Originally published In Press as doi:10.1074/jbc.M108079200 on January 17, 2002
J. Biol. Chem., Vol. 277, Issue 16, 13379-13388, April 19, 2002
Formation of Peroxynitrite from Reaction of Nitroxyl Anion with
Molecular Oxygen*
Michael
Kirsch
and
Herbert
de Groot
From the Institut für Physiologische Chemie,
Universitätsklinikum, Hufelandstrasse 55, D-45122 Essen, Germany
Received for publication, August 22, 2001, and in revised form, January 7, 2002
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ABSTRACT |
Peroxynitrite (ONOO
/ONOOH) is
generally expected to be formed in vivo from the
diffusion-controlled reaction between superoxide (O
) and
nitric oxide (·NO). In the present paper we show that under
aerobic conditions the nitroxyl anion (NO), released from
Angeli's salt (disodium diazen-1-ium-1,2,2-triolate, ON=NO
),
generated peroxynitrite with a yield of about 65%. Simultaneously,
hydroxyl radicals are formed from the nitroxyl anion with a yield of
about 3% via a minor, peroxynitrite-independent pathway. Further
experiments clearly underline that the chemistry of NO in
the presence of oxygen is mainly characterized by peroxynitrite and not
by HO· radicals. Quantum-chemical calculations predict that
peroxynitrite formation should proceed via intermediary formation of
·NO and O, probably by an electron-transfer mechanism.
This prediction is supported by the fact that
H2O2 is formed during the decay of
NO in the presence of superoxide dismutase
(Cu(II),Zn-SOD). Since the nitroxyl anion may be released endogenously
by a variety of biomolecules, substantial amounts of peroxynitrite
might be formed in vivo via NO in addition to
the "classical" ·NO + O pathway.
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INTRODUCTION |
Peroxynitrite (ONOO
1/ONOOH) can be formed
in vivo from the diffusion-controlled reaction
(k = 3.9-19 × 109
M1 s1) between superoxide
(O) and nitric oxide (nitrogen monoxide, ·NO) (1, 2).
The pathological activity of ONOO is closely related to
its reaction with CO2 (3), thereby producing the highly
reactive radicals CO
(4, 5) and
NO
(6) with a yield of
about 30-35% (7-9). Consequently, peroxynitrite can nitrate tyrosine
(10) and oxidizes protein and non-protein sulfhydryls (11), membrane
phospholipids (12), low density lipoproteins (13), ascorbate (14), and
NAD(P)H (15). Furthermore, there is increasing evidence that
peroxynitrite is able to induce nitrosation reactions (16-21). Thus,
formation of both mutagenic diazopeptides (22) and carcinogenic
N-nitrosoamines (23) are additionally expected to be
produced by peroxynitrite.
A further source of endogenous peroxynitrite may be the nitroxyl anion
(NO). This anion has been reported to be generated
in vivo from reduction of ·NO by Cu(I),Zn-SOD (24),
hemoglobin (25), and cytochrome c2+ (26),
respectively. There are also reports that the NOS-catalyzed oxidation
of L-arginine leads to initial formation of
NO and not ·NO radical (27), however, this finding
is subject to controversial discussion (28). Nitroxyl anion might be
further formed from reaction of S-nitrosothiols with thiols
(29-31), although this reaction is less well understood. Donald
et al. (32) have proven that the photochemical decomposition
of Angeli's salt, a chemical NO donor compound (33), in
fact yields peroxynitrite under aerobic conditions (Reaction 1). This
photochemical process is probably the reason why NO has
often been referred to as a peroxynitrite-yielding compound, even in
textbooks of inorganic chemistry (34).
Only very low yields of nitrated products have been observed from
NO-induced reactions (35, 36). From these facts it was
concluded that during thermal decomposition of Angeli's salt only a
small amount of peroxynitrite is generated (36). Recently, two research groups (37, 38) apparently disproved the capability of NO
to generate peroxynitrite under more physiological conditions. They
reported, for example, that typical peroxynitrite-mediated reactions,
e.g. nitrosation reactions, could not be observed (37) and
that NADPH could be oxidized by NO under hypoxic
conditions (38). Unfortunately, these experiments were performed in the
presence of organic buffer compounds (Good's buffer) which are known
to effectively react with peroxynitrite (39, 40). Thus, the formation
of peroxynitrite by NO very easily may have been masked.
In the present study, we demonstrate that in the presence of molecular
oxygen NO indeed mainly yields peroxynitrite and that
NO additionally produces HO· radicals via a minor,
peroxynitrite-independent pathway. Furthermore, we present a key
experiment which suggests the intermediacy of O during the
NO-mediated formation of peroxynitrite.
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EXPERIMENTAL PROCEDURES |
Materials--
Catalase from beef liver (EC 1.11.1.6),
copper-zinc superoxide dismutase from bovine erythrocytes (EC
1.15.1.1), and NADH were obtained from Roche Molecular Biochemicals
(Mannheim, Germany). Manganese dioxide, benzoic acid, and hydrogen
peroxide were from Sigma (Deisenhofen, Germany). Angeli's salt came
from Alexis-Deutschland (Grünberg, Germany). DHR and DAN were
obtained from Molecular Probes (Leiden, The Netherlands). Commercially
available mixtures of oxygen 5.0 and nitrogen 5.0 (20.5%
O2, 79.5% N2, "synthetic air") and
commercially available mixtures of oxygen 5.0, nitrogen 5.0, and carbon
dioxide 4.6 (20.5% O2, 74.5% N2, 5%
CO2) were purchased from Messer-Griessheim (Oberhausen,
Germany; 5.0 and 4.6 mean purities of 99.999 and 99.996%,
respectively). SIN-1 and its decomposition product SIN-1C
were generously provided by Drs. K. Schönafinger and J. Pünter (Aventis, Frankfurt/Main, Germany). NAT was synthesized as
described by Wheeler et al. (41). Peroxynitrite
(oxoperoxonitrate(1-)) (0.73 M) was prepared by isoamylnitrite-induced nitrosation of hydrogen peroxide (0.12 mol of
isoamylnitrite, 100 ml of H2O2 (1 M) plus diethylenetriaminepentaacetic acid (2 mM)) and purified (e.g. solvent extraction,
removal of excess H2O2, N2-purging)
as described by Uppu and Pryor (42) and stored at 
79 °C. All other
chemicals were of the highest purity commercially available.
Solutions--
Care was taken to exclude possible contamination
by both bicarbonate/carbon dioxide and transition metals. Doubly
distilled water was bubbled (2 liters/min) with synthetic air at room
temperature for 20 min. This water was used for synthesis of
oxoperoxonitrate(1-), NaOH (0.01-0.5 N) and for all other
solutions. Phosphate buffer solutions (50 mM) were treated
with the heavy metal scavenger resin Chelex 100 (0.3/0.5 g in 10 ml) by
gently shaking for 18 h in the dark. After low-speed
centrifugation for 5 min, the solutions were carefully decanted from
the resin. The resin treatment resulted in an increase in pH by about
0.25 units. Various additives (DHR, NADH, and benzoic acid) were then
added. The pH was adjusted to 7.5 at 37 °C and the solutions were
again bubbled (2 liters/min) with synthetic air or with the
CO2 mixture for 20 min. In the case of CO2
bubbling, the pH had to be readjusted to 7.5. SIN-1 and Angeli's salt
solutions were prepared as ×100 stock solutions at 4 °C in 50 mM KH2PO4 and in 10 mM NaOH, respectively, and used within 15 min.
Experimental Conditions--
SIN-1 was added to 1 ml of
phosphate buffer and incubated in 12-well cell culture plates (volume
of each well 7 ml, Falcon, Heidelberg, Germany). Under
HCO
/CO2-free conditions,
these plates were placed in an air-tight vessel (10 liters). During the
first 15 min of each experiment, these vessels were flushed (5 liters/min) with synthetic air in a warming incubator (Heraeus, Hanau,
Germany). In the presence of
HCO/CO2 the plates were
placed in an incubator for cell culture (37 °C, humidified
atmosphere of 95% authentic air and 5% CO2, Labotect, Göttingen, Germany). The experiments with authentic peroxynitrite (2 µl of 25-125 mM ONOO in 0.5 N NaOH was added to 1 ml of reaction solution) and with Angeli's salt (1 ml of reaction solution) were performed in reaction tubes (2.0 ml, Eppendorf, Hamburg, Germany) by using the drop-tube Vortex mixer technique as described previously (40). Under
HCO/CO2-free conditions,
the experiments with authentic peroxynitrite and Angeli's salt were
performed in a glove-bag (Roth, Karlsruhe, Germany) under synthetic air.
Determination of Peroxynitrite/SIN-1/Angeli's
Salt-driven Hydroxylation of Benzoic Acid--
Peroxynitrite, SIN-1,
and Angeli's salt (each 50-600
µM)-dependent hydroxylation of BA (5 mM) were employed. After vortexing, the samples were kept
for 2 min (in the case of peroxynitrite), 4 h (in the case of
SIN-1), and 30 min (in the case of Angeli's salt) at 37 °C,
respectively. The product formed was measured by reading its
fluorescence with excitation at 290 nm and emission at 410 nm (37).
Determination of Rhodamine 123--
Formation of RH was
quantified spectrophotometrically at 500 nm (
M = 78,000 M1 cm1) (43).
Determination of Angeli's Salt-driven Nitrosation
Reactions--
Angeli's salt (50-600
µM)-dependent nitrosation of DAN (200 µM) was employed. After vortexing, the samples were
incubated for 30 min at 37 °C. NaOH (0.5 M) was added
(5:1, v/v, final pH 11-11.5). The product formed, i.e. NAT,
was quantified by reading its fluorescence with excitation at 375 nm
and emission at 415 nm (44). Standard calibration curves were prepared
from known amounts of NAT.
Determination of NADH--
NADH was quantified by reading its
fluorescence with excitation at 339 nm and emission at 460 nm (45).
Standard calibration curves were prepared from known amounts of
NAD(P)H. Additionally, the oxidation of NAD(P)H was followed
photometrically at 340 nm using 
340 = 6200 M1 cm1 (45). Both methods gave
identical results, therefore, only one parameter, the decrease of
fluorescence, will be shown here.
Determination of H2O2 and of
O2--
Hydrogen peroxide was quantified by two
techniques. In peroxidase assays, horseradish peroxidase-catalyzed
formation of a colored product was measured. 4-Aminoantipyrine and
3,5-dichloro-2-hydroxybenzenesulfonic acid were used as peroxidase
substrates. The quinoneimine dye formed from these substrates was
measured spectrophotometrically at 546 nm (46) (peroxidase assay).
Alternatively, H2O2 was quantified by the
amount of O2 released upon addition of catalase (1,000 units/ml) (catalase assay). O2 was determined
polarographically with a Clark-type oxygen electrode (Saur, Reutlingen,
Germany). Both methods gave identical results, therefore, only one
parameter, the peroxidase assay, will be shown here.
Determination of Nitrate--
The nitrate yields from
decomposition of Angeli's salt (100 and 200 µM) were
quantified by the use of nitrate reductase in conjunction with the
Griess assay. The Griess assay was carried out as described elsewhere
(46).
Quantum-chemical Calculations--
Density functional theory and
ab initio calculations were performed with the Gaussian
98W (Revision A.9) suite of programs (47). Geometries were fully
optimized to stationary points, using the CBS-QB3 methodology in the
density functional theory calculations and single-excitation CI
calculations and second-order Møller-Plesset (48) (MP2) calculations
with the 6-311+G(d) basis set on the ab initio level.
Calculation of UV-VIS absorption spectra was performed by single point
energy calculations on the CBS-QB3-optimized structure using the
protocol of the time-dependent density functional theory
method (49). Aqueous solvent interactions were evaluated with the PCM
(50) procedure incorporated in Gaussian 98W. To verify whether an
electron transfer between NO and O2 would be
thermodynamically feasible, geometry optimizations and frequency
calculations were done using the MP2 approximation. Molecular
interactions were then evaluated with the IPCM (51) procedure.
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RESULTS |
Formation of Peroxynitrite from Nitroxyl Anion--
To
prove whether ONOO is formed during the decay of
NO in the presence of oxygen, we attempted to identify
peroxynitrite by UV-visible spectroscopy. As the half-life of
ONOO is short at physiological pH values and since UV
light induces the decomposition of Angeli's salt to peroxynitrite
(32), 8 samples of 5 mM Angeli's salt from the same stock
solution were incubated in parallel runs in the dark at 37 °C at pH
12.25 (Fig. 1A). The decay of
Angeli's salt is slow at these experimental conditions
(t1/2 ~12 h), thus, reaction times
of several hours were necessary to monitor the significant changes of
the optical density. The initial absorbance at the beginning of the
experiment was 0.35 ± 0.05 at 302 nm. With increasing reaction
time, the optical density at 302 nm increased continuously to reach a
maximum value of 0.88 ± 0.08 after 6 h, followed by a
further decrease of the absorption at longer reaction periods. After
24 h the extinction value had dropped to 0.4 ± 0.05, clearly
showing that a relatively long-lived intermediate has been formed
during the decay of Angeli's salt. The absorption at 302 nm had
decayed completely when the reaction solution was briefly (20-30 s)
bubbled with CO2 after 5 h of incubation (data not
shown). These observations strongly indicated the intermediary
formation of ONOO. In fact, when the initial UV spectrum
was subtracted from the one observed after 4 h of incubation, the
resulting difference spectrum exhibited an absorption spectrum with a
maximum at 302 nm (Fig. 1, B and C), similar to
what has been reported for peroxynitrite (52). This was verified by
comparison with the UV spectrum of authentic ONOO (Fig.
1C). The scatter of the difference spectrum at shorter wavelengths (
< 295 nm) derives from the strong absorption of Angeli's salt in this wavelength region. The absorbance at 302 nm has
been attributed to the cis-conformer of peroxynitrite (53, 54). This is excellently supported by time-dependent
density functional theory calculations (Table
I), which show that the trans-conformer of ONOO should absorb at
longer wavelengths (max = 374 nm). Thus,
cis-ONOO is produced from the
NO-donating compound Angeli's salt during its decay in
aerobic solution.
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Fig. 1.
Formation of peroxynitrite from
Angeli's salt. Eight samples of Angeli's salt (5 mM)
prepared from the same stock solution were incubated in the dark in
K3PO4 (50 mM, pH 12.25) for 0-24 h
at 37 °C in the presence of 100% oxygen. At selected time points an
UV-visible spectrum was read from one sample by using a diode array
spectrophotometer (scanning time 1 s). Afterward, the sample was
discarded. A, kinetic of the absorption at 302 nm. Each
value represents the mean ± S.D. of six experiments.
B, generation of a representative difference spectrum,
obtained by subtraction of the UV-visual spectrum which was observed
after 0 h of incubation from this spectrum observed after 4 h
of incubation. C, comparison between the UV-visual spectrum
of authentic peroxynitrite and the difference UV-visual spectrum from
B.
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Table I
Quantum chemically calculated UV-visable absorption of ONOO
isomers
Geometries of cis-ONOO,
trans-ONOO, and
2,4-cyclo-ONOO were fully optimized to stationary
points using the CBS-QB3 methodology. Calculation of UV-visible
absorption spectrum was performed by single point energy calculations
on the CBS-QB3 optimized structure using the protocol of the
time-dependent TDDFT (49) method. Aqueous solvent
interactions were evaluated with the PCM solvation model on the CBS-B3
basis set (TD PCM B3LYP/CBS-B3//CBS-QB3).
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Yield of Peroxynitrite Derived from Nitroxyl
Anion--
The above observations qualitatively prove the formation of
peroxynitrite from NO; the yield of this reaction
remained to be established. To this end, the potential of Angeli's
salt to oxidize both DHR and NADH were compared with those of the
·NO/O releasing compound SIN-1. Increasing concentrations (0-25 µM) of SIN-1 as well as of
Angeli's salt stimulated the oxidation of both DHR and NADH (each 50 µM) in a linear fashion (Fig.
2, A and B). While
SIN-1 oxidized DHR and NADH with yields of ~100 and 85%,
respectively, Angeli's salt was found to be significantly less
effective, oxidizing DHR and NADH with yields of ~60 and 65%,
respectively. Thus, the production of peroxynitrite from Angeli's salt
is only 65 ± 5% of that from SIN-1. This fact implied that in
the absence of suitable targets (i.e. HEPES, DHR, and NADH)
the formation of nitrate from Angeli's salt should be in the same
range. In fact, about 65 µM nitrate was formed during the
decay of 100 µM Angeli's salt at pH 7.5 irrespective of
the presence of CO2 (Fig. 2C). Interestingly, Angeli's salt-derived formation of nitrate was maximal at
physiological pH values. Control experiments revealed that nitrate was
not a contaminant of the applied Angeli's salt because nitrate could not be detected after decomposition of Angeli's salt (100 µM) at pH 12.25 in the presence of 20 µM
Cu2+(data not shown). Consequently, Angeli's salt had
consumed oxygen with an efficiency of about 65%. To verify this, the
oxygen uptake induced by Angeli's salt (50-200 µM) was
determined polarographically (Table II).
As expected, the amount of O2 consumed by NO
was found to be around 65% of the employed amount of Angeli's salt.
Thus, the nitroxyl anion yields peroxynitrite with a yield of about
65%. Our oxygen uptake experiments are in disagreement with data of
Miranda et al. (37), who observed a 1:1 stoichiometry between NO (or rather Angeli's salt) and O2.
However, it should be remembered that the experiments of Miranda
et al. (37) were performed in the presence of HEPES, which
is known to effectively react with peroxynitrite, thereby further
increasing the uptake of O2 (40).
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Fig. 2.
Capabilities of Angeli's salt to oxidize
both NADH and dihydrorhodamine 123 and to generate
nitrate. Various concentrations of either SIN-1 or Angeli's salt
(each 0-25 µM) in potassium phosphate buffer (50 mM, pH 7.5, HCO/CO2 (25 mM/5%), 37 °C) were incubated for 1 h to oxidize
either DHR or NADH (each 50 mM), respectively.
A, oxidation of DHR. B, oxidation of NADH.
Residual NADH was quantified by reading its fluorescence with
excitation at 339 nm and emission at 460 nm. These values were
corrected for autoxidation of NADH. In the absence of SIN-1/Angeli's
salt, about 97% of the initial NADH concentration (50 µM) could still be detected after 1 h of incubation.
C, Angeli's salt (100 µM) was incubated for
1 h in potassium phosphate buffer (50 mM, 37 °C) at
various pH values (6-8) in the absence and presence
HCO/CO2 (25 mM/5%). Formation of nitrate was quantified by using
nitrate reductase in conjunction with the Griess assay. Each value
represents the mean ± S.D. of three experiments performed in
duplicate.
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Table II
Yields of Angeli's salt-derived O2 consumption
Angeli's salt (0-200 µM) was added under normoxic
conditions to 50 mM potassium phosphate buffer (37 °C,
pH 7.5). O2 was measured polarographically with a Clark-type
electrode. Data are mean ± S.D. of 12 independent experiments.
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Peroxynitrite-independent Formation of Hydroxyl
Radicals from the Nitroxyl Anion--
Since only ~65% of the
generated NO was converted into peroxynitrite, the
question arose whether other (reactive) intermediates were formed from
the remaining 35% of NO. Recently, two research groups
(55, 56) reported that NO released from Angeli's salt
should generate hydroxyl radicals. As peroxynitrite is known to produce
HO· radicals (57, 58) with an efficiency of about 28% (9, 59),
it is unclear whether the HO· radicals detected by these groups
may have derived exclusively from peroxynitrite. To check on this
important point, the Angeli's salt (0-500 µM)-induced
hydroxylation of benzoic acid (5 mM) was studied and
compared with that of authentic peroxynitrite (0-500 µM)
(Fig. 3, A and B).
In the absence of CO2 peroxynitrite-dependent hydroxylation of BA increased in a linear manner with increasing concentrations of peroxynitrite. In the presence of CO2,
however, peroxynitrite-mediated hydroxylation of BA was inhibited by
about 99%. The effect of CO2 to strongly suppress
peroxynitrite-derived formation of hydroxyl radicals is in full
agreement with recent reports (57, 60). Moreover, this effect of
CO2 offers the possibility to distinguish between
HO· released from peroxynitrite and HO· radicals released
from other sources. Similar to peroxynitrite, Angeli's salt-mediated
hydroxylation of BA increased with increasing concentration of
Angeli's salt in the absence of CO2 although not in a
strictly linear fashion (Fig. 3B). The efficacy of Angeli's salt to hydroxylate BA decreased with increasing concentration compared
with authentic peroxynitrite. This result again is in disagreement with
observations by Miranda et al. (37) who found that Angeli's
salt was much more effective in hydroxylating BA than authentic
peroxynitrite. Again, the usage of HEPES as buffer compound and the
fact that no attention was given to exclude traces of CO2
may explain this discrepancy. Most interestingly, however, the presence
of CO2 did not completely inhibit the hydroxylation of BA
by Angeli's salt. Depending on the initial concentration of Angeli's
salt, about 14-29% of the amount of the hydroxylated product that was
found in the absence of CO2 was still formed in its
presence. In comparison to peroxynitrite generated in situ from SIN-1 (Fig. 3C), Angeli's salt was only slightly more
effective in hydroxylating BA. The observation that in the absence of
CO2 authentic peroxynitrite hydroxylates BA in a linear
manner, whereas peroxynitrite generated in situ from both
SIN-1 and Angeli's salt hydroxylates BA in an almost identical,
nonlinear manner implies the intermediary of the same reactive species
formed from these peroxynitrite generating systems. For instance, as
N2O3 is known to effectively react with
ONOO (61), one might speculate that such an intermediate
decreases the hydroxylating capabilities of in situ
generated peroxynitrite. To further verify that in fact HO·
radicals were produced independently from peroxynitrite, competition experiments with Angeli's salt (500 µM), BA (5 mM), and the hydroxyl radical scavenger Me2SO
(0-10 mM) were performed in the presence of
CO2 (Fig. 3D). These experiments demonstrated
that low concentrations of Me2SO effectively inhibited in
an apparently exponential manner the NO-derived
hydroxylation of BA. Thus, NO very likely generates
HO· radicals via a peroxynitrite-independent pathway. However,
as the Angeli's salt-induced hydroxylation of BA was only to ~71% inhibited by Me2SO, a hydroxylating species other than the
HO· radical may be additionally generated with low yields by the nitroxyl anion. Since the yield of HO· radicals from
peroxynitrite is ~28% (9, 59), and because NO
generates peroxynitrite with a yield of ~65%, and because the hydroxyl radicals from this in situ generated peroxynitrite
hydroxylated BA in a range from ~86 to 71%, and because the
peroxynitrite-independent hydroxylation of BA is only to 71% induced
by HO· radicals, it can be estimated that the
NO-derived yield of HO· radicals generated via the
peroxynitrite-independent pathway is about 3 ± 1.5%. To clarify
whether this pathway is additionally oxygen dependent, Angeli's salt
was decomposed at various pH values in the absence (hypoxia) or
presence of oxygen (normoxia) and CO2 (Fig. 3E).
In the absence of O2 the yield of HO· radicals from
Angeli's salt increased with increasing H+ concentration,
in agreement with findings of Stoyanovsky et al. (55).
Furthermore, at atmospheric O2 levels and in the presence of CO2, Angeli's salt was in regard to the
O2-free solution only about half as effective to
hydroxylate BA. Thus, under conditions where the nitroxyl anion cannot
react with oxygen, the NO-derived yield of HO·
radicals increased to ~8% at physiological pH values.
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Fig. 3.
Effect of Angeli's salt to hydroxylate
benzoic acid. Authentic peroxynitrite, Angeli's salt, or SIN-1
(each 0-500 µM) were added by vortexing to 5 mM BA in 50 mM potassium phosphate buffer
(37 °C, pH 7.5) in the absence and presence of
HCO/CO2 (25 mM/5%). In the case of SIN-1 the use of
HCO/CO2 was avoided. The
hydroxylation of benzoic acid was determined after an incubation of 2 min (A), 30 min (B), or 4 h (C),
respectively. D, Angeli's salt (500 µM) and
various concentrations of Me2SO (0-10 mM) were
incubated in potassium phosphate buffer (50 mM, pH 7.5, HCO/CO2 (25 mM/5%), 37 °C) for 30 min. E, Angeli's salt
(200 µM) was incubated for 1 h in potassium
phosphate buffer (50 mM, 37 °C) at various pH values
(6-8) in the absence and in the presence
HCO/CO2 (25
mM/5%). In all cases, the hydroxylation of benzoic acid
was subsequently quantified by reading its fluorescence with excitation
at 290 nm and emission at 410 nm. Each value represents the mean ± S.D. of three experiments performed in duplicate.
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Angeli's Salt-derived Nitrosation of
2,3-Diaminonaphthalene--
Since there is a peroxynitrite-independent
production of HO· radicals, one may question that in the
presence of oxygen the chemistry of NO is generally
dominated by peroxynitrite. A NO-mediated nitration of
tyrosine would, for instance, indicate that peroxynitrite is the
attacking species, but unfortunately, in situ generated
peroxynitrite does not effectively nitrate tyrosine (62, 63). On the
other hand, as in situ generated peroxynitrite is able to
induce nitrosation reactions (16, 17, 21), the nitroxyl anion released
from Angeli's salt is expected to provoke nitrosation reactions when
its chemistry is indeed governed by peroxynitrite. To verify this
assumption, the reaction between Angeli's salt (500 µM)
and DAN (200 µM) was studied in the presence of
CO2. The product of this reaction was identified as
2,3-naphthotriazole (NAT) because its emission spectrum (Fig.
4A) was found to be identical
with that of authentic NAT (Fig. 4B) (44). Thus,
peroxynitrite in situ generated from the nitroxyl anion is
able to induce nitrosation reactions. To rule out the possibility that
only minor amounts of NAT were formed from reaction of Angeli's salt
with DAN, the yield of this reaction was also determined (Table
III). Angeli's salt concentrations in
the range from 50 to 600 µM produced NAT with yields of
about 7 and 10% in the absence and presence of CO2,
respectively. Since such yields are very typical for
peroxynitrite-induced reactions, one must conclude that the chemistry
of the nitroxyl anion is mainly characterized by peroxynitrite and with
a minor contribution by independently generated HO·
radicals.
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Fig. 4.
Angeli's salt-mediated nitrosation of
2,3-diaminonaphthalene. Angeli's salt (500 µM) and
DAN (200 µM) were incubated for 30 min in potassium
phosphate buffer (50 mM, pH 7.5, HCO/CO2 (25 mM/5%), 37 °C). A, representative emission
spectrum of the product from the reaction between Angeli's salt and
DAN. B, emission spectrum of authentic
2,3-naphthotriazole.
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Table III
Yields of Angeli's salt-derived nitrosation of 2,3-diaminonaphthalene
Angeli's salt (0-600 µM) was added under normoxic
conditions to 50 mM potassium phosphate buffer (37 °C,
pH 7.5, 200 µM DAN) in the absence and presence of
HCO/CO2. After 30 min of incubation the
formation of NAT was quantified fluorimetrically (Ex = 375 nm, Em = 415 nm). Data are mean ± S.D. of three
experiments performed in duplicate.
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Putative Mechanism by Which Nitroxyl Anion Generates
Peroxynitrite--
The question arises how the formation of
cis-ONOO from NO and
O2 may proceed. It has often been suggested that these two
molecules, both having a triplet ground state (64), react with each
other, thereby directly generating ONOO (24, 30, 32, 36,
56, 65).
In fact, the reaction between 3NO and
3O2 appears to be thermodynamically feasible,
because MP2 ab initio calculations as well as CBS-QB3
calculations2 in conjunction
with the Isodensity Polarized Continuum Model (IPCM) for solvation
predicted both an exergonic reaction in the gas phase as well as in
aqueous solution (Table IV). These
MP2/IPCM calculations also support an outer sphere electron transfer
between 3NO and 3O2,
i.e. causing solvent-separated ·NO + O, to
be a thermodynamically feasible process.
The subsequent formation of cis-ONOO is,
of course, exothermic (Table IV).
To the best of our knowledge, Reaction 3 was first proposed in
1927 by Andrussow (66). In 1966 Fehsenfeld et al. (67) verified that Reaction 3 indeed proceeds in the gas phase at room temperature. Some years ago, the electron transfer was mentioned with
little modifications by the Ignarro group (68), but its significance
under physiological conditions has often been questioned by other
researchers. Although Reaction 3 appears to be thermodynamically feasible, any experimental indications that it really proceeds in
aqueous solution are as yet missing. Provided that in Reaction 3 ·NO is really released, the addition of superoxide dismutase is expected to strongly increase the yield of ·NO during the decay
of Angeli's salt.
As in case of Reaction 2 no free ·NO would be released, the
presence or absence of ·NO may be taken as a mechanistic probe.
In fact, there are several reports in the literature that superoxide
dismutase stimulates the formation of ·NO from NO
(e.g. Refs. 24 and 27). Contrary to our hypothesis, this superoxide dismutase-dependent formation of ·NO has
been exclusively interpreted in terms of NO being a
substrate for Cu(II),Zn-SOD, i.e. being oxidized by
Cu(II),Zn-SOD. However, if superoxide dismutase scavenges O rather than NO, H2O2 must be
formed as a product (see Reaction 5). To clarify this, the superoxide
dismutase-dependent formation of
H2O2 from Angeli's salt was studied in the
absence and presence of CO2 (Fig. 5). In the absence of Cu(II),Zn-SOD only
about 0.6 µM H2O2 was found
regardless of the presence of CO2. The addition of 100 units/ml superoxide dismutase, however, clearly stimulated the
formation of H2O2. In the absence of
CO2, 39.8 ± 2.4 µM
H2O2 was found, and in its presence a somewhat
lower amount of 30.6 ± 1.5 µM was detected.
Increasing the superoxide dismutase activity to 500 units/ml further
increased the yields of H2O2 to 46.3 ± 1.8 µM and 36.4 ± 2.3 µM
H2O2 in the absence and presence of
CO2, respectively. The Cu(II),Zn-SOD-induced formation of
H2O2 during the decay of Angeli's salt was
verified by two independent methods, namely peroxidase assay and
catalase assay. Either replacing Cu(II),Zn-SOD by an equivalent amount
of albumin (Fig. 5) or treating Cu(II),Zn-SOD with authentic
peroxynitrite (500 µM, data not shown) strongly decreased
H2O2 formation to 
4 µM. These
results strongly indicate that Reaction 3 is indeed operating.
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Table IV
Quantum chemically calculated reaction energies for reaction of the
nitroxyl anion with oxygen
Geometries of nitroxyl anion (3NO), oxygen
(3O2), nitric oxide (·NO), superoxide
radicals (O), and cis-ONOO were fully
optimized to stationary points using second-order Møller-Plesset
pertubation theory (MP2) on the 6-311 + G(d) basis set. Frequency
calculations were performed on the same level of theory. As these
calculations refer only to the conditions in the gas phase, single
point calculations were performed on the MP2/6-311 + G(d)//MP2/6-311 + G(d) level for water with the IPCM solvation
model.
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Fig. 5.
Effect of superoxide dismutase to stimulate
hydrogen peroxide formation from Angeli's salt. Cu,Zn-SOD (0-500
units/ml) was incubated with Angeli's salt (500 µM) in
potassium phosphate buffer (50 mM, pH 7.5, 37 °C) for 30 min in the absence and in the presence
HCO/CO2 (25 mM/5%). Formation of H2O2 was
quantified by using the peroxidase assay. Each value represents the
mean ± S.D. of eight experiments.
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Effect of Superoxide Dismutase on Nitroxyl Anion-mediated Oxidation
Reactions--
The above described action of Cu(II),Zn-SOD implied
that nitroxyl anion-related formation of peroxynitrite is inhibited by superoxide dismutase. To further support this assumption, the inhibitory effects of Cu(II),Zn-SOD on both SIN-1- and Angeli's salt-mediated oxidation of both DHR and NADH were studied in the absence and presence of CO2. As expected, Cu(II),Zn-SOD
inhibited these oxidations with increasing activity of superoxide
dismutase (Fig. 6, A-D).
Cu(II),Zn-SOD was generally somewhat more effective in inhibiting
SIN-1-dependent oxidation reactions than those mediated by
Angeli's salt. This might reflect either the likely differences in the
kinetics of O formation or the property of the nitroxyl anion
to additionally oxidize these target molecules by releasing HO·
radicals via superoxide-independent pathways.
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Fig. 6.
Effect of superoxide dismutase on both SIN-1-
and Angeli's salt-mediated oxidation reactions. Cu,Zn-SOD (0-500
units/ml) was incubated for 1 h with either DHR or NADH (each 50 µM) in 50 mM potassium phosphate buffer
(37 °C, pH 7.5) in the absence and presence of
HCO/CO2 (25 mM/5%). A, DHR was oxidized by SIN-1 (20 µM). B, DHR was oxidized by Angeli's salt (25 µM). C, NADH was oxidized by SIN-1 (30 µM). D, NADH was oxidized by Angeli's salt
(35 µM). Residual NADH was quantified by reading its
fluorescence with excitation at 339 nm and emission at 460 nm. These
values were corrected for autoxidation of NADH. In the absence of
SIN-1/Angeli's salt, about 97% of the initial NADH concentration (50 µM) could still be detected after 1 h of incubation.
Each value represents the mean ± S.D. of three experiments
performed in duplicate.
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Effect of Nitric Oxide on Nitroxyl Anion-mediated Oxidation
Reactions--
An anonymous referee mentioned that "NO
and nitric oxide would be present simultaneously" under various
conditions, therefore, the nitroxyl anion chemistry presented so far
might be of minor importance for biological systems because the
following reaction sequence is known to rapidly proceed (69,
70).
Provided that such a reaction sequence is indeed effectively
operating, nitroxyl anion-derived formation of peroxynitrite, and thus
of nitrate (see Fig. 2C), should be effectively inhibited by
nitric oxide. To check on this, the inhibitory effects of DEA-NONOate (0-150 µM) on Angeli's salt (200 µM)-mediated formation of nitrate were studied. Since
both compounds have nearly the same half-life at the selected
experimental conditions, nitric oxide and nitroxyl anion should be
generated with approximately the same rate, providing the optimum
conditions for the suggested reaction sequence. In line with the
reviewer's proposal, nitric oxide inhibited in a linear manner
nitroxyl anion-derived formation of nitrate (Fig. 7A). Keeping in mind that one
molecule of DEA-NONOate releases about two equivalents of ·NO it
can be estimated that the nitroxyl anion-induced formation of nitrate
is half-maximally inhibited at a relatively high
[·NO/[NO] ratio of 3.5. On the other hand, as
it is well known that the chemical power of ·NO/O
releasing systems is strongly diminished when the ·NO flux is
twice as much as the O flux (63, 71, 72), one might argue
that this should be especially true for
·NO/NO-releasing systems. This, however, is not
the case. As shown in Fig. 7B, low amounts of DEA-NONOate
(5-50 µM) strongly increase the capabilities of
Angeli's salt (250 µM) to hydroxylate benzoic acid (5 mM) in the presence of CO2. Even at high
concentrations of DEA-NONOate, i.e. 250 µM,
Angeli's salt-mediated hydroxylation of BA is only partially
inhibited. In contrast, the ·NO/O-generating system
SIN-1 did not effectively hydroxylate BA in the presence of
CO2, especially when nitric oxide was additionally released
by spermine-NONOate. Similar to the hydroxylation of benzoic acid,
Angeli's salt (75 µM) and low amounts of DEA-NONOate
(5-50 µM) synergistically acted on the oxidation of 150 µM NADH (Fig. 7C). Thus, the chemical power of
the nitroxyl anion is strongly increased by nitric oxide when the
[·NO/[NO] ratio is 1. On the other hand,
DEA-NONOate inhibited the Angeli's salt-mediated oxidation of NADH
when the [·NO/[NO] ratio is ~2,
i.e. under conditions where Reactions 6-R8 are favored.
Thus, the interplay between ·NO and NO is much
more complicated than hitherto believed, but Reactions 6-R8 may
contribute to the reduction of the (~65%) yield of peroxynitrite from Angeli's salt at sufficiently high nitric oxide
concentrations.
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Fig. 7.
Effect of nitric oxide on the Angeli's
salt-mediated oxidation reactions. Angeli's salt (200, 250, and
75 µM) and various concentrations of DEA-NONOate (0-250
µM) were incubated for 30 min in potassium phosphate
buffer (50 mM, pH 7.5, HCO/CO2 (25 mM/5%), 37 °C) to generate nitrate, to hydroxylate
benzoic acid (5 mM), or to oxidize NADH (150 µM), respectively. A, effect of DEA-NONOate on
Angeli's salt (200 µM)-mediated formation of nitrate.
B, effect of DEA-NONOate on Angeli's salt (250 µM)-mediated hydroxylation of benzoic acid. In
comparison, effect of spermine-NONOate on SIN-1 (250 µM)-mediated hydroxylation of benzoic acid (reaction time
4 h). C, effect of DEA-NONOate on Angeli's salt (75 µM)-mediated oxidation of NADH. Residual NADH was
quantified by reading its fluorescence with excitation at 339 nm and
emission at 460 nm. These values were corrected for autoxidation of
NADH. In the absence of Angeli's salt, about 98% of the initial NADH
concentration (150 µM) could still be detected after 30 min of incubation. Each value represents the mean ± S.D. of three
experiments performed in duplicate.
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DISCUSSION |
Contrary to the observations presented above,
formation of peroxynitrite during decay of Angeli's salt in the
presence of oxygen, i.e. from reaction of NO
and O2, has not been reported in previous studies (37, 38). In the present study we avoided the use of tertiary amines as both
buffer compounds and heavy metal chelators because it has been shown
that oxidizing species stimulate the artificial generation of
O in the presence of these amines (8, 40). The so formed
superoxide reacts in a diffusion-controlled manner with the
NO radicals produced from
peroxynitrite, thus yielding peroxynitrate
(O2NOO) (8, 14, 17). Peroxynitrate is at
physiological pH values of low reactivity in terms of
nitration/oxygenation of prototypical biomolecules (14, 17, 73). As a
consequence, NO-mediated nitration and nitrosation reactions are largely suppressed when O is simultaneously formed. Presumably because of these chain
reactions, the nitroxyl anion-derived formation of peroxynitrite was
not observed in the above mentioned studies, where 10 mM
HEPES or 500 mM triethanolamine (37, 38), respectively, were employed.
Due to spin conservation, the nitroxyl anion released from Angeli's
salt is initially produced in the singlet state (32). Reaction of
1NO with 3O2 is a
spin-forbidden process, thus, 1NO should not
react fast with 3O2. Furthermore, the reaction
of 1NO with 3O2 would
lead to triplet peroxynitrite, 3ONOO, which,
according to ab initio calculations is not a stable molecule (energy minimum).3 On the
other hand, 1NO is isoelectronic to singlet
oxygen. As singlet oxygen decays within a few ms to
3O2 (74), a similar behavior can be expected
for 1NO (Fig.
8). We favor an electron transfer from
3NO to 3O2 with
peroxynitrite being formed from the subsequent reaction of ·NO
with O. The present data do not allow one to say whether it
takes place via an outer sphere or an inner sphere electron transfer
mechanism. One may doubt that superoxide dismutase may effectively
scavenge the NO-derived O radicals, because
Liochev and Fridovich (65) estimated the rate constant for the
oxidation of 3NO by Cu(II),Zn-SOD to 4 × 109 M1 s 1,
i.e. this rate constant is about 100-fold higher than the
rate constant of the reaction between 3NO and
3O2 (75). On the other hand, Murphy and Sies
(24) demonstrated that Cu(I),Zn-SOD can re-reduce ·NO to
NO, and therefore, the reaction between
3NO and 3O2 may even
proceed in the presence of Cu(II),Zn-SOD. The work of Murphy and Sies
(24) is often referred to for demonstrating the direct reaction
between Cu(II),Zn-SOD and NO. These authors observed
that under aerobic conditions Cu(II),Zn-SOD stimulates the formation of
·NO from NO and hypothesized "that SOD accepts
an electron from NO, converting it to ·NO."
Although a reaction between Cu(II),Zn-SOD and NO seems to
be chemically feasible, there is yet no convincing proof that such a
reaction really takes place. According to our data, the superoxide
dismutase-dependent release of ·NO from
NO can satisfactorily be explained by the scavenging of
O by Cu(II),Zn-SOD. Schmidt et al. (27) reported
that the Cu(II),Zn-SOD to NO ratio must be about 50 under
aerobic conditions for achieving a quantitative conversion of
NO to ·NO. However, such a high ratio is in fact
required when superoxide dismutase would scavenge all O
formed to prevent the diffusion-controlled reaction between ·NO
and O.
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Fig. 8.
Proposed pathways of the formation of
peroxynitrite and of the hydroxyl radical from Angeli's salt.
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Our data support the conclusion of Stoyanovsky et al. (55),
that NO forms hydroxyl radicals via a pathway independent
of peroxynitrite. The mechanism offered by these authors appears
plausible (Fig. 8). A recent theoretical study indicates that the
pKa value of HNO is at about 7 (64) rather than at
4.7, as generally assumed (76). Provided that the theoretical
pKa value can be verified by experiment, the
dimerization of two HNO molecules and the subsequent generation of
HO· radicals should even occur at
physiological pH values. We estimated that NO yields
HO· radicals under normoxia with an efficiency of only ~3%
via the peroxynitrite-independent pathway. Given the fact that the
reaction of 3NO with
3O2 strictly depends on the availability of
O2, and as the O2 concentration in various
tissues is significantly lower than under our experimental conditions,
the NO-mediated production of hydroxyl radicals might be
expected to be increased under hypoxic conditions (up to 8% of the
NO yield at physiological oxygen concentrations).
Although there can be no doubt that peroxynitrite and radicals derived
from it effectively damage biomolecules and a variety of cell types,
there is still some uncertainty about the cytotoxic significance of
peroxynitrite in vivo, especially when ·NO and
O are generated from independent sources. There is evidence
that the chemical power of in situ generated peroxynitrite
is maximal, when the ratio (·NO]/[O) is about one
(63, 71, 72). An increase of either the ·NO flux or of the
O flux sharply limits the capability of peroxynitrite to
attack biomolecules. Therefore, it is most likely that the
(·NO]/[O) ratio also rules the cytotoxic potential
of in situ generated peroxynitrite. Since it is highly unrealistic that the ·NO fluxes in vivo are always
identical to the O fluxes, the formation of peroxynitrite
from independent sources of ·NO and O cannot fully
account for the damaging potential which is so far attributed to
peroxynitrite. Otherwise, when both radicals were produced from one
source, e.g. SIN-1, the ratio (·NO]/[O)
is about one and the damaging potential should then rise to a maximum.
This is the case when the nitroxyl anion transfers an electron to
molecular oxygen (Fig. 8). While increasing ·NO concentrations
continuously decrease the chemical power of a ·NO/O
flux (63, 71, 72), a similar behavior cannot simply be expected for
·NO/NO releasing systems, because several
additional reactions (e.g. Reactions 6-R8) may
simultaneously operate. For instance, the chemical reactivity of
NO (hydroxylation of BA, oxidation of NADH) was strongly
increased at a ratio (·NO]/[NO) 1. This might
be related to the reactivity of the protonated form of the intermediate
N2O (Reaction 9) or by further release of
HO· radicals (Reaction 10). In fact, quantum-chemical
calculations at the CBS-QB3 level of theory in conjunction with the
IPCM model for solvation (IPCM-B3LYP/CBS-B3//CBS-QB3) predict that the
homolysis of N2O2H· is an exergonic
reaction (RG(aq)= 28.1
kcal/mol).
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TOP
ABSTRACT
INTRODUCTION
