On the distinction between nitroxyl and nitric oxide using nitronyl nitroxides

Article abstract of DOI:10.1021/ja101945j

A better understanding of the origins of NO and HNO and their activities and biological functions requires accurate methods for their detection and quantification. The unique reaction of NO with nitronyl nitroxides such as 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (C-PTIO), which yields the corresponding imino nitroxides, is widely used for NO detection (mainly by electron paramagnetic resonance spectroscopy) and for modulation of NO-induced physiological functions. The present study demonstrates that HNO readily reacts with nitronyl nitroxides, leading to the formation of the respective imino nitroxides and hydroxylamines via a complex mechanism. Through the use of the HNO donor Angelis salt (AS) with metmyoglobin as a competing agent, the rate constant for C-PTIO reduction by HNO has been determined to be (1.4 ± 0.2) × 10⁵ M^-1 s^-1 at pH 7.0. This reaction yields the corresponding nitronyl hydroxylamine C-PTIO-H and NO, which is trapped by C-PTIO to form ^*NO₂ and the corresponding imino nitroxide, C-PTI. ^*NO₂ oxidizes the nitronyl and imino nitroxides to their respective oxoammonium cations, which decay mainly via comproportionation with the nitronyl and imino hydroxylamines. When [AS] > [C-PTIO], the reduction of C-PTI by HNO proceeds, eventually converting C-PTIO to the corresponding imino hydroxylamine, C-PTI-H. Similar results were obtained for 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO). It is concluded that nitronyl nitroxide is readily reduced by HNO to nitronyl hydroxylamine and is eventually converted into imino nitroxide and imino hydroxylamine. The yield of the imino hydroxylamine increases at the expense of the imino nitroxide as the ratio [AS]₀/[nitronyl nitroxide]₀ is increased. Since the reaction of NO with nitronyl nitroxide yields only the corresponding imino nitroxide, nitronyl nitroxide can discriminate NO from HNO only when present at a concentration much lower than the total production of HNO.

Full text of DOI:10.1021/ja101945j

Published on Web 05/26/2010
On the Distinction between Nitroxyl and Nitric Oxide Using
Nitronyl Nitroxides
Uri Samuni,† Yuval Samuni,‡ and Sara Goldstein*,§
Department of Chemistry and Biochemistry, Queens College, City UniVersity of New York,
Flushing, New York 11367, and Department of Prosthodontics, School of Dental Medicine, and
Institute of Chemistry, The Accelerator Laboratory, The Hebrew UniVersity of Jerusalem,
Jerusalem 91904, Israel
Received March 8, 2010; E-mail: sarag@vms.huji.ac.il
Abstract: A better understanding of the origins of NO and HNO and their activities and biological functions
requires accurate methods for their detection and quantification. The unique reaction of NO with nitronyl
nitroxides such as 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (C-PTIO), which yields
the corresponding imino nitroxides, is widely used for NO detection (mainly by electron paramagnetic
resonance spectroscopy) and for modulation of NO-induced physiological functions. The present study
demonstrates that HNO readily reacts with nitronyl nitroxides, leading to the formation of the respective
imino nitroxides and hydroxylamines via a complex mechanism. Through the use of the HNO donor Angeli’s
salt (AS) with metmyoglobin as a competing agent, the rate constant for C-PTIO reduction by HNO has
been determined to be (1.4 ( 0.2) × 105 M-1 s-1 at pH 7.0. This reaction yields the corresponding nitronyl
hydroxylamine C-PTIO-H and NO, which is trapped by C-PTIO to form •NO2 and the corresponding imino
nitroxide, C-PTI. •NO2 oxidizes the nitronyl and imino nitroxides to their respective oxoammonium cations,
which decay mainly via comproportionation with the nitronyl and imino hydroxylamines. When [AS] >
[C-PTIO], the reduction of C-PTI by HNO proceeds, eventually converting C-PTIO to the corresponding
imino hydroxylamine, C-PTI-H. Similar results were obtained for 2-phenyl-4,4,5,5-tetramethylimidazoline-
1-oxyl 3-oxide (PTIO). It is concluded that nitronyl nitroxide is readily reduced by HNO to nitronyl
hydroxylamine and is eventually converted into imino nitroxide and imino hydroxylamine. The yield of the
imino hydroxylamine increases at the expense of the imino nitroxide as the ratio [AS]0/[nitronyl nitroxide]0
is increased. Since the reaction of NO with nitronyl nitroxide yields only the corresponding imino nitroxide,
nitronyl nitroxide can discriminate NO from HNO only when present at a concentration much lower than
the total production of HNO.
ides.7-10 It has been argued that iron N-methyl-D-glucamine
Introduction
dithiocarbamate can distinguish NO from HNO,11 although the
Nitric oxide (NO) is an important mediator of both physi-
ological and pathophysiological processes.1 HNO, the 1-electron-
reduced and protonated product of NO, has been suggested to
be formed in cellular milieu via several routes.2 A better
understanding of the origins of NO and HNO and their activities
and biological functions requires accurate methods for their
detection and quantification. Electron paramagnetic resonance
(EPR) methods have been developed that enable the stabilization
of NO using endogenous and exogenous spin traps, such as iron
N-methyl-D-glucamine dithiocarbamate3-6 and nitronyl nitrox-
specificity of this method has been questioned.12
The most widely used nitronyl nitroxides are 2-phenyl-4,4,5,5-
tetramethylimidazoline-l-oxyl 3-oxide (PTIO) and its water-
soluble analogue 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimi-
dazoline-1-oxyl 3-oxide (C-PTIO). The unique reaction 1 of
nitronyl nitroxides with NO yields the corresponding imino
nitroxides, which are detectable and distinguishable by EPR
spectrometry.7–10
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† Queens College, City University of New York.
‡ School of Dental Medicine, The Hebrew University of Jerusalem.
§ Institute of Chemistry, The Hebrew University of Jerusalem.
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8428 J. AM. CHEM. SOC. 2010, 132, 8428–8432
10.1021/ja101945j  2010 American Chemical Society

On the Distinction between Nitroxyl and Nitric Oxide
A R T I C L E S
(Tempol),37 which at pH 7.0 has a lower reduction potential
than that of PTIO/C-PTIO (i.e., E1/2 ) 130 compared with 270
mV vs NHE, respectively).38,39
We previously reported that the mechanism of the reaction
of NO with C-PTIO and PTIO involves the oxidation of PTIO
and C-PTIO by •NO2 to their respective oxoammonium cations,
which can be reduced back to the parent nitroxides by NO.39
Here we demonstrate that C-PTIO and PTIO are readily reduced
by HNO and eventually converted into their respective imino
nitroxides or imino hydroxylamines.
Nitronyl nitroxides have also been widely employed as
specific NO scavengers in various experimental in vitro and in
vivo models13-21 to attenuate17,22-31 or even, surprisingly,
potentiate19,32 NO-induced physiological effects. Conversely,
the failure of C-PTIO to affect a system was taken as proof
excluding the intermediacy of NO.33 Several studies even
proposed a therapeutic potential of nitronyl nitroxides against
NO-induced neurotoxicity.15,34,35 However, some of the obser-
vations could not be readily settled with the reported selectivity
of nitronyl nitroxides toward NO.25,32 In cases where NO or
HNO donors induced similar biological effects, the use of
C-PTIO could not clearly distinguish NO from HNO.16,30 It was
therefore suggested that HNO is oxidized to NO by C-PTIO,
albeit slightly,16 but later that reaction was examined and
excluded.36 However, the feasibility of HNO oxidation by
nitronyl nitroxides is supported by the observation that HNO
readily reduces 4-hydroxyl-2,2,6,6 tetramethylpiperidine-N-oxyl
Materials and Methods
All of the chemicals were of analytical grade and used as
received. Water for preparation of the solutions was purified
using a Milli-Q purification system. Angeli’s salt (Na2N2O3, AS)
and C-PTIO were purchased from Cayman Chemical Co. and
PTIO from ALEXIS Biochemicals. A stock solution of AS was
prepared in 10 mM NaOH, and the concentration was determined
by the absorbance at 248 nm (ε ) 8 mM-1 cm-1). Metmyoglobin
(MbFeIIIOH2) was prepared by addition of excess ferricyanide
to myoglobin in 10 mM phosphate buffer (PB) at pH 7.0
followed by chromatographic separation through a Sephadex
G-25 column. The concentration of MbFeIIIOH2 was determined
spectrophotometrically using ε408 ) 188 mM-1 cm-1 40 NO was
.
purchased from Matheson Gas Products and purified by passing
it through a series of scrubbing bottles containing deaerated 50%
NaOH and purified water in that order. NO solutions were
prepared in gas-tight syringes containing 10 mM PB, and the
concentration of NO (1.9 mM/atm at 22 °C) was accurately
determined on the basis of its known temperature- and pressure-
dependent solubility in aqueous systems.
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Care Med. 1997, 25, 1019–1029.
C-PTI was prepared by mixing NO-saturated solution with a
deaerated solution of C-PTIO in 10 mM PB at pH 7.0. Oxidized
and reduced nitronyl and imino nitroxides were prepared electro-
chemically. Briefly, the electrochemical cell consisted of a working
electrode of graphite grains packed inside a porous Vycor glass
tube (5 mm i.d.) through which the solution of nitronyl or imino
nitroxides in 10 mM PB (pH 7.0) was pumped (2-4 mL min-1).
An outer glass cylinder contained 10 mM PB in which a Pt auxiliary
electrode and a Ag/AgCl (3.5 M) reference electrode were
immersed. A BAS100B Electrochemical Analyzer was used to
control the voltage.
Oxidation of imino hydroxylamines was achieved either elec-
trochemically or by addition of 1 mM ferricyanide as described
previously.41 In systems containing residual AS, a mixture of 10
mM H2O2 and 5 µM CuSO4 was added prior to the addition of
ferricyanide.
Cyclic voltammetry (CV) was performed with a BAS100B
electrochemical analyzer using a three-electrode system consisting
of a glassy carbon working electrode, a Pt wire auxiliary electrode,
and a Ag/AgCl (3.5 M) reference electrode.
Stopped-flow kinetic measurements were carried out using an
Applied Photophysics Bio SX-17MV sequential stopped-flow
apparatus having a 1 cm optical path.
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Kinetic measurements were carried out at 25 °C using an HP
8452A diode-array spectrophotometer coupled with a thermostat
(HP 89075C Programmable Multicell Transport).
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Samuni et al.
Figure 1. Conversion of C-PTIO to C-PTI upon exposure of 170 µM
C-PTIO to 350 µM AS in an aerated solution containing 40 mM PB at pH
7.0: (a) t ) 0, gain ) 7.9; (b) t ) 1 min, gain ) 7.9; (c) t ) 8 min, gain
) 16; (d) t ) 38 min, gain ) 20. A spectrum identical to (d) was observed
at t ) 57 min with and without the addition of 1 mM ferricyanide.
Figure 2. Conversion of C-PTIO to C-PTI-H upon exposure of 180 µM
C-PTIO to 1 mM AS in an aerated solution containing 100 mM PB at pH
7.0: (a) t ) 0, gain ) 12.5; (b) t ) 4 min, gain ) 12.5; (c) t ) 8 min, gain
) 40; (d) t ) 28 min, gain ) 400; (e) t ) 62 min, gain ) 12.5, obtained
after the addition of CuSO4/H2O2/ferricyanide.
EPR spectra were recorded using a Varian E-9 spectrometer and
repeated using JEOL X-band JES-RE3X and Bruker X-band
spectrometers operating at 9.25 GHz with the following settings:
center field, 3290 G; modulation frequency, 100 kHz; modulation
amplitude, 1 G; time constant, 0.128 s; field sweep, 25 G/min;
incident microwave power, 4-16 mW. The reaction mixture was
transferred to a gas-permeable Teflon capillary (Zeus Industries,
Orangeburg, SC) having an i.d. of 0.81 mm, a wall thickness of
0.38 mm, and a length of 15 cm. Each capillary was folded twice,
inserted into a narrow quartz tube that was open at both ends (2.5
mm inner diameter), and placed within the EPR cavity. All of the
experiments were carried out at room temperature.
Results and Discussion
The reaction of HNO with PTIO and C-PTIO was studied
using the HNO donor AS42,43 in aerated solutions at pH 7.0.
Upon exposure of 170 µM C-PTIO to 350 µM AS, the seven-
line EPR spectrum of C-PTI appeared at the expense of the
five-line EPR spectrum of C-PTIO (Figure 1). Figure 1b,c shows
the buildup of the nine-line EPR spectrum indicative of a
mixture of C-PTIO and C-PTI. Upon exposure of 180 µM
C-PTIO to 1 mM AS, the EPR spectrum of C-PTI appeared
and was progressively lost during the decay of AS (Figure
2a-d). However, C-PTI was fully restored upon oxidation
(Figure 2e), indicating that the EPR-silent final product was
C-PTI-H. The same results were observed in the case of PTIO.
Huang et al.36 reported that AS at 10 µM had no effect on the
EPR spectrum of C-PTIO at 4 µM. However, a closer examina-
tion of their findings clearly reveals the emergence of the C-PTI
signal.
Figure 3. UV-vis spectra of C-PTI, C-PTI-H, C-PTIO, C-PTIO-H, and
C-PTIO+. C-PTI, C-PTI-H, C-PTIO-H, and C-PTIO+ were prepared as
described in Materials and Methods.
Knowledge of the spectral characteristics of C-PTIO, C-PTI,
and their reduced and oxidized forms (Figure 3) also enables
the study of the reaction of HNO with C-PTIO through
monitoring of the UV-vis absorption of C-PTIO upon exposure
to AS. Figure 4 shows the decay of C-PTIO (190 µM) to C-PTI
upon exposure to 350 µM AS in an aerated solution at pH 7.0.
The addition of 1 mM ferricyanide did not restore the absorption
Figure 4. UV-vis spectra obtained upon exposure of 190 µM C-PTIO to
350 µM AS in an aerated solution containing 40 mM PB (pH 7.0, 25 °C)
at t ) 30, 60, 90, 130, 190, 250, 350, 450, 550, and 1100 s.
at 560 nm, indicating that C-PTIO-H did not accumulate during
this process. The AS-induced decay of C-PTIO followed at 560
nm and 25 °C obeyed first-order kinetics with a rate constant
of k ) (2.6 ( 0.2) × 10-3 s-1, whereas the decay of AS alone
followed at 240 nm demonstrated k ) (1.25 ( 0.05) × 10-3
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1404.
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On the Distinction between Nitroxyl and Nitric Oxide
A R T I C L E S
s-1. These rate constants were unaffected by the presence of
100 µM diethylenetriaminepentaacetic acid (DTPA).
The results described above demonstrate that C-PTIO and
PTIO are fully converted to the respective imino nitroxides and
imino hydroxylamines upon exposure to AS. We suggest that
the initiating reaction of nitronyl nitroxide with HNO yields
the corresponding nitronyl hydroxylamine and NO (reaction 2):
PTIO-H + PTI+ f PTIO + PTI + H+
(7)
We have previously shown that the comproportionation in the
case of cyclic nitroxides (e.g., Tempol) proceeds mainly through
the deprotonated form of the corresponding hydroxylamines.45
Since C-PTIO-H (pKa ) 3.5)39 is a stronger acid than Tempol-H
(pKa ) 6.9),45 the apparent rate constants of reactions 6 and 7
are expected to be significantly larger than that of Tempol-H
comproportionation with its corresponding oxoammonium cation
at pH 7.0 (i.e., 24 M-1 s-1 45).
In addition to reactions 6 and 7, C-PTIO-H having a nitronyl
group can react with NO similarly to C-PTIO, forming C-PTI-H
•
and NO2 (reaction 8):
The NO is then trapped by nitronyl nitroxide to form the
•
corresponding imino nitroxide and NO2 (reaction 1).
The conversion of the imino nitroxide to imino hydroxylamine
occurred when the nitronyl nitroxide was exposed to 1 mM AS
but not to 0.35 mM AS, suggesting that the imino nitroxides
are reduced by the excess HNO to their corresponding imino
hydroxylamines (reaction 3):
To support or exclude the proposed mechanism, the kinetics
of reactions 2, 6, 7, and 8 were studied. Reaction 2 was studied
using competition kinetics against metmyoglobin (reaction 9):
MbFeIIIOH2 + HNO f MbFeIINO + H+ + H2O (9)
The addition of 44 µM AS to 10 or 30 µM MbFeIIIOH2 in
aerated buffered solutions resulted in the same initial rate of
MbFeIINO formation, which was followed at 425 nm. This
C-PTIO was fully converted to C-PTI and C-PTI-H, indicating
that C-PTIO-H, which is formed via reaction 2, does not
accumulate. The possibility that NO2, which is formed via
observation implies that under such experimental conditions the
46
self-decomposition of HNO (k ) 8 × 106 M-1 s-1
)
and its
•
37
reaction with O2 (k ∼ 3 × 103 M-1 s-1
)
are negligible. The
reaction 1, oxidizes C-PTIO-H to C-PTIO has been proposed
initial rate of MbFeIINO formation was measured upon the
addition of 44 µM AS to aerated buffered solutions containing
10 µM MbFeIIIOH2 in the absence (V0) and presence (V) of
various concentrations of C-PTIO or PTIO. The effect of added
nitronyl nitroxide on the initial rate follows eq 10 if only
reactions 2 and 9 take place:
previously.41,44 However, this reaction proceeds slowly (k e 1
41,45
× 105 M-1 s-1
)
and cannot efficiently compete with the
•
oxidation of PTIO and C-PTIO by NO2 to their respective
oxoammonium cations PTIO+ and C-PTIO+ [reaction 4; k4 )
(1.5 - 2.0) × 107 M-1 s-1].39
k2
1
V
1
V0
[nitronyl nitroxide]
[MbFeIIIOH2]
)
+
(10)
(
)
k9V0
Accordingly, a plot of V0/V - 1 versus [nitronyl nitroxide]
should be linear, as demonstrated in Figure 5. The slope of the
line in Figure 5 yields k2/k9 ) 0.18 ( 0.02 for both PTIO and
The CV of an aerated solution containing 0.25 mM C-PTI, 0.1
M KNO3, and 10 mM PB (pH 7.0) exhibited a reversible
oxidation of C-PTI to C-PTI+ having E1/2 ) 550 ( 7 mV vs
NHE. This value is significantly lower than that for C-PTIO
(936 ( 21 mV vs NHE),39 suggesting that k5, the rate constant
for reaction 5, might be even larger than k4.
C-PTIO, and thus, k2 ) (1.4 ( 0.2) × 105 M-1 s-1 using k9 )
37
8 × 105 M-1 s-1
.
The kinetics of reactions 6 and 7 were studied under limiting
concentrations of C-PTIO+ or C-PTI+ using the stopped-flow
technique. The decays of C-PTIO+ and C-PTIO-H were
followed at 450 and 320 nm, respectively. Both reactions obeyed
pseudo-first-order kinetics, and kobs was linearly dependent on
[C-PTIO-H], yielding k6 ) (1.2 ( 0.1) × 106 M-1 s-1 and k7 )
(2.1 ( 0.3) × 107 M-1 s-1 (Figure 6).
The kinetics of the reaction of C-PTIO-H with NO was
studied using the stopped-flow technique under limiting con-
centrations of C-PTIO-H by following its decay at 320 nm. The
decay of the absorption obeyed pseudo-first-order kinetics, and
kobs was linearly dependent on [NO], yielding k8 ) (1.7 ( 0.2)
× 103 M-1 s-1 (Figure 7). The product of this reaction was
identified as C-PTI-H, as it was oxidized electrochemically to
C-PTI, where [C-PTI] ) [C-PTIO-H]0. This result indicates that
The failure of C-PTIO-H to accumulate could be due to its
reaction with C-PTIO+ (reaction 6) and/or C-PTI+ (reaction 7):
PTIO-H + PTIO+ f 2PTIO + H+
(6)
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A R T I C L E S
Samuni et al.
Figure 5. Effects of nitronyl nitroxide on the initial rate of MbFeIINO
formation analyzed according to eq 10. The rate of MbFeIINO formation
was measured at 425 nm upon exposure of 10 µM MbFeIIIOH2 to 44 µM
AS in aerated solutions containing 10 mM PB at pH 7.0 and 22 °C.
Figure 7. Dependence of the pseudo-first-order rate constant of the decay
of C-PTIO-H on [NO] at pH 7.0 and 22 °C. Equal volumes of C-PTIO-H
(120 µM, 10 mM PB, Ar-saturated) and NO (0.9-1.8 mM, 10 mM PB)
were mixed using the stopped-flow apparatus, and the decay of C-PTIO-H
was followed at 320 nm.
Scheme 1. Mechanism Underlying the Conversion of PTIO to PTI
and PTI-H by HNO at pH 7.0a
Figure 6. Dependence of the pseudo-first-order rate constants for the decays
of C-PTIO+ and C-PTI+ on [C-PTIO-H] at pH 7 and 22 °C. Equal volumes
of C-PTIO-H (9-356 µM) and C-PTIO+ (20 µM) or C-PTI+ (2-20 µM),
all in 10 mM PB, were mixed using the stopped-flow apparatus, and the
decay of the absorption was followed at 450 or 320 nm, respectively.
a Reaction numbers are the same as listed in the text; reactants are written
in enlarged bold text.
•NO2 formed in reaction 8 reacts with the excess NO to yield
N2O3, which hydrolyzes to nitrite.
In conclusion, the reaction of HNO with nitronyl nitroxide
forms the corresponding imino nitroxide and hydroxylamine.
The yield of the imino hydroxylamine increases at the expense
of the imino nitroxide with an increase in the ratio [AS]0/
[nitronyl nitroxide]0, where [AS]0 represents the total production
of HNO. Since the reaction of NO with nitronyl nitroxide yields
only the corresponding imino nitroxide, nitronyl nitroxide can
discriminate between NO and HNO only when used at a
concentration much lower than the total production of HNO.
We have shown that C-PTIO is converted into C-PTI and
C-PTI-H by HNO. The yield of C-PTI-H increases at the
expense of C-PTI as the ratio [AS]0/[C-PTIO]0 increases. The
mechanism of the reaction of HNO with PTIO and C-PTIO
involves the participation of several reactive species, including
•
NO, NO2, and oxoammonium cations, as demonstrated in
Scheme 1 for PTIO. Reaction 8 has not been included in Scheme
1, as it does not compete efficiently with the comproportionation
of PTIO-H with PTIO+ and PTI+. The mechanism is more
complex than what is shown in Scheme 1, and quantitative
analysis should also include the oxidation of the nitronyl and
imino nitroxides by the strongly oxidizing hyponitrite ion radical
N2O2•-, which is formed via the reaction of HNO with NO.47,48
JA101945J
(47) Poskrebyshev, G. A.; Shafirovich, V.; Lymar, S. V. J. Am. Chem.
Soc. 2004, 126, 891–899.
(48) Poskrebyshev, G. A.; Shafirovich, V.; Lymar, S. V. J. Phys. Chem. A
2008, 112, 8295–8302.
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8432 J. AM. CHEM. SOC. VOL. 132, NO. 24, 2010