed with the rate constant
Ϫ4
Ϫ1sϪ1 as determined at 25 °C. The rate of
Ϫ
of (2.5 Ϯ 0.2) ϫ 10
M
for ONOO that will enable us to understand the chemical and
.
ꢁ
decay of PAPA-NONOate was multiplied by a factor of two to
biological interactions of NO with O2.
ꢁ
obtain the rate of NO release, assuming that two molecules of
We have recently shown that boronic compounds (boronic
Ϫ
ꢁ
NO are released during the decomposition of one molecule of
acids and their esters) react stoichiometrically with ONOO ,
ꢁ
PAPA-NONOate (33). The stoichiometry of NO release was
yielding the corresponding hydroxylated compounds as the
major products (30). We have proposed that boronate groups
attached to the fluorogenic probes may be used in the detection
confirmed by performing the oxyhemoglobin assay (34) under
the conditions of complete decomposition of PAPA-NONOate
(10 and 20 ␮M) in the presence of excess of oxyhemoglobin (60
Ϫ
of ONOO both in cell-free and in cellular systems. As the rate
.
Ϫ
␮M). The flux of O2, generated by xanthine oxidase-catalyzed
constant of the reaction of arylboronates with ONOO is rela-
tively high (ϳ106
M
Ϫ1sϪ1 at pH 7.4), it can outcompete other
oxidation of xanthine to uric acid, was determined by monitor-
ing the ferricytochrome c reduction and the increase in absorb-
ance at 550 nm (using a difference in the values of the extinction
coefficients between reduced and oxidized cytochrome of 2.1 ꢁ
Ϫ
reactions resulting from the decay of ONOO and can be used
Ϫ
to monitor ONOO levels under different conditions.
Here we report the development of a novel fluorescent probe
104 Ϫ1cmϪ1 (35)).
M
Ϫ
for ONOO and resolve a long standing controversy with
Stopped-flow Measurements—Stopped-flow kinetic experi-
ments were performed on Applied Photophysics 18MX
stopped-flow spectrophotometer equipped with photomulti-
pliers for absorption and fluorescence measurements. The
thermostatted cell (25 °C) with a 10-mm optical pathway was
used for kinetic measurements. For determining the rate con-
stant, the reaction was carried out under pseudo first-order
conditions (greater than 10-fold excess of boronate probe over
regard to the identity, reaction profile, and yields of oxidant
.
ꢁ
formed from varying ratios of NO to O2 fluxes. We synthesized
and employed the boronate-based fluorogenic probe, namely
Ϫ
coumarin-7-boronic acid (CBA, Fig. 1), to monitor ONOO
.
ꢁ
formed under varying fluxes of O2 and NO. Contrary to previ-
ous results obtained with tyrosine and DHR probes (22–26), we
Ϫ
report in this study, using a boronate probe, that ONOO is the
.
ꢁ
major species formed under various flux ratios of NO and O2
Ϫ
Ϫ
ONOO ). For the fluorescence measurements, the cut-off fil-
and that there is no bell-shaped response in ONOO forma-
ter (transmitting the light longer than 400 nm) was placed
between the cell and the detector.
tion. We conclude that the bell-shaped response previously
ꢁ
reported during the reaction between co-generated NO and
.
HPLC Analysis—The CBA and 7-hydroxycoumarin (COH)
were separated on an HPLC system Agilent 1100 equipped with
fluorescence and UV-visible absorption detectors. Typically,
100 ␮l of sample was injected into the HPLC system equipped
with a C18 column (Alltech, Kromasil, 250 ϫ 4.6 mm, 5 ␮m)
O2 is due to the free radical chemistry of the probe employed
(tyrosine and dihydrorhodamine), which does not totally reflect
Ϫ
the actual yield of ONOO formation.
EXPERIMENTAL PROCEDURES
Materials—H2O2 was from Fluka, xanthine oxidase (XO), equilibrated with 10% acetonitrile (CH3CN) (containing 0.1%
and superoxide dismutase (SOD) from bovine erythrocytes (v/v) trifluoroacetic acid) in 0.1% trifluoroacetic acid aqueous
were from Roche Diagnostics, catalase was from Roche Applied solution. The compounds were separated by a linear increase in
MAY 7, 2010•VOLUME 285•NUMBER 19
JOURNAL OF BIOLOGICAL CHEMISTRY 14211

.
؊
ꢁ
Quantitation of ONOO from the Reaction between NO and O2
CH3CN phase concentration from 10 to 55% over 15 min at a
flow rate of 1 ml/min. Under those conditions, CBA eluted at
9.5 min, and COH eluted at 10.7 min. The concentrations of
CBA and COH were calculated based on the peak areas
detected by absorptions at 280 and 324 nm, respectively.
Although COH can also be detected with a high sensitivity
using the fluorescence detection (excitation at 332 nm and
emission at 475 nm), at the concentrations used, the absorption
detection was sufficient for reliable quantitation. Additionally,
under the HPLC conditions used, the concentration of uric acid
(2.8 min) and xanthine (3.7 min) could be also quantitated
based on the peak areas detected by monitoring the absorption
at 280 nm.
Kinetic Simulations—The simulation of the inhibitory effect
of SOD on the conversion of CBA into COH and on the steady-
ꢁ
state concentration of NO was carried out using a freely avail-
able software, Kintecus, version 3.95 (36). The kinetic model
used in this study is a modification of the published model of
peroxynitrite decay (37). The list of the chemical reactions, rate
constants, and major modifications used in the simulation is
shown in supplemental Table S1.
RESULTS
FIGURE 2. Fluorescentspectral changes during the reactionbetween CBA
and peroxynitrite. Fluorescence (Fl) spectra (excitation/emission) were
obtained from incubations containing CBA(50 ␮M)and DTPA(50 ␮M) in phos-
phate buffer (0.45 M, pH 7.4) after the addition of various amounts of ONOOϪ
(0–2 ␮M). Emission spectra were collected using excitation at 332 nm; excita-
tion spectra were collected by monitoring the emission intensity at 451 nm.
Insets, HPLC traces recorded from the incubation mixtures containing CBA
(100 ␮M), before (control, top trace) and after (bottom trace) the addition of
ONOOϪ (20 ␮M). The left panel represents the signal detected using the
absorption (Abs) detector set at 300 nm; the right panel represents the signal
Ϫ
Oxidation of Coumarin Boronate by ONOO and H2O2—
First, we investigated the stoichiometry and kinetics of the reac-
Ϫ
tion between CBA and ONOO or H2O2. Both oxidants con-
verted the boronate probe into a fluorescent product that was
visually examined under UV light illumination (supplemental
Fig. S1). The UV-visible absorption spectra (supplemental Figs. S2
and S3) of the product formed in both reactions indicated the for- detected using the fluorescence detector with the excitation set at 332 nm
and emission at 470 nm. HPLC peaks at 9.5 and 10.7 min correspond to CBA
and COH, respectively. a.u., arbitrary units.
mation of a single species with spectral characteristics similar to
that of COH. The fluorescence spectra observed upon oxidation of
Ϫ
CBA by ONOO were consistent with the formation of COH as
the major product (Fig. 2). Moreover, the intensity of both the
excitation and the emission bands increased linearly with increas-
Ϫ
ing ONOO concentration (Fig. 2). The identity of the product
was confirmed by HPLC analysis (Fig. 2, insets, and
supplementalFig. S4), showingthattheproductco-elutedwiththe
authentic standard, 7-hydroxycoumarin, under identical HPLC
conditions. The HPLC analysis enabled us to determine the stoi-
chiometry of the reaction, indicating that one molecule of CBA
Ϫ
reacts with a molecule of ONOO , producing COH with the over-
all yield of ϳ81% (Fig. 3 and supplemental Fig. S4). This finding is
similar to what has been previously reported for 4-acetylphenyl
and phenylalanine-4-boronic acids (30).
As can be seen in supplemental Fig. S1, the fluorescence of
the solutions was observed immediately after mixing with
Ϫ
ONOO ; however, no significant fluorescence was observed
even 30 min after mixing with H2O2, indicating that the reac-
tion was rather slow. As both oxidants resulted in the formation
FIGURE 3. Stoichiometry of the reaction between CBA and ONOO؊. The
of the same fluorescent product, this is attributed to vastly dif-
concentrations of CBA and COH calculated from HPLC data were obtained
from incubations containing CBA (100 ␮M), 20 ␮M DTPA, and ONOOϪ (0–200
ferent rates of oxidation of the probe. We monitored the reac-
␮M) in phosphate buffer (0.1 M, pH 7.4). Error bars indicate S.D.
tion progress with both oxidants by following the changes in the
UV-visible absorption spectra and the increase in the fluores-
cence intensity during oxidation of CBA to COH. The rate con- As shown in Fig. 4, the disappearance of the absorption band
stant of 1.5 Ϯ 0.2 MϪ1sϪ1 was determined for the reaction with responsible for CBA (as monitored at 286 nm) was accompa-
Ϫ
H2O2 at pH 7.4 (supplemental Fig. S5). With ONOO , the nied by the build-up of the absorption (monitored at 370 nm)
stopped-flow technique was used to measure the rate constant. and fluorescence (excitation at 332 nm, emission at Ͼ 400 nm)
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.
؊
ꢁ
Quantitation of ONOO from the Reaction between NO and O2
uric acid formed from xanthine oxidation interfered with tyro-
sine nitration assay (38). However, the reaction between
Ϫ
ONOO and CBA outcompetes not only the self-decomposi-
Ϫ
tion of ONOO but also its reaction with uric acid (the
reported value of the apparent rate constant for the reaction of
urate monoanion with ONOOH is 155 MϪ1sϪ1 at pH 7.4 (39)).
The slower reaction between CBA and H2O2 generated by XO
.
and by dismutation of O2 was completely mitigated using the
catalase enzyme. The oxidation of CBA to COH occurred only
.
ꢁ
in the presence of both O2 and NO, and virtually no oxidation
of the probe into fluorescent product was observed in the pres-
.
ꢁ
ence of either O2 or NO alone (Fig. 5). This is attributed to the
Ϫ
reaction between CBA and ONOO formed in situ. These
results suggest that it is feasible to monitor in “real time” the
Ϫ
formation of ONOO , using the boronate probe.
To investigate the relative contribution of H2O2 and
Ϫ
ONOO in the conversion of CBA into COH in this system, we
tested the effect of catalase and SOD on the fluorescence
increase in incubations containing X/XO system with or with-
ꢁ
ꢁ
out NO donor (Fig. 6). In the absence of the NO donor, the
fluorescence signal was inhibited by catalase but not by SOD.
This indicates that H2O2 is the primary oxidant responsible for
the observed increase in the fluorescence intensity obtained
FIGURE 4. Kinetics of the reaction between CBA and ONOO؊. A–C,
stopped-flow kinetic traces obtained after mixing CBA (20 ␮M) and ONOOϪ
(10 ␮M) in phosphate buffer (0.25 M, pH 7.4), using the absorption detector at
286 nm (A) and 370 nm (B) or using the fluorescence detector (C, excitation at
332 nm, emission at Ͼ 400 nm). a.u., arbitrary units. D, the dependence of the
pseudo first-order rate constant of COH formation (as detected by fluores-
cence) on the CBA concentration; [ONOOϪ] ϭ 0.1 ␮M.
ꢁ
ꢁ
in the absence of the NO donor. The addition of the NO donor
to the incubation caused an increase in the rate of formation of
the fluorescence product, which was partly inhibited by SOD
and not by catalase. This observation suggests that the oxida-
.
ꢁ
tion of CBA to COH was H2O2-independent and O2- and NO-
dependent oxidant formation in this system. In the presence of
ꢁ
the NO donor, maximal inhibition was observed when both
catalase and SOD were present in the system, indicating that
H2O2 produced by SOD is also contributing to the oxidation of
CBA to COH. For the quantitative analysis of the amount of
CBA consumed and COH formed, we used the HPLC method.
As can be seen in Fig. 6C, the effect of SOD and catalase on the
amount of COH formed during a 30-min incubation period
closely followed the fluorescence results. The concentration of
ꢁ
COH formed (50 ␮M) in incubations containing the NO donor
Ϫ
and X/XO system is lower than the theoretical yield of ONOO
(66 ␮M, based on the assumption of a steady flux of 2.2 ␮M/min
.
ꢁ
of O2 and NO), giving the net yield of 76%, which is reasonably
close to ϳ81% detected in the presence of a bolus addition of
Ϫ
FIGURE 5. Detection of ONOO؊ formed from co-generated superoxide
and nitric oxide fluxes. The fluorescence intensity (excitation at 332 nm,
emission at 450 nm) of the reaction mixtures consisting of CBA (100 ␮M) with
or without X/XO (O2 flux: 2 ␮M/min) and PAPA-NONOate ( NO flux: 2 ␮M/min)
was measured over a period of 30 min. The reaction mixtures contained cat-
alase (0.1 kU/ml) and DTPA (10 ␮M) in phosphate buffer (100 mM, pH 7.4). a.u.,
arbitrary units.
ONOO .
.
ꢁ
Effect of Variation of NO/O2 Flux Ratios on the Yield of
Ϫ
.
ꢁ
ONOO —The fluorescence intensity changes were monitored
over a 5-min period in incubation mixtures containing CBA
.
ꢁ
and a constant flux of O2 (2 ␮M/min) and different fluxes of NO
ꢁ
(0–12 ␮M/min) by varying the concentrations of NO donor,
ꢁ
bands for COH. Based on the rate of the product build-up, the PAPA-NONOate (Fig. 7A). At low rates of NO generation (Ͻ2
rate constant of (1.1 Ϯ 0.2) ϫ 106
M
Ϫ1sϪ1 was calculated for ␮M/min), the rate of increase in the fluorescence intensity was
Ϫ
Ϫ
ꢁ
ꢁ
oxidation of CBA by ONOO . Thus, ONOO reacts with the proportional to the NO flux as NO concentration was the lim-
Ϫ
ꢁ
coumarin boronate at least a million times faster than H2O2.
iting factor in ONOO generation. At rates of NO generation
.
ꢁ
Oxidation of Coumarin Boronate by Co-generated NO and higher than that of O2 (Ͼ2 ␮M/min), the rate of fluorescence
.
O2—To investigate whether the same probe can be oxidized increase reached a plateau and remained unchanged up to a
.
ꢁ
ꢁ
similarly by NO and O2 generated simultaneously, we moni- 6-fold excess of NO (Fig. 7B). At longer duration of incubation
.
ꢁ
tored the rate of oxidation of CBA to COH in incubation mix- (30 min) and gradually increasing ratios of NO to O2 fluxes
tures containing xanthine (X) and XO as a source of steady flux from 1 to 8, a slow decline in the yield of COH was noted as
.
ꢁ
of O2 and PAPA-NONOate as a source of NO flux (Fig. 5). The determined by HPLC (Fig. 7C). However, we also observed that
MAY 7, 2010•VOLUME 285•NUMBER 19
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.
؊
ꢁ
Quantitation of ONOO from the Reaction between NO and O2
ꢁ
under excess of NO, the extent of conversion of xanthine into
ꢁ
uric acid was also decreased with increasing NO flux (Fig. 7C).
ꢁ
This may be due to NO-dependent inhibition of the enzyme
.
(40–42). Assuming that the rate of O2 generation is propor-
tional to XO activity, and therefore, to the rate (and the yield) of
uric acid formation, we normalized the amount of COH (“cor-
.
rected”) to the yield of uric acid (and thus to O2). In the pres-
.
ꢁ
ence of excess NO, the rate of O2 production was the limiting
factor in COH formation. The dependence of the corrected
.
ꢁ
yield of COH on the ratio of NO/O2 fluxes is shown in Fig. 7D.
The HPLC data are in agreement with the fluorescence results,
Ϫ
indicating that the yield of ONOO is constant under condi-
.
ꢁ
tions of NO formation rate that exceeds the rate of O2 produc-
Ϫ
tion by 8-fold. From these results, we conclude that ONOO is
formed as a major species during simultaneous generation of
.
Ϫ
ꢁ
NO and O2 and that ONOO formation increased linearly up
.
ꢁ
to a 1:1 ratio of NO/O2 flux, which then began to plateau. In
contrast to results shown in Fig. 7, different results were
obtained when DHR was used as a detection probe for
Ϫ
ONOO . The rate of fluorescence increase of rhodamine 123
.
ꢁ
formed from DHR was monitored at different NO/O2 ratios
(supplemental Fig. S6). In agreement with the previous
reports (23–26), a bell-shaped response was observed under
these conditions. Clearly, the DHR-based detection method
Ϫ
is not reliable for quantitative analysis of ONOO formed
.
ꢁ
from NO/O2 reaction.
DISCUSSION
CBE has previously been reported to react with H2O2, result-
ing in the formation of a highly fluorescent COH (umbellifer-
one) (32). In this study, we show that coumarin-7-boronic acid
Ϫ
reacts rapidly and stoichiometrically with ONOO to give
COH as the major product (ϳ81%). This is similar to what had
been published with simple arylboronates, indicating that the
Ϫ
reaction between boronates and ONOO is quite general (30).
Ϫ
The rate of reaction between CBA and ONOO is at least a
million times faster than between CBA and H2O2, and even at
low micromolar concentrations, the boronate probes can effec-
Ϫ
tively compete with the self-decomposition of ONOO at neu-
tral pH. To our knowledge, this is the first report using a fluoro-
genic probe that reacts directly and stoichiometrically with
Ϫ
Ϫ
ONOO . The probe can successfully scavenge ONOO added
.
ꢁ
as a bolus or formed from co-generated NO and O2, thus allow-
Ϫ
ing for real-time monitoring of ONOO formation in biologi-
cal systems.
To confirm the identity of the oxidant trapped, we tested the
effects of SOD and catalase on the yield of the fluorescent prod-
uct formation (Fig. 6). The results are consistent with the oxi-
dation of the probe by an oxidant formed from interaction of
FIGURE 6. Detection of ONOO؊ formed from co-generated superoxide
and nitric oxide fluxes; effects of superoxide dismutase and catalase
(CAT). A, the time course of the fluorescence intensity increase (excitation at
332 nm, emission at 450 nm) measured in reaction mixtures containing CBA
(100 ␮M) and DTPA (10 ␮M) in phosphate buffer (100 mM, pH 7.4) with or
without X (200 ␮M), XO (O2 flux: 1.2 ␮M/min), and PAPA-NONOate ( NO flux:
2.7 ␮M/min). B, effects of SOD (1 mg/ml) and catalase (100 units/ml) on the
total change in the fluorescence intensity after 30 min of incubation. Error
bars indicate S.D. a.u., arbitrary units. C, COH concentrations in the reaction
.
ꢁ
NO and O2. From our earlier studies, we can conclude that
Ϫ
ꢁ
ꢁ
ONOO but not nitrogen oxides ( NO, NO2) is responsible for
oxidation of the boronate probe (30). The lack of a total inhibi-
tion of the oxidation by SOD, even in the presence of catalase
.
ꢁ
and at a high concentration of SOD (up to 1 mg/ml), can be
ꢁ
ꢁ
mixtures measured by HPLC after a 30-min incubation under similar condi- explained by the dynamic competition between NO and SOD
.
ꢁ
.
tions but with NO and O2 fluxes of 2.2 ␮M/min and SOD concentration of 0.2
mg/ml. Open and solid symbols/bars represent the values obtained from mix-
tures in the absence and presence of PAPA-NONOate, respectively.
for O2 as follows. SOD effectively competes with NO in remov-
.
ꢁ
ing O2, causing a rise in the steady-state concentration of NO
.
that can again effectively compete with SOD for O2 (43, 44).
14214 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 19•MAY 7, 2010

.
؊
ꢁ
Quantitation of ONOO from the Reaction between NO and O2
.
FIGURE 7. Detection of ONOO؊ formed from co-generated superoxide and nitric oxide fluxes; effects of varying NO/O2 ratios on product formation.
A, the time course of the increase in the fluorescence intensity (excitation at 332 nm, emission at 450 nm) obtained at room temperature from the incubation
.
mixtures containing CBA (100 ␮M), 10 ␮M DTPA, 100 units/ml catalase, xanthine (200 ␮M), XO (O2 flux: 2 ␮M/min), and different concentrations of PAPA-
ꢁ
ꢁ
NONOate( NOflux:0–12␮M/min)inphosphatebuffer(0.1M andpH7.4). NOfluxes(␮M/min)are:a, 0;b, 0.75;c, 1.5;d, 2.25;e, 3;f, 6;g, 9;h, 12. a.u., arbitraryunits.
ꢁ
B, rate of increase in the fluorescence intensity versus NO flux. Error bars indicate S.D. C, HPLC measurements of COH and uric acid formed after 30 min of
incubation under similar conditions but with an O2 flux of 2.2 ␮M/min. D, the concentration of COH after correcting for the inactivation of the enzyme, XO. Note
.
that in panels C and D, the x axis scale is not linear.
This proposal was indeed confirmed by the kinetic simulations nitration declined drastically with increasing flux of either spe-
of the inhibitory effects of SOD, yielding the same degree of cies (22, 24, 45). The net effect is a bell-shaped response of the
.
ꢁ
inhibition of CBA oxidation by the NO/O2 system, as noted yield of reaction product versus the flux of one species at the
experimentally (supplemental Figs. S7 and S8). Additionally, constant flux of the other co-reactant. A bell-shaped response
ꢁ
the kinetic simulations predicted an increase in the steady-state in DHR oxidation in the presence of various ratios of NO to
.
ꢁ
NO levels upon the addition of SOD, as reported previously O2 fluxes in cellular systems was also reported (26). These find-
(43). The reaction pathways involved in the oxidation of CBA to ings called into question the nature of the identity of the oxi-
.
ꢁ
COH and the inhibitory effects of SOD and catalase in the pres- dant formed from the reaction of NO with O2 under various
ent study are shown in Fig. 8.
ratios of both species. The lack of availability of a chemical
We used the coumarin boronate to resolve the long standing probe that would react quickly and stoichiometrically with
Ϫ
Ϫ
controversy regarding the yield of ONOO , formed under dif- ONOO hampered the progress in the understanding of the
.
.
ꢁ
ꢁ
ferent fluxes of NO and O2. Previous studies indicate that in chemistry of the interaction of NO with O2. Both tyrosine and
cell-free systems, tyrosine nitration/oxidation and dihydrorho- dihydrorhodamine react with the free radical product(s) of
Ϫ
Ϫ
damine oxidation are maximal only when the rates of genera- ONOO decomposition rather than with ONOO itself, com-
.
ꢁ
tion of NO to O2 are equal and that the probe oxidation and plicating the analysis of the primary events in the interaction
MAY 7, 2010•VOLUME 285•NUMBER 19
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.
؊
ꢁ
Quantitation of ONOO from the Reaction between NO and O2
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.
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.
.
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.
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