Nitroxyl (HNO) reacts with molecular oxygen and forms peroxynitrite at physiological ph: Biological implications

Article abstract of DOI:10.1074/jbc.M114.597740

Nitroxyl (HNO), the protonated one-electron reduction product of NO, remains an enigmatic reactive nitrogen species. Its chemical reactivity and biological activity are still not completely understood. HNO donors show biological effects different from NO donors. Although HNO reactivity with molecular oxygen is described in the literature, the product of this reaction has not yet been unambiguously identified. Here we report that the decomposition of HNO donors under aerobic conditions in aqueous solutions at physiological pH leads to the formation of peroxynitrite (ONOO^-) as a major intermediate. We have specifically detected and quantified ONOO^- with the aid of boronate probes, e.g. coumarin-7-boronic acid or 4-boronobenzyl derivative of fluorescein methyl ester. In addition to the major phenolic products, peroxynitrite-specific minor products of oxidation of boronate probes were detected under these conditions. Using the competition kinetics method and a set of HNO scavengers, the value of the second order rate constant of the HNO reaction with oxygen (k = 1.8 × 10⁴ M^-1 S^-1) was determined. The rate constant (k = 2 × 10⁴ M^-1 S^-1) was also determined using kinetic simulations. The kinetic parameters of the reactions of HNO with selected thiols, including cysteine, dithiothreitol, N-acetylcysteine, captopril, bovine and human serum albumins, and hydrogen sulfide, are reported. Biological and cardiovascular implications of nitroxyl reactions are discussed.

Full text of DOI:10.1074/jbc.M114.597740

Molecular Biophysics:
Nitroxyl (HNO) Reacts with Molecular
Oxygen and Forms Peroxynitrite at
Physiological pH: BIOLOGICAL
IMPLICATIONS
Renata Smulik, Dawid Debski, Jacek
Zielonka, Bartosz Michalowski, Jan Adamus,
Andrzej Marcinek, Balaraman Kalyanaraman
and Adam Sikora
J. Biol. Chem. 2014, 289:35570-35581.
doi: 10.1074/jbc.M114.597740 originally published online November 5, 2014
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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 51, pp. 35570–35581, December 19, 2014
2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
©
Nitroxyl (HNO) Reacts with Molecular Oxygen and Forms
Peroxynitrite at Physiological pH
□
S
*
BIOLOGICAL IMPLICATIONS
Received for publication, July 17, 2014, and in revised form, November 4, 2014 Published, JBC Papers in Press, November 5, 2014, DOI 10.1074/jbc.M114.597740
‡
‡
§
‡
‡
‡
Renata Smulik , Dawid D e˛ bski , Jacek Zielonka , Bartosz Michałowski , Jan Adamus , Andrzej Marcinek ,
§
1
‡2
Balaraman Kalyanaraman , and Adam Sikora
From the Institute of Applied Radiation Chemistry, Lodz University of Technology, 90-924 Lodz, Poland and the Department of
Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
‡
§
Background: Nitroxyl (HNO) is a reactive nitrogen species implicated in cardioprotection.
Results: Nitroxyl reacts with oxygen to form an oxidizing and nitrating species, peroxynitrite.
Conclusion: In the presence of oxygen, HNO donors may be a source of peroxynitrite.
Significance: Peroxynitrite formation should be taken into account in the extracellular milieu when exposing cells to HNO
donor under aerobic conditions.
Nitroxyl (HNO), the protonated one-electron reduction
Nitroxyl (1, 2) (HNO; nitrosyl hydride in the IUPAC nomen-
3
product of NO, remains an enigmatic reactive nitrogen species. clature), the protonated form of one-electron reduction prod-
ꢀ
Its chemical reactivity and biological activity are still not com- uct of nitric oxide ( NO), still remains a mysterious reactive
pletely understood. HNO donors show biological effects differ- nitrogen species. In the past, its chemistry received much less
ent from NO donors. Although HNO reactivity with molecular attention than the chemistry of other reactive nitrogen species
ꢀ
ꢀ
(
(
e.g. NO, nitrogen dioxide radical ( NO ), and peroxynitrite
oxygen is described in the literature, the product of this reaction
has not yet been unambiguously identified. Here we report that
the decomposition of HNO donors under aerobic conditions in
aqueous solutions at physiological pH leads to the formation of
2
Ϫ
ONOO )).
Significant and important pharmacological effects associated
with the use of nitroxyl donors have been reported. It has been
shown that HNO generated from Angeli’s salt acts as a vasore-
laxant (3–8). We reported that inclusion of Angeli’s salt in car-
dioplegic solution improved myocardial functional recovery
during ischemia and reperfusion in isolated rat hearts (9, 10).
؊
peroxynitrite (ONOO ) as a major intermediate. We have spe-
؊
cifically detected and quantified ONOO with the aid of boro-
nate probes, e.g. coumarin-7-boronic acid or 4-boronobenzyl
derivative of fluorescein methyl ester. In addition to the major
phenolic products, peroxynitrite-specific minor products of
oxidation of boronate probes were detected under these condi-
tions. Using the competition kinetics method and a set of HNO
scavengers, the value of the second order rate constant of the
ꢀ
We attributed cardioprotection to either nitric oxide ( NO) or
Ϫ
nitrous oxide (N O) generated from nitroxyl anion (NO ).
2
Because nitronyl nitroxides (also PTIO) reversed this cardio-
ꢀ
protective effect, we proposed that NO generated directly or
indirectly from Angeli’s salt was responsible for vasorelaxation
and cardioprotection. Additional research is needed to increase
our understanding of the chemical biology and reactivity of
HNO (11, 12). Several studies examining HNO in vitro demon-
strated that Angeli’s salt (HNO donor) exerts marked cytotox-
icity that is oxygen-dependent (13–16).
4
؊1 ؊1
HNO reaction with oxygen (k ؍ 1.8 ؋ 10
M
s
s
) was deter-
4
؊1 ؊1
mined. The rate constant (k ؍ 2 ؋ 10
M
) was also deter-
mined using kinetic simulations. The kinetic parameters of the
reactions of HNO with selected thiols, including cysteine,
dithiothreitol, N-acetylcysteine, captopril, bovine and
human serum albumins, and hydrogen sulfide, are reported.
The endogenous production of HNO in mammalian systems
Biological and cardiovascular implications of nitroxyl reac- has not been unambiguously shown to occur; however, there is
tions are discussed.
considerable in vitro evidence for the intermediacy of this spe-
cies from HNO donors. It has been suggested that HNO can be
␻
ꢀ
formed from NO synthases via the oxidation of N -hydroxy-L-
*
This work was supported, in whole or in part, by National Institutes of Health
Grant R01 HL073056 (to B. K.). This work was also supported by Grant
IP2011049271 from the Polish Ministry of Higher Education within the Iuven-
tus Plus program and by Grant POIG.01.01.02-00-069/09 from Jagiellonian
CentreforExperimentalTherapeutics(supportedbytheEuropeanUnionfrom
the resources of the European Regional Development Fund under the Inno-
vative Economy Programme).
S
This article contains supplemental Table S1 and supplemental references.
To whom correspondence may be addressed: Dept. of Biophysics, Medical
College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226.
Tel.: 414-955-4000; Fax: 414-955-6512; E-mail: balarama@mcw.edu.
To whom correspondence may be addressed: Inst. of Applied Radiation
Chemistry, Lodz University of Technology, Zeromskiego 116, 90-924 Lodz,
Poland. Tel.: 48-42-631-31-70; E-mail: adam.sikora@p.lodz.pl.
3
The abbreviations used are: HNO, nitroxyl; CBA, coumarin-7-boronic acid; COH,
7-hydroxycoumarin; CPTIO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazo-
line-1-oxyl-3-oxide; DPTA-NONOate, ((Z)-1-[N-(3-aminopropyl)-N-(3-ammo-
niopropyl)-amino]diazen-1-ium-1,2-diolate); dtpa, diethylenetriaminepenta-
acetic acid; FBBE, 7-benzylboronic derivative of fluorescein methyl ester; FME,
□
fluorescein methyl ester; iPrOH, isopropanol; MitoPh, benzyltriphenylphos-
1
Ϫ
phonium cation; ONOO , peroxynitrite, p-MitoPhB(OH) , (4-boronobenzyl)-
2
triphenylphosphonium cation; p-MitoPhOH, (4-hydroxybenzyl)-triphenyl-
phosphonium cation; p-MitoPhNO , (4-nitrobenzyl)-triphenylphosphonium
2
2
cation; PMT, photomultiplier; PTIO, 2-phenyl-4,4,5,5-tetramethylimidazoline-
1-oxyl-3-oxide; TEMPOL, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl;
UPLC, ultra performance liquid chromatography.
3
5570 JOURNAL OF BIOLOGICAL CHEMISTRY
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Peroxynitrite Formed from HNO Reaction with Molecular Oxygen
molecule in biological systems (30–32). Moreover, HNO is
capable of reacting with metals and metalloproteins (e.g. super-
oxide dismutase, HRP, catalase, and cytochrome c) (33–39).
Although the high reactivity of HNO toward cellular thiols and
metal centers could classify this species as a potential signaling
molecule (32), the lack of information concerning its endoge-
nous biosynthetic pathways questions its biological relevance.
Nitroxides and nitronyl nitroxides are also commonly used
HNO scavengers (36, 40, 41).
Ϫ
One of the most intriguing aspects of nitroxyl (HNO/NO )
chemistry is its reaction with molecular oxygen (42–46). It is
commonly accepted that the reaction between nitroxyl anion
Ϫ
(
NO ) and molecular oxygen leads to the formation of
Ϫ
ONOO (45, 46). Despite numerous studies on the reactivity of
HNO with molecular oxygen, the product of this reaction has
not been unequivocally identified (42, 44). The possibility of
peroxynitrite formation from the reaction between HNO and
O remained skeptical and understudied (42, 44). This is, in
2
part, due to the lack of availability of specific probes that
Ϫ
directly react with ONOO to form specific diagnostic prod-
Ϫ
ucts. Here, we present unambiguous evidence for ONOO for-
mation during the reaction between HNO and molecular oxy-
gen using the recently developed assays for specific detection
and quantification of peroxynitrite (47, 48).
We have reported previously that boronic acids and their
Ϫ
6
Ϫ1
esters react directly and rapidly with ONOO (k ϭ 10
M
FIGURE 1. Chemical structures of HNO donors (A) and the boronate
probes (B) used in this study.
Ϫ1
s
), with the formation of corresponding phenols as major
products (85–90%) (49). Although boronates also react with
arginine (17–19). Moreover, it has been shown in vitro that
Ϫ1 Ϫ1
H O , the rate of reaction is rather sluggish (k ϭ ϳ1 M
s
).
2
2
ꢀ
HNO can be formed via the reduction of NO by mitochondrial
An array of pro-fluorescent boronate-based probes has been
cytochrome c, xanthine oxidase, or ubiquinol (2, 20–22).
Reports indicate that HNO is also presumably formed from
nitrosothiol reaction with other thiols or ascorbate (23, 24).
Although based on kinetic considerations, their biological rel-
evance remains doubtful (23, 24).
reported, providing now the opportunity for noninvasive, real
Ϫ
time monitoring and imaging of ONOO in cell-free and cel-
lular systems (50–54). In this study two profluorescent probes
(
CBA and FBBE) and a nonfluorescent probe, phenylboronic
acid with a bulky triphenylphosphonium group (p-MitoPhB
More recently, new fluorescent probes for quantitative
detection of HNO in aqueous solution were reported (25, 26).
Ϫ
(
OH) ) (47) were used (Fig. 1B). The reaction between ONOO
2
Ϫ
and boronate leads to the formation of ONOO adduct, fol-
lowed by the cleavage of the O-O bond, which may proceed via
a nonradical, major pathway (heterolytic cleavage of O-O bond)
or radical-mediated, minor pathway (homolytic cleavage of the
O-O bond). The minor pathway yields unique nitrated products
Ϫ
The ground state of HNO is a singlet, but NO anion, which is
formed upon HNO deprotonation, similarly to the isoelec-
tronic O molecule, has the triplet ground state. HNO is a weak
2
acid (pK ϭ 11.4) (27), and its deprotonation is a spin-forbidden
a
reaction and an exceptionally slow process. It has been reported
Ϫ
that are highly specific for ONOO reaction with boronate. The
Ϫ
that nitroxyl anion (NO ) reacts very quickly with molecular
Ϫ
detailed mechanism of the reaction of boronates with ONOO
oxygen forming peroxynitrite.
has been recently reported (47, 48). We demonstrated that the
formation of the products of both major and minor pathways in a
specific ratio can be used as a “peroxynitrite fingerprint” and
applied this strategy to unambiguously determine the identity of
the product of the reaction of HNO with molecular oxygen.
Ϫ
Ϫ
NO ϩ O ¡ ONOO
2
REACTION 1
In the absence of scavengers, HNO spontaneously dimerizes in
aqueous solutions with the second order rate constant of k ϭ
EXPERIMENTAL PROCEDURES
6
Ϫ1 Ϫ1
8
ϫ 10
M
s
to yield hyponitrous acid, which upon water
Chemicals—Boronate probes CBA and p-MitoPhB(OH)
2
elimination yields N O (28).
2
were synthesized according to the published procedure (50, 55).
FBBE was synthesized by benzylation of fluorescein methyl
ester (FME) with the use of 4-(iodomethyl)phenylboronic acid
2
HNO ¡ ͓HONNOH͔ ¡ N O ϩ H O
2 2
REACTION 2
4
pinacol ester. In all experiments Angeli’s salt was used as a
The most frequently used HNO donors are Angeli’s salt and
Piloty’s acid or its derivatives (29) (Fig. 1A). HNO is highly reac-
tive toward thiols, which are considered major targets of this
4
K. D e˛ bowska, D. D e˛ bski, B. Michałowski, J. Adamus, and A. Sikora, manu-
script in preparation.
DECEMBER 19, 2014•VOLUME 289•NUMBER 51
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Peroxynitrite Formed from HNO Reaction with Molecular Oxygen
HNO donor, synthesized according to the published procedure quantified by monitoring the absorption at 300 Ϯ 5 nm for CBA
(
29). The concentration of Angeli’s salt stock solutions was solutions and 268 Ϯ 5 nm for p-MitoPhB(OH) solutions,
2
determined by measuring the absorbance at 248 nm (⑀ ϭ 8.3 ϫ respectively. The mass spectrometer was operated in W mode
3
Ϫ1 Ϫ1
1
0
M
s
) (29) in 1 mM aqueous NaOH solutions. Another with a lock mass correction. Lock mass solution (leucine-en-
ϩ ϩ
HNO donor, 2-bromo-N-hydroxybenzenesulfonamide, was kephalin, reference mass: [M ϩ H]
556.2771; [M ϩ H]
C12
C13
synthesized according to the published procedure (56). Per- 557.2801) was prepared at a concentration of 0.5 ng/␮l in 50:50
oxynitrite was prepared according to the published procedure (v/v), water/acetonitrile solution and stored in 4 °C until further
(
57), by reacting nitrite with H O . The concentration of use. Data acquisition was performed using a MassLynx 4.1 data
2
2
Ϫ
ONOO was determined by measuring the absorbance at 302 software (Waters).
3
Ϫ1 Ϫ1
nm (⑀ ϭ 1.7 ϫ 10 M
s
) (57) in 1 mM aqueous NaOH solu-
Deoxygenation and Oxygenation of CBA and Angeli’s Salt
tions. Stock solution of hydrogen sulfide was prepared by dis- Solution—Samples consisting of 250 ␮M CBA and 200 ␮M
solving sodium sulfide in water. HNO scavengers, and all other Angeli’s salt were incubated in phosphate buffer (50 mM, pH
reagents (of the highest purity available) were obtained from 7.4) containing dtpa (100 ␮M) for 90 min at 25 °C under vacuum
Sigma-Aldrich, whereas DPTA-NONOate was purchased from or purged with O to obtain anaerobic and O -saturated solu-
2
2
Cayman Chemicals, and catalase was from Boehringer. Each tions, respectively.
.
ꢀ
ꢀ
solution was prepared using deionized water (Millipore Milli-Q
system).
Determination of O and NO Fluxes— NO fluxes were deter-
2
mined from the measured rate of the decomposition of DPTA-
UV-visible Absorption Measurements—The UV-visible NONOate by following the decrease in its characteristic
3
Ϫ1 Ϫ1
absorption spectra were collected with Agilent 8453 spectro- absorbance at 250 nm (⑀ ϭ 8 ϫ 10 M
s
) (58, 59). This rate
ꢀ
photometer equipped with photodiode array detector and ther- was multiplied by a factor of 2 to obtain the rate of NO release
ꢀ
mostated (25 °C) cell holder.
(assuming that two molecules of NO are released from one
.
Stopped Flow Measurements—Stopped flow kinetics experi- molecule of DPTA-NONOate). The flux of O was determined
2
ments were performed using Applied Photophysics SX 20 by monitoring of cytochrome reduction following the increase
stopped flow spectrophotometer equipped with a fluorescence in absorbance at 550 nm (using molar absorption coefficient of
4
Ϫ1 Ϫ1
detector. Angeli’s salt (3 ␮M in 1 mM NaOH) was rapidly mixed 2.1 ϫ 10
M
s
) (60).
with the solution containing CBA (25 ␮M), phosphate buffer (25
Kinetic Simulations—The kinetic simulation was carried out
mM, pH 7.4), dtpa (50 ␮M), 5% CH CN, and HNO scavenger (at using a freely available software, Kintecus 4.55 (61). The kinetic
3
an appropriate concentration). The reaction mixtures were model used in this study is a modification of the published
excited at 332 nm, and the emitted light intensity was measured model (62). The list of the chemical reactions, rate constants,
at 470 nm (PMT voltage ϭ 850 V, emission/excitation slit ϭ 2.5 and major modifications used in the simulation is shown in
nm). The thermostated cell (25 °C) with a 10-mm optical path- supplemental Table S1.
way was used for kinetic measurements.
RESULTS
Fluorescence Measurements—Fluorescence measurements
were performed using Varian Cary Eclipse spectrofluorometer
The Oxidation of CBA Probe in O -saturated Angeli’s Salt
2
equipped with a plate reader accessory. The solutions contain- Solutions—Angeli’s salt slowly decomposed at pH 7.4, releasing
ing FBBE were excited at 494 nm, and the emitted light intensity HNO. As shown in Fig. 2A, its decay resulted in the disappear-
was measured at 518 nm (PMT voltage ϭ 580 V, emission slit ϭ ance of the absorption band maximum at 235 nm. In the pres-
2
.5 nm, excitation slit ϭ 5 nm). Reactions were performed at ence of CBA the decomposition of Angeli’s salt was accompa-
room temperature. Typically, the boronate probe (25 ␮M) was nied by 7-hydroxycoumarin (COH) formation as reflected by
incubated in phosphate buffer (50 mM, pH 7.4), dtpa (100 ␮M), the disappearance of the absorption band of boronate probe at
5
% CH CN, and HNO donor and scavenger (at the appropriate 287 nm and the build-up of absorption of COH at 370 nm (Fig.
3
concentration), and the extent of FBBE oxidation to FME was 2B). The kinetics of CBA decay and COH appearance mirror
monitored over time.
the dynamics of Angeli’s salt decomposition observed in the
UPLC/MS Analyses—Boronate probes and products of their absence of CBA (Fig. 2C). These data indicate that decomposition
oxidative conversion were analyzed using Acquity UPLC of Angeli’s salt resulted in the formation of an oxidant capable of
(
Waters) system coupled online with LCT Premier XE (Waters) rapid conversion of CBA probe into COH. The other HNO donor,
mass spectrometer with a time-of-flight mass detector. One 2-bromo-N-hydroxybenzenesulfonamide, also oxidized CBA in
microliter of sample was injected into the UPLC system the same manner as Angeli’s salt (data not shown).
Ϫ
equipped with the C column (Waters Aquity BEH C18, 100 ϫ
To test the possibility of the formation of ONOO via the
18
2
.1 mm, 1.7 ␮m) maintained at 40 °C and equilibrated with 30% reaction of HNO with O , it was essential to confirm that
2
CH CN (containing 0.1% (v/v) TFA) in 0.1% TFA solution. The Angeli’s salt itself does not trigger the oxidation of CBA. As
3
compounds were separated by a linear increase of CH CN con- expected, neither COH formation was observed in the absence
3
centration in mobile phase from 30 to 60% using a flow rate of of Angeli’s salt (Fig. 3, line d) nor in the absence of oxygen (Fig.
0
.35 ml/min. Under these conditions, the following retention 3, line c). Similar yields of COH were observed when CBA was
times were observed: CBA, 2.79 min; COH, 3.04 min; coumarin incubated with Angeli’s salt in aerated (line b) or oxygenated
(
CH), 3.66 min; 7-nitrocoumarin (CNO ), 3.86 min; p-MitoPhB (line a) conditions. These data indicate that both HNO and
2
(
OH) , 2.75 min; p-MitoPhOH, 3.10 min; p-MitoPhNO , 3.71 molecular oxygen are essential for oxidative conversion of
2
2
min; MitoPh, 3.91 min. The compounds were detected and boronate probe. Only a minimal increase was observed after the
3
5572 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 289•NUMBER 51•DECEMBER 19, 2014

Peroxynitrite Formed from HNO Reaction with Molecular Oxygen
FIGURE 2. A and B, spectral changes observed during decomposition of Angeli’s salt alone (A) and Angeli’s salt-induced oxidation of CBA to COH (B). C,
comparison of the kinetics of decomposition of Angeli’s salt alone and of Angeli’s salt-induced conversion of CBA to COH. Incubation mixture consisted of 150
␮
M Angeli’s salt in phosphate buffer (pH 7.4, 50 mM) containing dtpa (100 ␮M) and 5% CH CN (without CBA, A; or with CBA 100 ␮M, B). The decay kinetics of
3
Angeli’s salt and CBA were monitored spectroscopically at 235 and 287 nm, respectively. The COH formation kinetic trace was recorded at 370 nm.
compounds by the nitro group strongly supports an intermedi-
ary role of peroxynitrite (47, 48). Mechanism of the reaction of
Ϫ
CBA with ONOO is shown in Fig. 5. The profile of products
formed during Angeli’s salt-induced oxidation closely resem-
Ϫ
bles the profile observed for ONOO -induced oxidation of
CBA (Fig. 4). The addition of glutathione, a known scavenger of
HNO, almost completely inhibited oxidation of boronate probe
in the solutions containing Angeli’s salt, but not when a bolus
Ϫ
ONOO was used. Although glutathione is known to react
3
Ϫ1 Ϫ1
with peroxynitrite (k ϭ 1.36 ϫ 10
M
s
) (63), 100 ␮M GSH
Ϫ
cannot compete with 100 ␮M CBA probe for ONOO (the rate
constant of the CBA reaction with peroxynitrite is equal to k ϭ
6
Ϫ1 Ϫ1
1
.1 ϫ 10
M
s
) (50).
The quantitative analysis of substrate depletion and product
FIGURE 3. The effect of oxygen on COH formation from Angeli’s salt-in-
duced oxidation of CBA. Incubation mixtures consisted of 250 ␮M CBA and
2
formation during the incubation of CBA probe in aerated
00 ␮M Angeli’s salt (lines a–c) in phosphate buffer (pH 7.4, 50 mM) containing Angeli’s salt solution and in the reaction between CBA and
dtpa (100 ␮M), 5% CH CN, and the spectra were recorded after 1.5 h of incu-
Ϫ
3
authentic ONOO , added as bolus or generated from superox-
bation at 25 °C. Line a, O -saturated solution; line b, solution in equilibrium
2
ide and nitric oxide fluxes, is shown in Fig. 6.
with the air; line c, solution deaerated using vacuum; line d, solution without
Angeli’s salt addition.
.
Previous studies indicated that neither O produced by xan-
2
ꢀ
thine/xanthine oxidase, nor NO (DPTA-NONOate), caused
Ϫ
ϳ5-fold increase in O concentration (from 20% O in air to the oxidation of boronate. Only ONOO generated in situ from
2
2
1
00% O ).
these systems was able to cause an oxidative conversion of the
2
Profiling of CBA Products Formed in Aqueous Solutions of probe (47, 49, 50, 52).
Ϫ
The titration profiles are very similar for ONOO and for
Angeli’s Salt: UPLC Study—For detailed characterization of
oxidant formed in the reaction between HNO and molecular Angeli’s salt, indicating that the reaction between HNO and
Ϫ
oxygen, the profiles of major and minor oxidation products of molecular oxygen results in the formation of ONOO , which
6
Ϫ1 Ϫ1
CBA were examined. For qualitative and quantitative determi- reacts rapidly with CBA (k ϭ 1.1 ϫ 10 M
s
) yielding COH
nation of these products, we used the UPLC analysis. As shown as a major product (50) and the minor products formed via the
Ϫ
in Fig. 4, in the presence of authentic ONOO , CBA was con- radical pathway (Fig. 5).
verted to COH as the major product, and coumarin and 7-ni-
As shown previously (47), the ratios of rate constants for the
trocoumarin (CNO ) as minor products formed in the radical radical (k ) and nonradical (k ) pathways can be determined
2
r
nr
pathway. This result is consistent with previous reports for sim- from the plot of the sum of the concentrations of minor prod-
ple arylboronates (49) and mitochondria-targeted boronate ucts versus the concentration of the major, phenolic product.
probes (47). Replacement of the boronate moiety in boronic Fig. 6 shows the k /k ratio determined for CBA oxidation.
r
nr
DECEMBER 19, 2014•VOLUME 289•NUMBER 51
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Peroxynitrite Formed from HNO Reaction with Molecular Oxygen
Clearly, the oxidation of CBA probe in the aerated solution of equal k /k ratio. The yields of both major (COH) and minor
r
nr
Angeli’s salt and in xanthine/xanthine oxidase/DPTA NONO- (coumarin and CNO ) products were the same (within the
2
.
ꢀ
ate system results in the same product formation profiles and experimental error) for Angeli’s salt- and O / NO-induced oxi-
2
dation of CBA (Table 1). The oxidative transformation of CBA
by peroxynitrite added as bolus leads to the higher yield of COH
and, as a consequence, a lower k /k ratio. A possible explana-
r
nr
tion of that phenomenon is the formation of peroxynitrate
Ϫ
Ϫ
(
O NOO ) in the reaction between ONOO and its proto-
2
nated form (ONOOH) at the high local concentration of
Ϫ
ONOO , when added by bolus addition (64). Peroxynitrate
anion is also able to oxidize CBA to COH (data not shown), but
not through the radical pathway.
As discussed previously, the profile of the products formed
from the reaction between peroxynitrite and boronate probes is
highly specific for this oxidant, providing a “peroxynitrite fin-
gerprint.” Therefore, the similar profiles of the products formed
during the peroxynitrite (both bolus and generated in situ from
.
ꢀ
co-generated O and NO fluxes) and Angeli’s salt-dependent oxi-
2
dation of CBA, both qualitatively and quantitatively (Table 1) pro-
vide strong evidence that the oxidant formed during the reaction
Ϫ
between HNO and molecular oxygen is ONOO .
Ϫ
Because the minor products of CBA oxidation by ONOO
have not been previously identified, we used another boronate
probe, p-MitoPhB(OH) (47), from which the minor products
2
Ϫ
of reaction with ONOO have been well characterized (47).
The detailed mechanism of the reaction of this probe with
Ϫ
ONOO is similar to the previously mentioned CBA oxidation
mechanism (Fig. 5) (49). The major pathway leads to the for-
mation of corresponding phenol (p-MitoPhOH), whereas
under the conditions used the minor pathway yields two prod-
ucts: MitoPh (dominant) and p-MitoPhNO . Titration of p-
2
MitoPhB(OH) probe with Angeli’s salt results in the product
2
profiles shown in Fig. 7, indicating the formation of both phe-
nolic (major) and radical-mediated products (minor). The pro-
files of the product formation resemble the profiles described
FIGURE 4. The chromatograms recorded during UPLC analyses of prod-
ucts formed during oxidation of CBA by peroxynitrite and Angeli’s salt.
Incubation mixtures consisted of 100 ␮M CBA in phosphate buffer (pH 7.4, 50
previously for authentic peroxynitrite (47). Detection of
Ϫ
mM) containing dtpa (100 ␮M), catalase (100 units/ml), 10% iPrOH, and per- ONOO -specific products using two different boronate probes in
oxynitrite or Angeli’s salt and glutathione at the indicated concentrations.
aerobic reaction mixtures of Angeli’s salt undoubtedly shows that
CBA and its corresponding oxidation products were detected using UV
absorption detection at 300 Ϯ 5 nm.
the reaction of HNO with O leads to formation of peroxynitrite.
2
؊
FIGURE 5. The mechanism of the reaction between ONOO and CBA.
3
5574 JOURNAL OF BIOLOGICAL CHEMISTRY
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Peroxynitrite Formed from HNO Reaction with Molecular Oxygen
FIGURE 6. Relationship between substrate depletion and major/minor product formation during Angeli’s salt (A) or peroxynitrite titration of CBA
solutions (C) and xanthine/xanthine oxidase/DPTA NONOate-induced oxidation of CBA (B) over the time. Incubation mixtures consisted of 90 ␮M CBA
inphosphatebuffer(pH7.4, 50mM)containingdtpa(100␮M), catalase(100units/ml), 10%iPrOH, andAngeli’ssalt/peroxynitriteattheindicatedconcentration.
After 1.5 h of incubation at 25 °C of Angeli’s salt or bolus addition of peroxynitrite, the reaction mixtures were analyzed using the UPLC/UV method. CBA and
its corresponding oxidation products were detected using UV absorption detection at 300 Ϯ 5 nm. Each point represents the average value of three samples.
The ratio of the rate constants of homolytic (radical, k ) and heterolytic (nonradical, k ) cleavage pathways was estimated. Xanthine (1 mM) and xanthine
r
nr
oxidase (2.25 milliunits/ml) were mixed and added to 600 ␮M DPTA-NONOate in phosphate buffer (pH 7.4, 50 mM) containing CBA (90 ␮M), dtpa (100 ␮M),
catalase (100 units/ml), and 10% iPrOH, resulting in 1.55 Ϯ 0.05 ␮M/min superoxide and Ͼ1.6 ␮M/min nitric oxide flux at 25 °C. CBA and its corresponding
oxidation products were detected and analyzed as previously.
TABLE 1
dation to COH (Fig. 3), indicating that reaction with oxygen
Yields of products formed upon oxidation of CBA in different systems outcompetes the self-decomposition of nitroxyl. HNO released
yielding peroxynitrite
from Angeli’s salt reacts with molecular oxygen and HNO scav-
Angeli’s salt (0–160 ␮M) was incubated for 1.5 h at 25 °C in the solution of 90 ␮M of
engers, when they are present (Fig. 8). Reaction of HNO with
CBA in phosphate buffer (pH 7.4, 50 mM) containing dtpa (100 ␮M), catalase (100
units/ml), and 10% iPrOH. Xanthine (1 mM) and xanthine oxidase (2.25 milliunits/ molecular oxygen leads to the formation of peroxynitrite, and
ml) were mixed and added to 600 ␮M DPTA-NONOate in phosphate buffer (pH 7.4,
there are two pathways of peroxynitrite-derived CBA oxidation
5
0 mM) containing CBA (90 ␮M), dtpa (100 ␮M), catalase (100 units/ml), and 10%
iPrOH, resulting in 1.55 Ϯ 0.05 ␮M/min superoxide and Ͼ1.6 ␮M/min nitric oxide (Fig. 5). The nonradical (nr) pathway of that reaction leads to
Ϫ
flux at 25 °C. ONOO (0–160 ␮M) added by bolus addition to phosphate buffer (pH
the formation of COH, and the rate of COH accumulation over
7
1
.4, 50 mM) containing 90 ␮M of CBA dtpa (100 ␮M), catalase (100 units/ml), and
time is expressed by Equation 1,
0% iPrOH. The presented yields represent the average values of three independent
experiments. The Student’s t test was used to determine the statistical significance
of differences for presented values of products yields.
d͓COH͔
Ϫ
v ϭ
ϭ kCBA,nr͓CBA͔͓ONOO ͔
(Eq. 1)
COH
%
83.8 Ϯ 0.2
82.7 Ϯ 0.3
85.8 Ϯ 0.3a
Coumarin
CNO2
%
1.8 Ϯ 0.2
2.0 Ϯ 0.1
1.7 Ϯ 0.1
dt
%
Angeli’s salt
14.4 Ϯ 0.2
15.3 Ϯ 0.2
12.5 Ϯ 0.3a
where k
is the rate constant for the nonradical pathway of
CBA,nr
X/XO/DPTA NONOate
ONOOϪ bolus addition
p values of Ͻ 0.005 were considered as significant.
CBA oxidation. Changes in the concentrations of HNO and
a
Ϫ
ONOO over time are expressed by Equations 2 and 3,
d͓HNO͔
Determination of the Rate Constant of the Reaction of HNO
ϭ k ͓AS͔ Ϫ k ͓S͔͓HNO͔ Ϫ k ͓O ͔͓HNO͔
AS
S
O2
2
dt
with Oxygen—To determine the second order rate constant of
the reaction between HNO and molecular oxygen, the compe-
tition kinetics approach was used. In this study, the HNO
dimerization was not taken under consideration as in the aer-
ated aqueous solutions, and at low Angeli’s salt concentration
(
Eq. 2)
Ϫ
d͓ONOO ]
ϭ k ͓O ͔͓HNO͔
O2
2
dt
(
0–10 ␮M) this process is negligible. In fact, a 5-fold increase in
Ϫ
Ϫ
oxygen concentration did not enhance the extent of CBA oxi-
Ϫ kCBA,nr͓CBA͔͓ONOO ͔ Ϫ k ͓CBA͔͓ONOO ͔ (Eq. 3)
CBA,r
DECEMBER 19, 2014•VOLUME 289•NUMBER 51
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Peroxynitrite Formed from HNO Reaction with Molecular Oxygen
tion with peroxynitrite. To solve the equations steady state
approximation was made (Equation 1.4).
Ϫ
d͓ONOO ͔ d͓HNO͔
ϭ
ϭ 0
(Eq. 4)
dt
dt
Finally, the solution of aforementioned equations leads to
Equations 5 and 6.
kAS͓AS͔
͓
HNO͔ ϭ
(Eq. 5)
k ͓S͔ ϩ k ͓O ͔
S
O2
2
k ͓O ͔
O2
2
Ϫ
͓
ONOO ͔ ϭ
͓HNO͔
(Eq. 6)
͑
kCBA,nr ϩ kCBA,r͓͒CBA͔
The initial rate of the COH formation is expressed by Equation
7
, whereas in the absence of HNO scavenger it is expressed by
relationship Equation 8.
kCBA,nr
kO2͓O ͔
2
vi ϭ
kAS͓AS͔
(Eq. 7)
(Eq. 8)
kCBA,nr ϩ kCBA,r
k ͓S͔ ϩ k ͓O ͔
S 1 O2 2
kCBA,nr
kCBA,nr ϩ kCBA,r
v0 ϭ
kAS͓AS͔
Comparison of the equations expressing initial rates results in
Equation 9.
v0
vi
kS ͓S͔i
ϭ 1 ϩ
(Eq. 9)
FIGURE 7. Relationship between substrate depletion and major/minor
product formation during the Angeli’s salt-induced oxidation of p-
MitoPhB(OH) . Incubation mixtures consisted of 90 ␮M p-MitoPhB(OH) in
phosphate buffer (pH 7.4, 50 mM) containing dtpa (100 ␮M), catalase (100
units/ml), 10% iPrOH, and Angeli’s salt at the indicated concentrations. After
K ͓O ͔
O2
2
2
2
The ratios of the rate constants of the second order reaction of
HNO with its scavenger (k ) and molecular oxygen (k ) were
S
O2
1
5 min of incubation at 40 °C with Angeli’s salt, the reaction mixtures were
estimated using Equation 9. Two fluorogenic probes, CBA and
analyzed using the UPLC/UV method. p-MitoPhB(OH) and its corresponding
2
oxidation products were detected using UV absorption detection at 268 Ϯ 5 FBBE, were used, which upon oxidation yield blue fluorescent
nm. Each point represents the average value of three samples. The concen-
trations were determined based on the calibration curves obtained for the
authentic standards.
(
(
COH) and green fluorescent (FME) products, respectively
Fig. 8). In this survey, an excess of boronate probe (25 ␮M CBA
or FBBE) was used over Angeli’s salt (3 or 10 ␮M) and HNO
scavenger (0–10 ␮M). The oxidation products of those two
probes are fluorescent; therefore, changes in the initial rate of
the formation of the corresponding phenols were monitored
with the use of spectrofluorometer or the stopped flow spectro-
photometer. As shown in Fig. 9 (A and B), the addition of glu-
tathione, an efficient HNO scavenger, inhibits the initial rates
of oxidation of both probes. The kinetic traces were obtained
from at least two independent measurements. A linear function
was fitted to data obtained within first 3 min of steady state
measurements (Fig. 9, B and E). In the stopped flow experi-
ments, the initial period of the reaction was reduced to 60 s. The
results were plotted versus the ratio of the HNO scavenger to
oxygen concentration (225 ␮M) and fitted linearly. The slope of
the dashed lines in Fig. 9 (C and F) is equal to the value of the
ratio of rate constants of the reactions of HNO with glutathione
and with molecular oxygen.
FIGURE8. Reactionmodelusedforthedeterminationoftherateconstant
of the reaction between HNO and oxygen using competition kinetics
approach. S, scavenger.
The values of the second order rate constant published in the
literature for the reaction of HNO with molecular oxygen
where k is the rate constant of Angeli’s salt decomposition, vary. Liochev and Fridovich (31) estimated that this rate con-
AS
4
Ϫ1 Ϫ1
and k and k are the constants of HNO reactions with scav- stant equaled Ϸ1 ϫ 10 M
s
, whereas others reported the
S
O2
3
Ϫ1 Ϫ1
enger and molecular oxygen, respectively. The rate constant rate constant to be ϳ3 ϫ 10 M
s
(36). We calculated the
k
is the constant for the radical (r) pathway of CBA reac- values of the k from the k /k ratios obtained experimen-
CBA,r
O2
S
O2
3
5576 JOURNAL OF BIOLOGICAL CHEMISTRY
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Peroxynitrite Formed from HNO Reaction with Molecular Oxygen
FIGURE 9. The effect of glutathione on the initial rate of corresponding phenol formation from the boronate probes. A, incubation mixture consisted of
CBA (25 ␮M), Angeli’s salt (10 ␮M), and GSH (line a, 0 ␮M; line b, 2 ␮M; line c, 4 ␮M; line d, 6 ␮M; line e, 10 ␮M) in phosphate buffer (pH 7.4, 25 mM) containing dtpa
(50 ␮M). D, incubation mixture consisted of FBBE (25 ␮M), Angeli’s salt (10 ␮M), and GSH (line a, 0 ␮M; line b, 2 ␮M; line c, 4 ␮M; line d, 6 ␮M; line e, 10 ␮M) in
phosphate buffer (pH 7.4, 25 mM) containing dtpa (50 ␮M). B and E, initial period of the reaction presented in A or D, respectively. C and F, the effect of
glutathioneontheinitialrateofCOHorfluoresceinmethylesterformation, respectively. ThesolutionscontainingCBAwereexcitedat332nm, andtheemitted
light intensity was measured at 470 nm (PMT voltage ϭ 585 V, emission/excitation slit ϭ 5 nm), whereas the solutions containing FBBE were excited at 494 nm,
and the emitted light intensity was measured at 518 nm (PMT voltage ϭ 580 V, emission slit ϭ 2.5 nm, excitation slit ϭ 5 nm). Each point represents the average
value of two samples. The error bars represent standard deviations.
tally using the published rate constants for selected scaven- but slightly higher values of the rate constants of HNO scaveng-
6
Ϫ1 Ϫ1
gers of HNO (CPTIO, PTIO, TEMPOL, superoxide dismu- ing, namely 3.1 Ϯ 0.6 ϫ 10
M
s
for glutathione and 1.4 Ϯ
6
Ϫ1 Ϫ1
tase, and HRP) (36, 40, 41). From obtaining the mean of 0.3 ϫ 10
M
s
for N-acetylcysteine (Table 3).
values (Table 2), we determined this rate constant to be
Kinetic Simulations—The value of the rate constant of HNO
4
Ϫ1 Ϫ1
ϳ(1.8 Ϯ 0.3) ϫ 10
M
s
.
reaction with O was also determined by kinetic simulations,
2
Reactivity of HNO toward Thiols—Next, we determined the which mirrored the kinetic experiments. In computational cal-
rate constants for the reactions between HNO and selected culations, we were changing the value of k to find the best fit
O2
thiols. The calculated values of the rate constant of selected of the computed values of k /k ratios to the experimental
S
O2
thiols are shown in Table 3. With small molecular weight thiols values. The resulting values of second order rate constant for
(
except H S), there is a correlation between the rate constants the reaction of HNO with molecular oxygen are presented in
2
4
Ϫ1 Ϫ1
and pK values of SH groups. The higher the value of pK , the the Table 2, and the averaged value is equal to 2 ϫ 10 M
s
.
a
a
lower the reaction rate constant of HNO with thiol. The rate In the reaction with HNO all selected scavengers: CPTIO,
constants for the reactions of HNO with glutathione and with PTIO, TEMPOL, superoxide dismutase, and HRP yield nitric
6
N-acetylcysteine have been previously reported (2 ϫ 10 and oxide, which subsequently reacts with nitroxyl (Reaction 3, k ϭ
5
Ϫ1 Ϫ1
6
Ϫ1 Ϫ1
5
ϫ 10 M
s
, respectively) (36). Our analysis leads to similar (5.8 Ϯ 0.4) ϫ10
M
s
) (27).
DECEMBER 19, 2014•VOLUME 289•NUMBER 51
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Peroxynitrite Formed from HNO Reaction with Molecular Oxygen
TABLE 2
The determined values of the k /k ratio (average value of three independent experiments; pH 7.4, 25 °C) for selected HNO scavengers and the
S
O2
calculated values of kO2
kO2a ؋ 10
؊4
kO2b ؋ 10
؊4
Compound
kS/kO2
kS
s
Ϫ1 Ϫ1
Ϫ1 Ϫ1
Ϫ1 Ϫ1
M
M
s
M
s
c
d
c
5
CPTIO
PTIO
6.9 Ϯ 0.1
(1.4 Ϯ 0.2) ϫ 10 (40)
(2.0 Ϯ 0.3)
(2.1 Ϯ 0.3)
(1.7 Ϯ 0.3)
(1.3 Ϯ 0.3)
(1.7 Ϯ 0.3)
(1.6 Ϯ 0.4)
2.5
2.2
6
.8 Ϯ 0.1
2.1
1.8
1.4
2.0
1.6
2.8
2.0
5
8.3 Ϯ 0.2
(1.4 Ϯ 0.2) ϫ 10 (40)
d
1
0.5 Ϯ 0.5
c
5
TEMPOL
SOD
8.1 Ϯ 0.2
(1.4 Ϯ 0.2) ϫ 10 (41)
d
9
Ϯ 1
c
d
5
28 Ϯ 1
7 ϫ 10 (36)
3
6 Ϯ 2
1.9
1.6
d
6
HRP
125 Ϯ 1
2 ϫ 10 (36)
1.8
a
b
4
Ϫ1 Ϫ1
Mean values
k
k
ϭ (1.8 Ϯ 0.3) ϫ 10
M
M
s
O2
O2
ϭ (2.0 Ϯ 0.4) ϫ 104
Ϫ1 Ϫ1
s
a
b
c
Value of kO2 obtained by dividing k
S
from the literature by the determined k
S
/kO2 ratio.
Value of kO2 obtained from kinetic simulations.
Steady state measurements.
d
Stopped flow measurements.
TABLE 3
The determined values of the k /k ratio (average value of three independent experiments; pH 7.4, 25 °C) for selected HNO scavengers and the
S
O2
calculated values of kS
؊
6a
؊6b
Compound
kS/kO2
k ؋ 10
k ؋ 10
pK (reference)
S
S
a
Ϫ1 Ϫ1
Ϫ1 Ϫ1
M
s
M
s
Thiols
c
c
c
Cysteine
244 Ϯ 7
171 Ϯ 1
155 Ϯ 9
4.5 Ϯ 0.9
3.1 Ϯ 0.6
2.8 Ϯ 0.7
1.4 Ϯ 0.3
0.6 Ϯ 0.1
1.2 Ϯ 0.3
5.0
3.5
3.2
1.5
0.7
1.3
8.3 (67)
8.8 (67)
9.1 (67)
9.5 (67)
9.8 (67)
7.05 (68)
Glutathione
Dithiothreitol
N-Acetylcysteine
Captopril
c
75 Ϯ 1
c
33 Ϯ 1
c
H S
63 Ϯ 3
2
Proteins
c
BSA
75 Ϯ 2
1.4 Ϯ 0.3
1.4 Ϯ 0.4
1.5
1.6
7.86–8.00 (69)
8.6 (70)
c
HSA
79 Ϯ 8
a
b
c
a
b
4
Ϫ1 Ϫ1
Value of k
Value of k
S
S
obtained by multiplying determined k
S
S
/kO2 ratio by kO2 in Table 2 ϭ (1.8 Ϯ 0.3) ϫ 10
M
M
s
.
/kO2 ratio by kO2 in Table 2 ϭ (2.0 Ϯ 0.4) ϫ 104
Ϫ1 Ϫ1
s
obtained from kinetic simulations.
obtained by multiplying determined k
Stopped flow measurements.
ꢀ
.
2
formed from the reaction between HNO and molecular oxygen
was not characterized. It was also shown that Angeli’s salt,
HNO ϩ NO ¡ HN O
2
REACTION 3
Ϫ
unlike ONOO , is unable to oxidize phenols to their dimeric
and fluorescent products. This led to the conclusion that
Because this reaction cannot be separated from experimentally
Ϫ
ONOO is not formed in the reaction of HNO with molecular
observed effect of HNO scavengers, the obtained k rate con-
O2
oxygen, and different products of the reaction of HNO with O
2
stant differs from the value obtained via classical kinetic analy-
sis. Similar simulations were performed to determine the rate
constants of HNO reaction with thiols (cysteine, glutathione,
dithiothreitol, N-acetylcysteine, and captopril), bovine and
were proposed (42). However, the dimeric phenols are formed
via recombination of phenoxyl radicals. Because phenoxyl rad-
icals are moderate one-electron oxidants, they can be readily
Ϫ
reduced by HNO formed in this system. Contrary to Miranda
human serum albumin, and HS . In these simulations, the rate
Ϫ
(
42), Kirsch and de Groot (43) have shown that ONOO and
constant for the reaction of HNO with O was set to be equal to
2
4
Ϫ1 Ϫ1
oxidant formed in aerated solutions of Angeli’s salt exhibit a
similar pattern of the reactivity, suggesting formation of per-
2
ϫ 10
M
s
. During kinetic simulation, we were changing
the value of k to get the best agreement with experimentally
S
oxynitrite in the reaction of HNO with O . Several studies
determined k /k ratios. The calculated rate constants are pre-
2
S
O2
examining HNO in vitro demonstrated that Angeli’s salt (HNO
sented in Table 3. Fig. 10 (A and B) shows the comparison of the
experimentally recorded increase of fluorescence correspond-
ing to CBA oxidation to COH and the simulated kinetic traces
of COH build-up for two HNO scavengers: TEMPOL and
glutathione.
donor) exerted marked and oxygen-dependent cytotoxicity
Ϫ
(
13–16). The present results show that ONOO is formed as a
major product from HNO reaction with O , and thus, the cyto-
2
toxicity of HNO donors can be at least partially explained
through formation of peroxynitrite.
DISCUSSION
The rate constant for the reaction of HNO with molecular
Ϫ
4
Ϫ1 Ϫ1
Chemical Considerations—Here we have shown that ONOO
oxygen was determined to be ϳ(1.8 Ϯ 0.3) ϫ 10 M
s
at pH
is the major product of the reaction between HNO and molecular 7.4, and this value is two times higher than the reported value by
3
Ϫ1 Ϫ1
oxygen. Previously Miranda (42) has shown that Angeli’s salt, like Liochev and Fridovich et al. (31) (8 ϫ 10 M
s
) and approx-
Ϫ
ONOO , is able to oxidize dihydrorhodamine in the presence imately five times higher than the value proposed by other
3
Ϫ1 Ϫ1
of oxygen. However, the structure of the reactive oxidant authors (36) (3 ϫ 10
5578 JOURNAL OF BIOLOGICAL CHEMISTRY
M
s
). Based on the rate constant
3
VOLUME 289•NUMBER 51•DECEMBER 19, 2014

Peroxynitrite Formed from HNO Reaction with Molecular Oxygen
function) of HNO donor (CXL-1020) in patients with heart
failure was recently demonstrated (66). CXL-1020 is synthe-
sized from Piloty’s acid and decomposes to HNO more slowly
than Angeli’s salt (66). Published results show that CXL-1020
decomposes to form N O through a mechanism similar to that
2
of Angeli’s salt (66). Although CXL-1020 was shown to be safe
and well tolerated in patients with heart failure (66), prolonged
administration of this drug at slightly higher doses caused
inflammatory reactions in dogs (66). The authors indicated that
this toxic side effect could derail its use as a human therapeutic
(
66). Based on the present results, it is abundantly clear that in
Ϫ
the presence of oxygen, HNO donors will form ONOO as a
major intermediate in the extracellular milieu. This finding may
be relevant in explaining their toxic side effects.
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FIGURE 10. The effect of HNO scavengers on the oxidative conversion of
CBA into COH by ONOO formed in the reaction of HNO with molecular
oxygen: the comparison of experimental data with the kinetic simula-
tion. The experimental conditions were as follows: A, CBA (25 ␮M), Angeli’s
salt (10 ␮M), TEMPOL (0 ␮M, 5 ␮M, 10 ␮M, 20 ␮M), phosphate buffer (pH 7.4, 50
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2
(0 ␮M, 2 ␮M, 4 ␮M, 8 ␮M), phosphate buffer (pH 7.4, 50 mM), and dtpa (100 ␮M),
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2
vivo. Hypertension 49, 885–892
solid lines represent the calculated COH concentrations.
Ϫ
7
8
9
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determined for the reaction of HNO with O and the kinetic
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HNO with selected thiols of possible biological and/or pharma-
cological relevance. The proposed methodology is easy to con-
duct and could be used for further HNO reactivity assessment.
As previously shown for glutathione, we observed that other
thiols also react with HNO significantly faster than does molec-
ular oxygen, with the rate constants 2 orders of magnitude
higher. These results indicate that it is very unlikely that HNO
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