Oxidation of N-hydroxy-L-arginine by hypochlorous acid to form nitroxyl (HNO)

Article abstract of DOI:10.1016/j.jinorgbio.2012.09.024

Recent research has shown that nitroxyl (HNO) has important and unique biological activity, especially as a potential alternative to current treatments of cardiac failure. HNO is a reactive molecule that undergoes efficient dimerization and subsequent deh

Full text of DOI:10.1016/j.jinorgbio.2012.09.024

Journal of Inorganic Biochemistry 118 (2013) 148–154
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Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Oxidation of N-hydroxy-L-arginine by hypochlorous acid to form nitroxyl (HNO)
⁎
Meredith R. Cline, Tyler A. Chavez, John P. Toscano
Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 June 2012
Received in revised form 1 September 2012
Accepted 1 September 2012
Available online 5 October 2012
Recent research has shown that nitroxyl (HNO) has important and unique biological activity, especially as a
potential alternative to current treatments of cardiac failure. HNO is a reactive molecule that undergoes effi-
cient dimerization and subsequent dehydration to form nitrous oxide (N2O), making its detection in solution
or biologically relevant preparations difficult. Due to this limitation, HNO has not yet been observed in vivo,
though several pathways for its endogenous generation have been postulated. Here, we investigate the oxi-
dation of N-hydroxy-L-arginine (NOHA) by hypochlorous acid (HOCl), which is generated in vivo from hydro-
gen peroxide and chloride by the heme enzyme, myeloperoxidase. NOHA is an intermediate in the enzymatic
production of nitric oxide (NO) by NO synthases, and has been shown previously to be chemically oxidized
to either HNO or NO, depending on the oxidant employed. Using membrane inlet mass spectrometry and
standard N2O analysis by gas chromatography, we find that NOHA is oxidized by excess HOCl to form
HNO-derived N2O. In addition, we also observe the analogous production of HNO from the HOCl oxidation
of hydroxylamine, hydroxyurea, and (to a lesser extent) acetohydroxamic acid.
Keywords:
Nitroxyl
HNO
N-Hydroxy-L-arginine
Hypochlorous acid
Myeloperoxidase
Membrane inlet mass spectrometry
© 2012 Elsevier Inc. All rights reserved.
1. Introduction
NOHA is a well-studied biosynthetic intermediate in the
NOS-mediated production of NO (and L-citrulline) from L-arginine
(Scheme 1) [23,24]. NOHA, which can be uncoupled from NOS at levels
up to 15 μM both in vitro and in vivo [25,26], has also been shown to be a
potent inhibitor of arginase, regulating the production of L-arginine
[27,28]. In addition, research has demonstrated that NOHA can be
chemically oxidized to produce either NO or HNO depending on the ox-
idant used [29–31]. Likewise, related N-hydroxyguanidines have been
shown to form either HNO or NO depending upon the oxidative condi-
tions [32]. The HNO-producing pathway presumably involves a nitroso
intermediate and the corresponding production of a cyanamide deriva-
tive (Scheme 2).
A biologically relevant oxidant that may be able to generate HNO
via the oxidation of NOHA is hypochlorous acid (HOCl). HOCl, a strong
oxidant that has been shown to be both bactericidal and virucidal
[33,34], is generated in vivo by myeloperoxidase (MPO) from the re-
action of hydrogen peroxide (H2O2) with chloride ion. MPO is re-
leased by neutrophils, monocytes, and tissue macrophages at sites
of inflammation [35–37], and may play a role in cardiovascular dis-
ease [38–40]. Activated neutrophils have been shown to produce
HOCl at concentrations up to 100 μM in vitro and similar or higher
levels may be possible at inflammatory sites in vivo [41–43]. The reac-
tion of HOCl with biologically relevant thiols such as glutathione
(GSH) generates oxidized thiol products such as sulfonamides, gluta-
thione disulfide, and thiolsulfonates [44], while reaction of HOCl with
amines produces chloramines.
Nitroxyl (HNO), the protonated and one-electron reduced sibling
of nitric oxide (NO), has been shown to be involved in chemistry
that has a positive impact on vasorelaxation and contractility in the
cardiac system making it a potentially novel therapeutic agent for
use in the fight against heart disease [1–6]. HNO is a reactive species
that dimerizes rapidly (k=8×106M−1 s−1) and subsequently dehy-
drates to form nitrous oxide (N2O) [7]. As a result, HNO cannot be
used directly and must be generated in situ using donor molecules.
Given its inherent reactivity, HNO has proven difficult to detect directly.
Recent methods have been developed for HNO detection [8–15]; how-
ever, due to a lack of sensitivity and/or selectivity, HNO has not yet
been observed in vivo.
Despite the above limitation, numerous reports support the
idea that HNO can be produced endogenously. Research has shown
that nitrosothiols can react with thiols to form HNO (and disulfide)
[16,17]. HNO can also be produced from nitric oxide synthases
(NOS) under conditions where the tetrahydrobiopterin cofactor is
absent or in low concentrations [18–21]. In addition, recent work by
Donzelli et al. examined whether the oxidation of hydroxylamine or
N-hydroxy-L-arginine (NOHA) by a number of heme peroxidases pro-
duces HNO [22]. Although heme peroxidases were found to oxidize
hydroxylamine to HNO, the analogous oxidation of NOHA did not
produce HNO.
To our knowledge, the oxidation of NOHA by HOCl has not yet
been reported. Herein, we examine the reaction of NOHA with HOCl
and show that HNO is generated under conditions where HOCl is in
⁎
Corresponding author. Tel.: +1 410 516 6534; fax: +1 410 516 7044.
E-mail address: jtoscano@jhu.edu (J.P. Toscano).
0162-0134/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jinorgbio.2012.09.024

M.R. Cline et al. / Journal of Inorganic Biochemistry 118 (2013) 148–154
149
headspace vial using a gastight syringe, yielding a final HNO donor
or substrate concentration of 100 μM. Samples were then incubated
for 1.5 h to ensure complete equilibration of N2O in the headspace.
Headspace (60 μL) was then sampled five successive times using a
gastight syringe with a sample lock. HNO yields are reported relative
to the HNO donor, Angeli's salt, which by comparison to standard N2O
samples we find provides an 80% absolute yield of HNO.
2.2.2. Membrane inlet mass spectrometry design and methods
Scheme 1. Nitric oxide synthase (NOS) production of NO and L-citrulline from L-arginine
via N-hydroxy-L-arginine.
Mass spectra were obtained using a Balzers Prisma QMS-200 mass
spectrometer. Our membrane inlet probe is modeled after the mem-
brane inlet design of Silverman and co-workers with a few modifica-
tions [51]. The membrane probe is comprised of a length of Silastic
tubing (1.5 mm i.d. and 2.0 mm o.d.) that is sealed at one end by a
glass bead and attached at the other end to a piece of glass tubing
that is attached via swagelok to an external vacuum chamber
containing a dosing line that leads directly into the source of the
Balzers Prisma QMS-200 mass spectrometer located in a main vacu-
um chamber (Fig. 1). The length of the Silastic tubing between the
glass bead and the glass tubing is 1 cm. The Silastic tubing is
prevented from collapsing in the partial vacuum of the inlet by a
coiled piece of fine stainless steel wire. The membrane is immersed
in the solution to be analyzed in a sealable 4 mL glass sample cell
modified for the introduction of samples. The buffer solution and
the sample cell are degassed and purged with a continuous flow of
argon for 15 min before experimentation. The deaerated buffer solu-
tion (4 mL) is then injected into the deaerated sample cell and at-
tached to the system via an ultra torr swagelok connection. The
external vacuum chamber is evacuated and then opened via a leak
valve to the main chamber where the mass spectrometer is located.
A dry ice/acetone ice trap is placed between the sample cell and the
leak valve to trap water vapor.
Injections of oxidants occurred after several minutes of back-
ground signal collection. When additional reactants were added,
they were injected prior to the oxidant and given 10 min to equili-
brate. Ion currents were measured for at least 30 min and data were
collected at m/z 30 (NO+) and 44 (N2O+). Data are reported follow-
ing baseline subtraction of the observed ion current at the start of an
experiment. Scans from m/z 0 to m/z 100 were also collected. Mass
spectra were obtained by electron impact (EI) ionization (70 eV) at
an emission current of 1 mA; source pressures were approximately
5×10−7–1×10−6 Torr.
excess of NOHA. HNO production is confirmed by both membrane
inlet mass spectrometry (MIMS) techniques and standard N2O analysis
by gas chromatography. The analogous production of HNO from the
HOCl oxidation of hydroxylamine, hydroxyurea, and (to a lesser extent)
acetohydroxamic acid is also observed.
2. Materials and methods
2.1. Materials
Unless otherwise noted chemicals were obtained from Aldrich
Chemical Company or Fisher Scientific and used without further puri-
fication. N-hydroxy-L-arginine was purchased from Chemdea. Hydro-
gen peroxide (30% v/v; Merck) was quantified by absorbance at
240 nm using a molar absorption coefficient of 39.4 M−1 cm−1
[45]. Hypochlorous acid (4–5%) was quantified by injecting NaOCl into
a 0.01 M NaOH and measuring the absorbance at 292 nm where the
molar absorption coefficient of the hypochlorite ion is 350 M−1 cm−1
[46]. Dimethyl-sulfoxide-d6 and chloroform-d were obtained from Cam-
bridge Isotope Labs. Nitrous oxide was obtained from Airgas. Angeli's
salt (AS) and sodium 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate
(DEA/NO) were synthesized according to literature procedure [47–49].
The HNO donor, 2-bromo-N-hydroxybenzenesulfonamide (2-bromo-
Piloty's acid, 2BrPA), was synthesized following a recent report [50].
Phosphate-buffered saline (PBS) solutions (0.1 M) were prepared with
140 mM NaCl, and 3 mM KCl, and adjusted to pH 7.4. Since we observed
that the metal chelator, diethylenetriaminepentaacetic acid (DTPA), was
oxidized by HOCl, it was not included in our buffer solutions. However,
presumably because all experiments were conducted under anaerobic
conditions, representative MIMS and gas chromatographic (GC) head-
space analysis experiments in the presence or absence of 100 μM DTPA
provided similar results.
3. Results and discussion
2.2. Methods
3.1. Utility of MIMS to study possible endogenous pathways for HNO
production
2.2.1. Gas chromatographic (GC) headspace analysis of N2O
Gas chromatographic headspace analysis of N2O was performed
using a Varian CP-3800 gas chromatograph instrument equipped
with a 1041 manual injector, electron capture detector, and a Restek
packed column. Samples were prepared in 15 mL vials with volumes
pre-measured for uniformity. Vials were filled with 5 mL PBS buffer,
sealed with a rubber septum, and then purged with argon for
10 min. The vials were equilibrated at 37 °C for 10 min in a block
heater. An aliquot of an HNO donor solution or other relevant sub-
strate species was then introduced into the thermally equilibrated
MIMS is a method used to detect gases dissolved in liquid phases
through the use of a semipermeable hydrophobic membrane that
Scheme 2. Oxidation of N-hydroxyguanidines to produce HNO and a cyanamide deriv-
ative via a nitroso intermediate.
Fig. 1. Schematic representation of membrane inlet mass spectrometry (MIMS) method.

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M.R. Cline et al. / Journal of Inorganic Biochemistry 118 (2013) 148–154
2.5
2.0
1.5
1.0
0.5
0.0
Table 2
Oxidation of NOHA by HOCl.
m/z = 30, NH2OH
m/z = 44, NH2OH
m/z = 30, NOHA
m/z = 44, NOHA
HOCl (μM)
% HNOa
20
3
11
22
60
65
100
250
500
1000
a
NOHA (100 μM) was incubated at 37 °C in 0.1 M
PBS, pH 7.4 with varying amounts of HOCl. HNO yields
are reported relative to the HNO donor, Angeli's salt,
as determined by N2O headspace analysis (SEM
5%; n≥3).
0
400
800
1200
1600
Time (s)
to substrate) was selected for future experiments. Although it is unlike-
ly that HOCl is present in vivo at this concentration, a 5:1 oxidant to sub-
strate ratio is potentially plausible since HOCl can be generated at
concentrations up to 100 μM and NOHA has been observed at concen-
trations of 15 μM in blood samples [25,26,41–43].
Fig. 2. The oxidation of 500 μM hydroxylamine by 10 μM HRP and 100 μM H2O2 to
produce HNO-derived N2O compared to the lack of oxidation observed for the same
reaction carried out with 500 μM NOHA at 37 °C in 0.1 M PBS, pH 7.4.
MIMS experiments following the addition of 500 μM HOCl to a PBS
solution containing 100 μM NOHA reveal a rise in both the m/z 30 and
44 signals (Fig. 3). The m/z 44:30 intensity ratio is 2:1 for standard
N2O; however, in Fig. 2 the observed m/z 44:30 ratio is 3:1. We pos-
tulate that some of the m/z 44 signal is due to the detection of carbon
dioxide (CO2), which originates from another oxidative process. Pre-
vious work has shown that HOCl is capable of oxidizing amino
acids, including L-arginine, to form first monochloramines and ulti-
mately aldehydes along with CO2 (Scheme 3) [59,60].
allows the dissolved gases, but not the liquid phase, to enter a mass
spectrometer [52–54]. It has proven useful for the study of NO in
aqueous and blood solutions [51,55–57]. Recently, we have reported
its utility for the study of HNO in aqueous solutions [13]. Although
the low intensity of the parent m/z 31 MIMS signal for HNO currently
precludes its unambiguous detection, its EI fragmentation product
can be detected at m/z 30 (NO+). (The relative intensity of the m/z
30 vs 31 in the reported EI mass spectrum of HNO is greater than
35:1 [58].) At high enough precursor concentrations, MIMS signals
for N2O, the product of HNO dimerization, can also be observed at
m/z 44 (N2O+ parent ion) and 30 (NO+ fragment ion), with a 44:30
intensity ratio of approximately 2:1.
3.3. Reaction of hypochlorous acid with L-arginine and L-arginine methyl
ester
To demonstrate that MIMS is a viable technique for the study of
possible endogenous pathways for HNO production, we examined
the oxidation of hydroxylamine and NOHA by horseradish peroxidase
(HRP) and H2O2 following the conditions reported by Donzelli et al.
(10 μM HRP with 100 μM H2O2 and either 500 μM hydroxylamine
or NOHA) [22]. As shown in Fig. 2, under these conditions we detect
MIMS signals at m/z 44 and 30 in a 2:1 ratio characteristic of the pro-
duction of N2O for hydroxylamine, but not for NOHA, consistent with
the previous findings.
To determine if the free carboxylic acid group of NOHA was re-
sponsible for the observation of a larger than expected m/z 44:30
ratio, we investigated the oxidation of L-arginine and L-arginine
methyl ester by HOCl. The addition of 500 μM HOCl to a solution of
100 μM L-arginine generated a large increase in the m/z 44 signal.
4
These results were corroborated using standard N2O headspace
analysis by GC. The incubation of hydroxylamine with HRP and
H2O2 produced N2O at a comparable percentage to that previously
reported [22], while the analogous incubation of NOHA produced no
N2O under the same 5:1 substrate to oxidant conditions (Table 1).
When the H2O2 concentration was increased, a small amount of N2O
was observed from the oxidation of NOHA.
3
m/z = 44
2
m/z = 30
1
3.2. Reaction of N-hydroxy-L-arginine with hypochlorous acid
The reaction of NOHA with HOCl was first examined by GC head-
space analysis to quantify the amount of N2O produced following the in-
cubation of NOHA (100 μM) with varying concentrations of HOCl.
Significant N2O was produced only with excess HOCl (Table 2). From
these results, a concentration of 500 μM HOCl (and 5:1 ratio of oxidant
0
0
400
800
1200
1600
Time (s)
Fig. 3. MIMS response at m/z 30 and 44 following the addition of 500 μM HOCl to a
solution containing 100 μM NOHA at 37 °C in 0.1 M PBS, pH 7.4.
Table 1
Oxidation of NH2OH and NOHA by the HRP/H2O2 system.
Substrate
H2O2 (μM)
% HNOa
NH2OH
NOHA
NOHA
20
20
200
12
0
2
a
Substrates (100 μM) were incubated with 2 μM HRP at 37 °C in 0.1 M PBS, pH 7.4.
HNO yields are reported relative to the HNO donor, Angeli's salt, as determined by N2O
headspace analysis (SEM 5%; n≥3).
Scheme 3. The reaction of HOCl with amino acids.

M.R. Cline et al. / Journal of Inorganic Biochemistry 118 (2013) 148–154
151
1.2
1.0
0.8
0.6
0.4
0.2
0.0
40
5.0 µM NOHA, 25 µM HOCl
2.5 µM NOHA, 12.5 µM HOCl
1.0 µM NOHA, 5.0 µM HOCl
5.0 µM HOCl
(a)
30
m/z = 30, Arginine
m/z = 44, Arginine
m/z = 30, Arginine Methyl Ester
m/z = 44, Arginine Methyl Ester
20
10
0
0
400
800
1200
1600
0
500
1000
1500
Time (s)
Time (s)
Fig. 4. The MIMS responses observed at m/z 30 and 44 following the addition of
500 μM HOCl to either 100 μM L-arginine or 100 μM L-arginine methyl ester at 37 °C
in 0.1 M PBS, pH 7.4.
150
100
50
5.0 µM NOHA, 25 µM HOCl
2.5 µM NOHA, 12.5 µM HOCl
1.0 µM NOHA, 5.0 µM HOCl
5.0 uM HOCl
(b)
When the same experiment was performed with 100 μM L-arginine
methyl ester, a significant decrease in the m/z 44 signal is observed
(Fig. 4). (The small m/z signal (ca. 0.3 nA) remaining with L-arginine
methyl ester suggests an additional oxidative pathway that also forms
CO2.) These results indicate that the reaction shown in Scheme 3 is
likely responsible for the additional signal observed in Fig. 3 at m/z
44 via generation of CO2. Further support of the production of aldehyde
and CO2 is the observation of hydrazone formation upon reaction of the
L-arginine–HOCl product mixture with 2,4-dinitrophenylhydrazine
and barium carbonate formation upon reaction with barium chloride,
respectively.
0
0
400
800
1200
1600
Time (s)
To confirm that the m/z 44 signal observed for L-arginine upon ox-
idation by HOCl is not due to N2O, we performed GC headspace anal-
ysis experiments (100 μM L-arginine plus 500 or 1000 μM HOCl) and
observe no evidence for N2O formation. Additional experiments using
H2O2 (500 μM) as the oxidant instead of HOCl, as well an experiment
where both HRP and H2O2 (2 and 20 μM, respectively) were added to
L-arginine also did not result in the production of N2O.
Fig. 5. MIMS response for the reaction of varying concentrations of NOHA and HOCl
(maintaining a 5:1 substrate to oxidant ratio) at (a) m/z 30 and (b) m/z 44 at 37 °C
in 0.1 M PBS, pH 7.4.
To overcome this limitation, we turned to the use of a physical
trap. NO has a boiling point of −152 °C, while the boiling point of
HNO is not known. We estimate that the boiling point of HNO is
higher than that of NO, and hypothesized that we could selectively
trap HNO and N2O (bp=−88 °C) using an appropriate cold trap.
Cold traps with boiling points between −90 °C and −160 °C were
initially attempted, but since our system is under vacuum, they all
allowed N2O to pass through unaffected. Control experiments with
standard N2O gas and HNO (produced from 500 nM 2BrPA), however,
show that both species can be completely trapped by liquid nitrogen
(bp=−196 °C) (Fig. 6). In contrast, most of the NO (generated from
DEA/NO) is observed to flow through the liquid nitrogen cold trap.
When a liquid nitrogen cold trap is used for the MIMS examination
of the reaction between 100 μM NOHA and 500 μM HOCl, no signal
at m/z 30 is detected, indicating that NO is not produced (Fig. 7).
3.4. Confirmation that HNO is produced by the hypochlorous acid oxida-
tion of N-hydroxy-L-arginine
GC headspace analysis shows that N2O is produced upon the oxi-
dation of NOHA by HOCl. To demonstrate that the N2O formed is
the result of HNO dimerization rather than direct production, we ex-
amined low concentrations of NOHA and HOCl by MIMS maintaining
the same 5:1 HOCl to NOHA ratio, which was determined to be opti-
mal for N2O production. Using the HNO donor 2BrPA, we have previ-
ously shown that we can detect HNO directly by lowering the
concentration of 2BrPA to a level (low micromolar) where dimeriza-
tion to N2O is not observed by MIMS at m/z 44. Only the m/z 30 sig-
nal, corresponding to NO+ formed from the fragmentation of HNO, is
observed. As shown in Fig. 5, at 5 μM NOHA and 25 μM HOCl the same
3:1 m/z 44 to m/z 30 signal is observed as in the higher concentration
experiment (Fig. 2). As the concentrations of NOHA and HOCl are
lowered, we observe a decrease in the m/z 44:30 ratio indicating
that we are producing less N2O and CO2. Below 2.5 μM NOHA we ob-
serve only the m/z 30 MIMS signal, corresponding to the direct detec-
tion of HNO.
In previous work, to rule out the possibility that the observed m/z
30 MIMS signal is not due to the parent ion of NO (rather than HNO
fragmentation) we have used selective chemical traps for HNO (e.g.,
thiols under anaerobic conditions or phosphines under anaerobic or
aerobic conditions) [13]. Unfortunately, we were unable to use the
thiols or phosphines used previously because they react readily
with HOCl.
3.5. Oxidation of other substrates by hypochlorous acid
In addition to NOHA, we have also examined the HOCl oxidation of
other substrates. Hydroxylamine, hydroxyurea, and acetohydroxamic
acid have all been shown to be capable of being oxidized to produce
HNO (Scheme 4) [22,61]. The reaction of HOCl with these substrates
was studied by both MIMS and GC headspace analysis. For the HOCl
oxidation of hydroxylamine, MIMS experiments show the character-
istic 2:1 ratio for the m/z 44 and 30 signals, indicative of clean N2O
production (Fig. 8a). HOCl also readily oxidizes hydroxyurea to gener-
ate an increase in the m/z 30 and 44 signals, although the 44:30 in-
tensity ratio is larger here (Fig. 8b), presumably the result of CO2
production from the carbamic acid byproduct (Scheme 4). HOCl
only minimally oxidizes acetohydroxamic acid under the conditions
of our experiments (Fig. 8c). The larger m/z 44:30 ratio observed

152
M.R. Cline et al. / Journal of Inorganic Biochemistry 118 (2013) 148–154
4
10
8
m/z = 30, -78 °C
m/z = 44, -78 °C
m/z = 30, -196 °C
m/z = 44, -196 °C
m/z = 30, -78 °C
m/z = 44, -78 °C
m/z = 30, -196 °C
m/z = 44, -196 °C
(a)
3
2
1
0
6
4
2
0
0
400
800
1200
1600
0
400
400
400
800
1200
1600
1600
1600
Time (s)
Time (s)
Fig. 7. MIMS response at m/z 30 and 44 following the injection of 500 μM HOCl into a
0.1 M PBS solution at pH 7.4 and 37 °C containing 100 μM NOHA using either a liquid
nitrogen (bp=−196 °C) trap or the standard dry ice/acetone (bp=−78 °C) trap.
8
6
(b)
m/z = 30, -78 °C
m/z = 30, -196 °C
to 0.1 M PBS solution containing 143 mM chloride ion and 100 μM
NOHA or hydroxylamine. To initiate the generation of HOCl, 500 μM
H2O2 was added to this solution. Reaction results were examined by
N2O headspace analysis (Table 4). Control experiments examining
the reaction of NOHA with MPO, NOHA with H2O2, and NOHA with
MPO plus H2O2 did not result in the production of N2O.
With 15 nM MPO, large yields of N2O are not observed from the
oxidation of NOHA or hydroxylamine; however, N2O yields increase
when 75 nM MPO is used. Although the effects of authentic HOCl
have been shown to be identical to those of HOCl generated via
MPO [62], significantly more N2O is observed with hydroxylamine
compared with NOHA in these MPO experiments (Table 4) in contrast
to results with HOCl itself (Tables 2 and 3). Since HOCl is likely gener-
ated more slowly from MPO than when it is added bolus, we suspect
that unreacted NOHA may be quenching HNO as it is produced, con-
sistent with the previous observation that N-hydroxyguanidines can
react with HNO [32].
4
2
0
-2
0
800
1200
Time (s)
8
6
4
2
0
(c)
m/z = 30, -78 °C
m/z = 44, -78 °C
m/z = 30, -196 °C
m/z = 44, -196 °C
4. Conclusions
MIMS and GC headspace analysis have been used to observe the
formation of HNO-derived N2O via the oxidation of NOHA by the bio-
logically relevant oxidant, HOCl. HOCl is also able to generate HNO
from the oxidation of hydroxylamine, hydroxyurea, and (to a lesser
extent) acetohydroxamic acid. These results provide support for the
possible endogenous generation of HNO via the oxidative chemistry
of MPO-generated HOCl.
0
800
1200
Time (s)
Fig. 6. MIMS response for control experiments using a liquid nitrogen (bp=−196 °C)
trap compared to standard experimental conditions in which dry ice/acetone
a
(bp=−78 °C) trap is used for injection of (a) 30 μL N2O, (b) 500 nM of the HNO
donor 2BrPA, and (c) 25 μM DEA/NO, which corresponds to 50 μM NO, into a 0.1 M
PBS solution at pH 7.4 and 37 °C.
here suggests the generation of CO2 derived from secondary oxidative
pathways. Analogous GC headspace analysis shows that both hydrox-
ylamine and hydroxyurea produce a significant amount of N2O upon
reaction with HOCl, whereas acetohydroxamic acid generates rela-
tively little N2O (Table 3).
3.6. Oxidation of N-hydroxy-L-arginine by myeloperoxidase
Since MPO is the enzyme responsible for the generation of HOCl in
vivo, we also examined the oxidation of NOHA and hydroxylamine
(for comparison) by the MPO catalyzed reaction of H2O2 with chlo-
ride ion. For these experiments, either 15 or 75 nM MPO was added
Scheme 4. Potential pathways to HNO following oxidation of hydroxylamine, hydroxyurea,
and acetohydroxamic acid.

M.R. Cline et al. / Journal of Inorganic Biochemistry 118 (2013) 148–154
153
5
4
3
2
1
0
Table 4
(a)
Oxidation of NOHA and hydroxylamine by MPO.
Substrate
MPO (nM)
% HNOa
NOHA
NH2OH
NOHA
NH2OH
15
15
75
75
2
21
8
m/z = 44
m/z = 30
73
a
Substrates (100 μM) were incubated with MPO and H2O2 (500 μM) at 37 °C in
0.1 M PBS (containing 143 mM Cl−), pH 7.4. HNO yields are reported relative to the
standard HNO donor, Angeli's salt, as determined by N2O headspace analysis (SEM
5%; n≥3).
Disclosures
0
400
800
1200
1600
Time (s)
J.P.T. is a co-founder, stockholder, and serves on the Scientific
Advisory Board of Cardioxyl Pharmaceuticals.
6
5
4
3
2
1
0
(b)
Acknowledgment
We gratefully acknowledge the National Science Foundation
(CHE-1213438) for the generous support of this research. We also
thank Professor Howard Fairbrother, Samantha Rosenberg, and Michael
Barclay of the Johns Hopkins Chemistry Department for their assistance
and valuable advice.
m/z = 44
m/z = 30
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