Dependence of product formation from decomposition of nitroso-dithiols on the degree of nitrosation. Evidence that dinitroso-dithiothreitol acts solely as an nitric oxide releasing compound

Article abstract of DOI:10.1039/b801583j

Hitherto, the decay mechanisms of nitrosated dithiols as well as formation of related products have not been conclusively elucidated. In this paper, we demonstrate that nitrosated dl-dithiothreitol (DTT) decays via two independent pathways, that is, one producing exclusively nitric oxide and one producing (initially) nitroxyl (HNO/³NO^-). The importance of the two decomposition pathways depends on the degree of nitrosation of DTT. Dinitroso-dithiothreitol (NODTTNO) generates quantitatively nitric oxide, whereas mononitroso-dithiothreitol (NODTT) yields initially nitroxyl. Since NODTT and DTT are both targets for nitroxyl, their availability governs the HNO-derived formation of nitric oxide (with NODTT as reactant) or hydroxyl amine and ammonium ion (with DTT as reactant). The formation of NH₄ ⁺ from the HNO-DTT reaction probably proceeds by a stepwise, NH ₂OH-independent mechanism, because DTT-derived sulfinamide was identified by N-15 NMR spectrometry as an intermediate. Our data are in line with the assumption that triplet nitroxyl (³NO^-) is formed by a unimolecular decay of the deprotonated (thiolate) form of NODTT, because CBS-QB3 calculations predict the existence of a low-lying triplet state of the latter species. The identified pathways are proposed to be of general importance for physiological systems because control experiments showed that the physiological dithiol thioredoxin reacts in a similar manner. The Royal Society of Chemistry 2008.

Full text of DOI:10.1039/b801583j

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Dependence of product formation from decomposition of nitroso-dithiols on
the degree of nitrosation. Evidence that dinitroso-dithiothreitol acts solely as
an nitric oxide releasing compound
Sonja Liebeskind,a Hans-Gert Korth,b Herbert de Groota and Michael Kirsch*a
Received 29th January 2008, Accepted 31st March 2008
First published as an Advance Article on the web 7th May 2008
DOI: 10.1039/b801583j
Hitherto, the decay mechanisms of nitrosated dithiols as well as formation of related products have not
been conclusively elucidated. In this paper, we demonstrate that nitrosated DL-dithiothreitol (DTT)
decays via two independent pathways, that is, one producing exclusively nitric oxide and one producing
(initially) nitroxyl (HNO/3NO−). The importance of the two decomposition pathways depends on the
degree of nitrosation of DTT. Dinitroso-dithiothreitol (NODTTNO) generates quantitatively nitric
oxide, whereas mononitroso-dithiothreitol (NODTT) yields initially nitroxyl. Since NODTT and DTT
are both targets for nitroxyl, their availability governs the HNO-derived formation of nitric oxide (with
NODTT as reactant) or hydroxyl amine and ammonium ion (with DTT as reactant). The formation of
+
NH4 from the HNO–DTT reaction probably proceeds by a stepwise, NH2OH-independent
mechanism, because DTT-derived sulfinamide was identified by N-15 NMR spectrometry as an
intermediate. Our data are in line with the assumption that triplet nitroxyl (3NO−) is formed by a
unimolecular decay of the deprotonated (thiolate) form of NODTT, because CBS-QB3 calculations
predict the existence of a low-lying triplet state of the latter species. The identified pathways are
proposed to be of general importance for physiological systems because control experiments showed
that the physiological dithiol thioredoxin reacts in a similar manner.
skeletal and cardiac isoforms of the ryanodine receptor, which
Introduction
suggests that •NO modulates muscle activity.10,11
Nitric oxide (•NO), which is an important mediator in
vasodilatation,1 neurotransmission,2 and immune reactions, is a
The trans-nitrosation reaction competes with the thiol-mediated
reduction of the nitroso-function to NH3 at excess thiol
concentrations,12 reaction (2).
short-lived intermediate in blood because it is rapidly trapped by
the heme iron of hemoglobin. Therefore, it has been suggested
that in vivo nitric oxide uses so-called “transporters” in order to
prolong its life-time.3 Thiols have been suggested to be involved in
•NO transport, and protein-bound cysteine residues may regulate
activities of a variety of enzymes after S-nitrosation in a fashion
that parallels protein phosphorylation and dephosphorylation.4,5
For instance, experimental evidence has been found that both ex-
ogenously and endogenously produced •NO impedes apoptosis via
S-nitrosation of caspases.6–8 One characteristic chemical feature of
S-nitrosothiols is the transfer of the nitroso-function to another
thiol (“transnitrosation”), reaction (1).
2 RSNO + 8 RSH →→→ 5 RSSR + 2 H2O + 2 NH3
(2)
In addition, Wong et al.13 reported that the reaction between
thiols and S-nitrosothiols may yield nitroxyl (HNO), reaction (3),
R1SNO + R2SH → R1SSR2 + HNO
(3)
•
which further produces NO by reaction with a second molecule
of S-nitrosothiol, reaction (4).
RSNO + HNO → RSH + 2 •NO
(4)
However, the situation is less clear when the nitroso function
is transferred to a dithiol in which the two thiol groups can
interact with each other. The S-nitrosoglutathione (GSNO)-
dependent nitrosation of the physiological dithiols dihydrolipoic
acid (DHLA) and thioredoxin (Trxn) has been studied by several
research groups, as overviewed by Stoyanovski et al.14 However,
conflicting reaction mechanisms have been proposed for this
reaction (Scheme 1).
R1SNO + R2SH ꢀ R1SH + R2SNO
(1)
By this reaction, low-molecular weight S-nitrosothiols can
nitrosate cysteine residues in proteins. For instance, S-
nitrosocysteine inhibits responses mediated by the N-methyl-D-
aspartate subtype of the glutamate receptor of rat cortical neurons,
presumably via trans-S-nitrosation of the channel.9 Further, low-
molecular weight RSNOs trans-S-nitrosate vital thiols in both
Arnelle and Stamler15 reported that DHLA and the frequently
used dithiol dithiothreitol (DTT) reduce GSNO to glutathione
(GSH) and nitroxyl (HNO) via the intermediate formation of
mononitroso-dihydrolipoic acid (1 → 2 → 3). This view was
supported by data of Stoyanovsky et al.14 On the contrary,
Petit et al.16 suggested that 2 decomposes to •NO and the
aInstitut fu¨r Physiologische Chemie, Universita¨tsklinikum Essen, Hufeland-
strasse 55, 45122, Essen, Germany. E-mail: michael.kirsch@uni-duisburg-
essen.de; Fax: +49 201-723 5943; Tel: +49 201-723 4108
bInstitut fu¨r Organische Chemie, Universita¨t Duisburg-Essen, Univer-
sita¨tsstrasse 5, 45117, Essen, Germany
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dithiothreitol as a model, in the absence and presence of certain
targets, as a degree of nitrosation. These results are presented here.
In contrast to all previous studies we clearly identify a HNO-
•
dependent and a HNO-independent reaction channel of NO
production. The HNO-independent pathway decisively involves
the intermediacy of dinitroso-dithiols. The thermochemistry of all
reactions is computationally established at the CBS-QB3 level of
theory.
Scheme 1 Decomposition of mononitroso-dihydrolipoic acid according
to Stoyanovski et al.14
Results
Mono- and dinitroso-DTT show very similar electronic ab-
S-centered radical 4. The latter thiyl radical has been shown to
undergo a rapid intramolecular ring closure to the radical anion
5. Such radicals are known to react with oxygen at the diffusion-
sorptions at kmax = 332 and 336 nm, respectively, with molar
absorbtivities (NODTTNO: e336 = 1685 M −1 cm−1, NODTT: e332
=
−1
825 M cm−1) differing by about a factor of two.23 The current
literature does not give a conclusive answer to the question whether
NODTTNO is only generated after complete mono-nitrosation of
DTT because the nitrosation of the two thiol functions appears
to proceed with the same rate constant.23 Since quantum chemical
calculations performed at the CBS-QB3 level of theory predicted
both that the nitrosation of the second thiol group is a little
more exergonic than the nitrosation of the first one and that
the trans-nitrosation reaction between two NODTT molecules is
additionally an exergonic reaction (reaction (5), Table 4, entry 5),
•
controlled limit to yield the superoxide radical anion (O2 −).17–19
Similar to the conversion 2 → 4, Niktovic and Holmgren19
reported that the key physiological dithiol thioredoxin (Trxn)
catalyzes the denitrosation of GSNO with release of •NO via the
intermediate formation of mononitroso-thioredoxin (HS-Trxn-
SNO). However, the occurence of reaction 2 → 4 is in question
as S-nitrosothiols such as GSNO do not spontanousely homolyze
in the absence of any Cu2+/Cu+-ions.20,21 Somewhat surprisingly,
•
the suggestion of Wong et al.13 that HNO releases NO from S-
nitrosothiols via reaction (4) has not been taken into account
for mononitrosated dithiols, although this reaction apparently
explains the occurrence of both HNO and •NO.
In contrast to all the foregoing conclusions, Brock et al.22
reported that S,Sꢀ-dinitrosated dithiols, but not mononitrosated
ones, exhibited pharmacological effects typical for nitric oxide.
In physiological systems, clean mono- or dinitrosation of the
available dithiols cannot be expected. From the above, it is obvious
that the way in which the degree of nitrosation of the dithiols
correlates with their chemical activity was unresolved so far.
Therefore, we elucidated the chemical pathways of decomposi-
tion of nitrosated dithiols (see Scheme 2), using primarily DL-
(5)
NODTTNO is expected to be present even in such nitroso-DTT
solutions which were generated at a [NO2−]/[DTT] ratio lower
than 2.
Nitrosation was performed by reacting constant concentrations
of DTT (60 lM) with varying amounts of sodium nitrite at
pH 2 and rapidly adjusting the pH to 7.4 in order to slow
down the spontaneous decomposition of nitrosated DTT. Since
the decomposition at pH 7.4 is still relatively fast (see below),
quantificationofthe yields of nitrosated species was not attempted,
and the degree of nitrosation was expressed as the [NO2−]/[DTT]
ratio, assuming almost quantitative conversion of the minor
reactant at all [NO2−]/[DTT] ratios.
The validity of this assumption is demonstrated by Fig. 1A,
showing an excellent linear dependence of the optical density at
336 nm on the [NO2−]/[DTT] ratio, with a slope of 0.487 for
[NO2−]/[DTT] values ≤ 2. This would correspond to a nitrosation
yield of 97.5
1%. At [NO2−]/[DTT] values ≥ 2, the optical
density remained constant, clearly demonstrating that both thiol
functions were essentially quantitatively nitrosated under the
applied experimental conditions. The latter finding is in line with
data of Brock et al.22 Therefore, such solutions were generally
applied for all further experiments.
The oxidation of dihydrorhodamine-123 (DHR-123) is, in the
absence of catalytically active transition metals, a known test for
the intermediacy of oxidizing nitrogen-oxide species.24,25 From
Fig. 1B it is understood that with increasing [NO2−]/[DTT]
ratio the oxidation of DHR-123 increased as well, until at
[NO2−]/[DTT] = 2 a constant yield of rhodamine-123 is reached,
i.e., the same dependence as has been observed for the optical
Scheme 2
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Fig. 2 Nitrosation of DAF-2 by in situ generated S-nitroso DTT. Mix-
tures of 60 lM GSNO and the corresponding concentration of DTT were
allowed to react with 10 lM DAF-2 for 180 min at 37 ◦C in the dark.
In the absence of dithiol, GSNO is rather inefficient in convert-
ing DAF-2 to DAF-2T because only 0.14 0.03 lM DAF-2T was
detected. Since the nitric oxide-releasing decomposition of GSNO
requires catalysis by transition metal ions (preferably copper and
iron),20 this low yield can be attributed to efficient complexation
of traces of such metal ions by the added EDTA. Nitrosation of
DAF-2 increased with increasing dithiol concentration, until a
[GSNO]/[DTT] ratio of 2 is reached, that is, at the same ratio as
was found from preformed nitroso-DTT (see Fig. 1). At higher
[GSNO]/[DTT] ratios the fluorescence of DAF-2T decreased
slightly, the reason for which was not further explored.
The foregoing observations strongly suggested that NODTTNO
acts as a general nitrosating agent in the presence of oxygen.
For further corroboration, the nitrosation of N-acetyltryptophan
(NAT) by dinitroso-DTT was studied by H-1 NMR (Fig. 3).
Decomposition of NODTTNO was complete after a reaction
period of 90 min (vide infra). The H-1 NMR spectrum then showed
only the presence of N-nitroso-N-acetyltryptophan (NANT)
and some residual NAT, thus clearly proving the capability of
NODTTNO to nitrosate NAT.
Fig. 1 Degree of DTT nitrosation. A: Dependence of the optical density
at kmax = 336 nm on the degree of DTT nitrosation expressed as
[NO2−]/[DTT] ratio. The absorbance was measured immediately after
mixing the reactants ([DTT] = 60 lM) and adjusting the pH to 7.4 (T =
25 ◦C). B: Oxidation of dihydrorhodamine-123 as a function of DTT
nitrosation expressed as [NO2−]/[DTT] ratio. Mixtures of solutions of
preformed nitroso-DTT (25 lM) and DHR-12 (50 lM) were allowed to
react for 180 min at 37 ◦C.
density (Fig. 1A) is followed. This observation was somewhat
unexpected, because hitherto it was generally believed that the
reactive species responsible for oxidation of DHR-123 were
exclusively released from NODTT.14–16,19,26
In order to verify a similar chemical activity of in situ nitrosated
dithiols, the GSNO-mediated nitrosation of the primary aromatic
diamine 4,5-diaminofluorescein (DAF-2) in the presence of DTT
was studied (Fig. 2). Formation of the corresponding fluorescent
triazole product DAF-2T indicates the intermediacy of •NO
whose autoxidation in the presence of air produces the nitrosating
species •NO2/N2O3.
In order to find evidence for the kind of the reactive intermedi-
ates whichareresponsiblefor thenitrosating/oxidizingcapabilities
of NODTTNO, the yields of products from reaction with NAT,
melatonin, DAF-2, DHR-123, aminophenyl fluorescein (APF),
and hydroxyphenyl fluorescein (HPF), respectively, were quanti-
fied (Table 1). Possible reactive intermediates for conversion of
these targets as discussed in the literature are also listed.
Nitrosation of NAT, melatonin, and DAF-2 by NODTTNO
proceeded uniformly with a yield of about 22%. It should be noted
that all experiments were performed in phosphate buffer which is
known to catalyse the hydrolysis of N2O3,30 and this fact might
Table 1 Nitrosation/oxidation by dinitroso-dithiothreitol
Target
Substrate nitrosation/oxidation (%)
Possible intermediates
References
NAT
23.7 7.5
21.6 1.5
22.5 0.6
6.0 0.3
1.6 0.1
0.0 0.0
N2O3, HNO
N2O3, HNO
24,27
24,27
24,27
25,28
29
Melatonin
DAF-2
DHR-123
APF
N2O3, HNO
•NO2, HNO, ONOOH
HOCl, ONOOH
•
HO•, CO3
29
−
HPF
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Fig. 3 H-1 NMR analysis. H-1 NMR (500-MHz) detection of nitrosation of N-acetyltryptophan (NAT) by NODTTNO (bottom). The spectrum (region
of aromatic resonances) was recorded immediately after mixing of preformed NODTTNO (10 mM) and NAT (10 mM). Spectra of preformed NODTTNO
(10 mM), N-nitroso-N-acetyltryptophan (NANT) (5 mM) and NAT (10 mM), respectively, are shown for comparison.
have limited the yields of the nitrosation products. Oxidation of
DHR-123 and APF was significantly lower, and no oxidation was
observed for HPF. APF and HPF are only converted to fluorescein
by strongly oxidizing species like peroxynitrite and the hydroxyl
radical. Since hydroxyl radicals can be generated from HNO,31 the
failure of NODTTNO to oxidize HPF disfavours the intermediacy
of nitroxyl. Thus, the product yields of Table 1 strongly suggest
the intermediacy of N2O3.
presence of NAT, even though NANT is produced (monitored
photometrically at kmax = 335 nm; data not shown). However, in
the absence of NAT, the decay of NODTTNO does not follow a
clean first-order rate law as evident from the logarithmic plot of
the decay data. At longer reaction times, the decay of NODTTNO
appears to be somewhat retarded, probably due to a second-order
component. In fact, the best fit of the data points was obtained
by assuming a superposition of a first-order and a second-order
decay process. Noteworthy, in the presence of NAT, the decay
of NODTTNO excellently followed a single first-order rate law
In order to discriminate whether NODTTNO nitrosates its
targets via a reactive intermediate (N2O3) or directly by a
bimolecular trans-nitrosation process, the influence of NAT on the
decay of NODTTNO was monitored. The decay of NODTTNO
was followed by reading its optical density photometrically at k =
545 nm in order to avoid interference with the formation of NANT
(kmax = 335 nm).
with a rate constant of k1 = (4.91
0.07) × 10−3 s−1 (298 K),
corresponding to a half-life of 2.3
0.1 min. The fact that in
the presence of NAT, NODTTNO decays by a first-order process
clearly indicates that the NODTTNO-mediated nitrosation of
NAT proceeds via a reactive intermediate (vide infra), most
reasonably N2O3. The intermediacy of N2O3 also would explain
the retarded decay in the absence of NAT (Fig. 4) as N2O3
is known to catalyze the decay of mono-S-nitrosothiols.32 The
catalysis of decomposition of only one S-nitrosothiol group then
inhibited the alternative (faster) decay, i.e., the simultaneous bond
breaking of the two RS–NO bonds in NODTTNO (see Discus-
sion).
Inspection of Fig. 4 reveals that the decay of NODTTNO,
with a half-life of about 2.4 min, is only slightly affected by the
Since NANT is highly effective in nitrosating thiols,33 the decay
of NODTT ([NO2−]/[DTT] = 1) was only measured in the absence
of NAT. The decay of NODTT has been previously analyzed by
Arnelle and Stamler15 who reported a half-life of ca. 9 min at
pH 7.4 and room temperature. We observed a moderately faster
decay with a half-life of about 7 min at pH 7.4 and 25 ◦C (Fig. 5A).
However, as evident from the regression line, the decay of NODTT
is not of pure first-order. The decay was also dependent on the pH
value, showing a maximum around pH 9 (Fig. 5B). This finding
indicated that the decomposition of NODTT depended on the
availability of the deprotonated thiol (thiolate) form. The increase
of the half-life time at higher pH values is discussed below on the
basis of a quantum-chemically supported mechanism.
Fig. 4 Kinetics of NODTTNO decay. Decay of NODTTNO (2 mM)
in the absence (closed circles) and in the presence (open circles) of
N-acetyltryptophan (5 mM) at pH 7.4 (T = 25 ◦C). The optical density
of NODTTNO was read at 545 nm. The solid lines through the left-hand
data points are least-squares fits to a first-order rate law (open circles) and
a linear combination of a first- and second-order rate law (closed circles).
In order to confirm the proposed release of HNO as the reac-
tive intermediate from NODTT,3 we applied a MnIII–porphyrin
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Although NODTTNO does not release any HNO during
decomposition (Fig. 6E and F) but is nevertheless more reactive
than NODTT (compare, e.g., Fig. 1B), an intermediate other than
nitroxyl has to be released. Petit et al.16 claimed •NO to be the main
intermediate from decomposition of NODTT. Therefore, •NO was
measured polarographically from preformed and in situ generated
nitroso-DTT, prepared at [NO2−]/[DTT] or [GSNO]/[DTT] ratios
of 0.25–2. As the enzyme superoxide dismutase (SOD) is supposed
•
to increase the yield of NO in the presence of HNO37 as
•
well as from •NO/O2 −-releasing systems,38 these experiments
were performed in the absence and in the presence of SOD
(100 units ml−1). (Note that the foregoing experiments were carried
out in an open experimental setup, thus only the equilibrium
concentration of •NO with respect to diffusion into the gas phase
and autoxidation could be determined.)
As shown in Fig. 7, a maximum equilibrium concentration
•
of NO of 12.4 lM was released from preformed nitroso-DTT
•
synthesized at a [NO2−]/[DTT] ratio = 2. The NO equilibrium
concentration decreased with decreasing [NO2−]/[DTT] ratio to
about 50% at a ratio of 0.25. Noteworthy, at this ratio the presence
of SOD increased the •NO equilibrium concentration from 6.2 lM
to 8.3 lM, thus indicating the intermediacy of either HNO or
•
O2 −. In order to discriminate between these two intermediates,
the H2O2 concentration was measured in the absence and in the
presence of SOD, because only a SOD-catalyzed dismutation of
•
O2 − would lead to an increased level of H2O2. However, the H2O2
concentration in the presence of SOD (100 units ml−1) (15.4
1.6 lM) was within the error margins the same as in the absence
of SOD (17.4 0.7 lM). Thus, the SOD-mediated increase in the
•NO equilibrium concentration most likely derived from reaction
Fig. 5 Kinetics of NODTT decay. Decay of NODTT (2 mM, plus 100 lM
EDTA) at 25 ◦C. A: Decay trace at pH 7.4. The optical density was
monitored at kmax = 336 nm. Solid line: least-squares fit to a first-order
rate law. B: pH dependence of the half-life of NODTT (2 mM). The half-life
values were estimated from simple first-order fits.
•
with nitroxyl. The influence of SOD on the equilibrium NO
concentration was even more pronounced when nitroso-DTT was
in situ generated from the GSNO-DTT reaction, despite the fact
that in this case the equilibrium concentration of nitric oxide was
generally lower than from preformed nitroso-DTT (Fig. 7). These
complex (manganese(III)-tetrakis(1-methyl-4-pyridyl)porphyrin
pentachloride = MnIIITMPyp) as a scavenger for nitroxyl as
proposed by Marti et al.,34 who used a related MnIII–porphyrin
derivative, manganese(III)meso-tetrakis-(N-ethylpyridinum-2-yl)-
porphyrin, for the detection of nitroxyl. The HNO-trapping capa-
bility of MnIIITMPyp was tested with the HNO donor Angeli’s salt
•
results further confirmed that both NO and HNO are released
when DTT is only partially S-nitrosated.
To clarify whether a physiological dithiol can act in a sim-
ilar manner, a few experiments were performed with nitroso-
thioredoxin (nitroso-Trxn) (56.7 lM). Since preformed nitroso-
Trxn cannot be synthesized at pH values lower than 4, it was
(AS). On addition of AS, the Soret band of MnIIITMPyp (e463
=
9.2 × 104 M−1 cm−1 35) decreased instantaneously with concomitant
build-up of the absorption of the nitroso complex MnIITMPyp-
NO at kmax = 435 nm (e435 = 1.1 × 105 M−1 cm−1 36) (Fig. 6A).
Nitroso-DTT preparations at various [NO2−]/[DTT] ratios were
analyzed.
−
synthesized at pH 5.4 by using an excess amount of NO2
(133.4 mM). Release of •NO was initiated by adjusting the solution
to pH 7.4. From such a solution, an equilibrium nitric oxide
concentration of 2.9 1.5 lM was determined, which corresponds
to about 27% of the level that was achieved from DTT under
the same conditions (•NO production could not be detected in
the absence of dithiol, thus ruling out inadvertent •NO formation
from nitrite via the HNO2/N2O3 pathway). A similar relationship
was found when the related nitroso-dithiols were generated
in situ at pH 7.4 from the reaction of GSNO (250 lM) with Trxn
or DTT (55.6 lM each). Here, the Trxn-dependent nitric oxide
equilibrium concentration (1.21 0.1 lM) amounted to 20% of
that produced by DTT. Due to the presence of EDTA (100 lM), a
GSNO-dependent •NO release was not detected in the absence of
dithiols. Thus, dinitroso-Trxn also releases nitric oxide at pH 7.4,
although the yields and kinetics differ from that of DTT.
In fact, an identical shift of the Soret band was observed in the
presence of preformed nitroso-DTT synthesized at [NO2−]/[DTT]
ratios of 0.25, 0.5, and 1 (Fig. 6B–D), which indicated the release
of HNO during the decay of nitroso-DTT. Most remarkably,
preformed NODTTNO did not cause any change of the 463-nm
absorption (Fig. 6E). The production of HNO as a function of
nitrosation of DTT (400 lM) was analyzed in more detail after a
reaction period of 300 s (Fig. 6F). A constant amount of about
3.9 lM nitroxyl was trapped from [NO2−]/[DTT] ratios 0.75–1.25.
The amount of trapped nitroxyl decreased to about 50% at a
[NO2−]/[DTT] ratio of 1.5, and the MnIIITMPyp assay failed to
detect any nitroxyl at [NO2−]/[DTT] ratios ≥ 1.75. These data
clearly demonstrate that the type of intermediate released from
nitroso-DTT crucially depends on the level of nitrosation of DTT.
With respect to the suggested mechanism of HNO formation
from mononitroso-dithiols (Scheme 1, reaction (2) → (3)), one
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Fig. 6 Determination of HNO release from Angeli’s salt and nitroso-DTT. A: Addition of AS (400 lM). B, C, D, E: Addition of preformed nitroso-DTT
(500 lM) from various [NO2−]/[DTT] ratios (at a fixed NO2-concentration). F: Amount of trapped nitroxyl after a reaction period of 90 s from preformed
−
nitroso-DTT (500 lM) that were generated from various [NO2−]/[DTT] ratios (at a fixed NO2 concentration).
would expect that per mole released HNO one mole of thiol
function is consumed. In order to check on this point, the
concentration of the thiol functions of preformed nitroso-DTT
prepared from various [NO2−]/[DTT] ratios (at a fixed amount of
DTT) was quantified (Fig. 8).
Fig. 7 Release of •NO. Equilibrium •NO production from preformed
and in situ generated nitroso-DTT in the absence (closed symbols) and
presence (open symbols) of SOD. Circles: Preformed ([NO2−]/[DTT])
nitroso-DTT (at a constant concentration of 250 lM SH groups). Squares:
In situ generated nitroso-DTT from reaction of 250 lM GSNO with varying
concentrations of DTT.
Fig. 8 Yields of thiol oxidation. Consumption of thiol functions of
nitroso-DTT depending on the [NO2−]/[DTT] ratio.
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In the absence of NO2−, 980
20 lM thiol groups were
to [N-15]NO2− and [N-15]NO3−. Neither typical HNO-derived de-
composition products (N2O, N2) nor NH2OH, a product expected
from reduction of HNO by DTT,15 could be detected (Fig. 9B). So
far, NH4+ has not been reported as a product from decomposition
of nitroso-dithiols, but it has been reported from reaction of
GSNO with GSH.12 At [NO2−]/[DTT] = 0.5, [N-15]NH4+ was the
found from 500 lM DTT 30 min after dissolution of DTT. This
observation clearly demonstrates that DTT is not artificially
consumed under our reaction conditions. In line with experiments
described below and from literature data, all thiol functions were
oxidized after complete decay of preformed nitroso-DTT prepared
at [NO2−]/[DTT] ratios ≥ 1. Surprisingly, however, about 98%
of the thiol groups were already oxidized after decomposition
of nitroso-DTT prepared at a [NO2−]/[DTT] ratio of 0.5, i.e.,
−
−
major product. Here, the yields of [N-15]NO2 and [N-15]NO3
were strongly diminished, but new signals of [N-15]NH2OH (d =
−277 ppm) and a second, unknown product with d = −300.8 ppm
were detected (Fig. 9C). Sulfinamides are characterized by N-15
NMR resonances around 300 ppm,39 therefore, it was assumed
that this resonance is due to the monosulfinamide of DTT
(DTTSONH2). In order to support this assumption, the N-15
NMR chemical shifts of [N-15]DTTSONH2 and of the other
products were quantum-chemically computed with the individual
gauges for atoms in molecules (IGAIM) protocol at the B97-
2/aug-cc-pVDZ level of theory. Since the reliability of the applied
model to predict N-15 NMR resonances is not well reported in
the literature, a small set of N-15 NMR resonances was selected
as benchmark for comparing the IGAIM-B97-2/aug-cc-pVDZ
method (Table 2).
986
4 lM thiol were consumed by 250 lM NO2−, whereas a
thiol depletion of only 500 lM had to be expected in the case
of an exclusive HNO-producing pathway. A similar situation
was observed for a [NO2−]/[DTT] ratio of 0.25, because now
125 lM nitrite mediated the oxidation of 368
11 lM thiol,
instead of 250 lM theoretically. These observations made clear
that the reaction mechanism is more complicated than hitherto
assumed.3,14,16 This further suggests that the enhanced oxidation
of thiols may be accompanied by the release of a nitrogen species
other than •NO and HNO, respectively.
In order to identify the nitrogen products from preformed
nitroso-DTT, N-15-enriched nitrite was applied at [NO2−]/[DTT]
ratios of 2, 1, 0.5, and 0.25, and the reaction mixtures were ana-
lyzed by N-15 NMR spectrometry after complete decomposition
of nitroso-DTT (Fig. 9A–D).
The applied model predicted N-15 NMR resonances with a
mean absolute deviation of 8.3 ppm and the N-15 chemical shift
of [N-15]DTTSONH2 is calculated to be 295.8 ppm, i.e., 5 ppm
lower than the N-15 NMR resonance of the unkonwn compound
(Table 2). Thus, the calculations were not in conflict with the
assignment of the resonance at −300.8 ppm to [N-15]DTTSONH2.
At a 4-fold excess of DTT, i.e. at [[N-15]NO2−]/[DTT] = 0.25
From fully nitrosated DTT (NODTTNO; [[N-15]NO2−]/
[DTT] = 2) only [N-15]nitrite (d = 229 ppm) and [N-15]nitrate
(d = −4.2 ppm) were detected after decomposition at pH 7.4
(Fig. 9A). At [[N-15]NO2−]/[DTT] = 1, viz. NODTT, a prominent
+
+
peak of [N-15]NH4 (d = −359.3 ppm) was recorded in addition
(Fig. 9D), only [N-15]NH2OH and [N-15]NH4 were found as
Fig. 9 N-15 NMR spectrometric product analysis of decomposed nitroso-DTT solutions. Preformed nitroso-DTT at various [[N-15]NO2−]/[DTT]
ratios (100 mM [N-15]nitrite, 100 lM EDTA, final pH = 7.4) were allowed to decompose for 6 h at room temperature. Chemical shifts (d) are given in
parts per million (ppm) relative to nitromethane (d = 0) as an external standard.
2566 | Org. Biomol. Chem., 2008, 6, 2560–2573
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Table 2 Quantum-chemically calculated isotropic absolute shielding constants and N-15 chemical shifts (d, in ppm)
Isotropic shielding constanta
B97-2
Isotropic chemical shift
B97-2
Compound
Exp.40
0.0
228.9
184.0
Deviation
[N-15] CH3NO2
−125.8
−356.1
−334.4
−280.5
−120.9
−55.3
1.0
0.0
230.3
208.6
−
[N-15]NO2
+1.4
+24.6
−0.5
−1.4
−0.9
−24.3
+3.0
[N-15]CH3ONO
[N-15](CH3)2NNO
154.7
155.2
−
[N-15]NO3
−4.9
−3.5
[N-15]N2
−70.5
−69.6
[N-15]CN−
−126.8
−128.5
−148.2
−145.9
−137.2
−189.3
−234.6
−275.9
−294.2
−293.9
−320.3
−367.3
−102.5
−131.5
−135.8
−147.3
−148.7
−174.1
−235.5
−278.1
−280.6
−290.9
−302.9
−359.6
[N-15][−N=N+=N−]
[N-15]CH3CN
[N-15]NNO
2.7
22.4
20.2
11.4
−12.4
+1.3
[N-15](CH3)2NNO
+11.5
−15.2
+0.9
[N-15]SCN−
63.5
[N-15]NNO
108.8
150.1
168.4
168.1
194.5
241.5
[N-15]NH2OH
+2.2
−
+
−
=
[N-15][ N N =N ]
[N-15]CH3NCS
[N-15]OCN−
−13.6
−3.0
−17.4
−7.7
−3.0
8.3
+
[N-15]NH4
mean deviation
mean abs. deviation
unknown
−300.8
[N-15]DTTSONH2
170.0
−295.8
a Isotropic absolute shielding constants were calculated using the IGAIM protocol at the B97-2/aug-cc-pVDZ//B97-2/aug-cc-pVDZ level of theory.
Solvation corrections (nitromethane for nitromethane, acetonitrile for acetonitrile and CH3ONO, acetone for (CH3)2NNO and CH3NCS, none for NNO,
H2O for all others) with the CPCM solvation model were performed at the same level of theory.
products. In conclusion, the N-15 NMR spectra confirmed that
the products from nitrosated DTT strongly depend on the degree
of nitrosation of DTT, or, vice versa, the availability of free SH
goups.
to about 2.1% at a [NO2−]/[DTT] ratio of 0.25. The yield of
NH4 changed inversely to the nitrite yield. About equimolar
+
amounts (96 12 lM) of nitrite and ammonium were produced
+
at [NO2−]/[DTT] = 0.5, until the yield of NH4 approached a
Since product yields are difficult to be quantified by N-15
NMR spectrometry, the foregoing experiments were repeated at
much lower concentrations and three of the four products, namely
limiting value of about 43% (107 lM) at [NO2−]/[DTT] ratios ≤
0.25. Production of NO3− did not exceed 2% at any [NO2−]/[DTT]
ratios. The observed changes of product yields paralleled those
observed in the N-15 NMR experiments. According to the N-15
NMR spectra, the missing nitrogen content is due to formation
of NH2OH and DTTSONH2 (see Fig. 9A–D,) which were not
independently quantified. As NH4+ is formed from nitroso-DTT as
long as unnitrosated DTT is available, the question arose whether
+
NO2−, NO3−, and NH4 , were independently quantified (Fig. 10).
Percentage yields are given relative to the concentration of the
thiol groups.
+
NH4 is formed from reaction of nitroso-DTT with DTT or from
reduction of NH2OH by DTT. In fact, in text books of inorganic
+
chemistry it is mentioned that NH2OH can be reduced to NH4 .
+
NH2OH + 2 e− + 3 H+ → NH4 + H2O
(6)
As DTT is well known for its reducing capabilities, the yields of
+
NH4 from the NH2OH–DTT reaction were determined by N-15
NMR spectrometry. Experiments were performed with solutions
of [N-15]NH2OH (200 mM) in the absence and in the presence
of DTT (400 mM). In both cases, however, only the resonance of
[N-15]NH2OH was detected after a reaction period of 6 h, proving
Fig. 10 Quantification of nitrite, nitrate, and ammonium from de-
+
that the yield of [N-15]NH4 must be lower than 3% with respect
composed nitroso-DTT solutions. Nitroso-DTT prepared from various
NO2−/DTT ratios (250 lM SNO function) was allowed to react for 3 h at
to the applied [N-15]NH2OH concentration (spectra not shown).
25 ◦C. Closed circles: NO2−, open circles: NH4+, closed diamonds: NO3
.
Another possibility of NH4 formation would be the reduction
+
−
+
of HNO by DTT. We therefore quantified the NH4 production
From NODTTNO ([NO2−]/[DTT] = 2) almost quantitative
amounts of nitrite (248 13 lM) were produced. With decreasing
[NO2−]/[DTT] ratio, the yield of nitrite decreased continuously
from the NH2OH–DTT reaction as well as from the HNO–DTT
reaction by means of the glutamate dehydrogenase assay (Table 3).
Angeli’s salt (250 lM) was employed as HNO source.
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Table 3 Yield of ammonium ion from DTT reactionsa
NH2OH + DTTb
AS + DTTc
[NH2OH]/[DTT], [AS]/[DTT]
Yield/lM
Yield (%)
Yield/lM
Yield (%)
0.25
0.5
1.0
2.0
No DTT
22.7 8.0
19.0 3.8
40.2 6.5
39.4 6.1
14.3 2.2
0.9
0.8
1.6
1.6
0.6
7.8 1.0
9.2 2.1
2.9 1.8
1.3 1.8
1.0 1.2
3.1
3.7
1.2
0.5
0.4
a Concentrations of NH4 were quantified after a reaction period of 3 h (100 lM EDTA, T = 37 ◦C, pH 7.4). b 2.5 mM NH2OH. c 250 lM AS.
+
+
In agreement with the above data, the production of NH4
from the NH2OH–DTT reaction did not exceed 2% with respect
to the applied amount of NH2OH. A slightly higher yield of
approximately 3.5% was observed from the HNO–DTT reaction.
Control experiments demonstrated that neither NH2OH nor
the decomposition products of Angeli’s salt inhibited glutamate
dehydrogenase at the applied concentrations (data not shown).
Thus, neither the NH2OH–DTT reaction nor the HNO–DTT one
can explain the 43% yield of NH4+ from decomposition of nitroso-
DTT in the presence of excess DTT, i.e. at [NO2−]/[DTT] = 0.25
volatile product. Consequently, nitrite, formed via autoxidation of
•NO41,42 and subsequent hydrolysis of N2O3,43 is essentially the only
stable product (yield > 98%) in the presence of oxygen (Fig. 9).
Although DTTox is the only detectable other product, it is not
•
clear whether formation of NO from NODTTNO proceeds by
simultaneous bond-breaking of the two RS–NO bonds in the
dinitroso-dithiothreitol molecule (reaction (7)) or via a stepwise
process (reaction (8)).
+
(Fig. 9 and 10). Therefore, it must be concluded that NH4 is
formed via a pathway independent of the presence of HNO and
(7)
NH2OH, respectively.
In order to demonstrate that NH4 may also be formed
NODTTNO → NODTT• + •NO → DTT + 2 •NO
(8)
+
from an in situ generated nitroso-dithiol, additional experiments
were performed with GSNO in the presence of Trxn and DTT,
respectively. Under roughly physiological conditions (100 lM
EDTA, pH 7.4, T = 37 ◦C), Trxn (170.8 lM) reacted with GSNO
Quantum-chemical calculations performed at the CBS-QB3
level of theory predicted that reaction (7) is exergonic by
−20.9 kcal mol−1 (Table 4, entry 1). The stepwise mechanism
(reaction (8)) is predicted to proceed via a moderately endergonic
reaction (21.8 kcal mol−1, Table 4, entry 2) followed by a strongly
exergonic one (−42.8 kcal mol−1, Table 4, entry 3). The Gibbs free
energy for homolytic bond dissociation of the S–NO function in
NODTTNO is predicted to be 31 kcal mol−1. This value is very
similar to data experimentally observed for many simple mono-S-
nitrosothiols.44 Since the CBS-QB3 methodology is very reliable
in predicting S–NO dissociation energies of S-nitrosothiols45 and
because such S-nitrosothiols do not spontaneously dissociate,42
we doubt that the decomposition of NODTTNO proceeds via
a stepwise homolytic dissociation mechanism. Nevertheless, a
homolytic route cannot be ruled out with certainty.
+
(341.6 lM) to give 20.7 0.2 lM NH4 , about 20% of the amount
that could be generated from DTT under the same conditions.
+
Thus, NH4 production cannot be ignored on decomposition of
physiological nitroso-dithiols.
Discussion
In the present paper we proved for the first time that nitroso-
dithiols decay through various independent pathways, the relative
importance of which depends crucially on the degree of nitro-
sation. A fully dinitrosated dithiol yields solely nitric oxide as
Table 4 Quantum-chemically calculated Gibbs energies and aqueous solvation energies
Entry
Reactiona
DrGgb/kcal mol−1
DrGsolvc/kcal mol−1
DrGaqd/kcal mol−1
1
2
3
4
5
6
7
8
9
NODTTNO
NODTTNO
NODTT-S•
NODTT + HNO
2 NODTT
→
→
→
→
→
→
→
→
→
→
→
→
DTTox + 2 •NO
NODTT-S• + •NO
DTTox + •NO
−21.5
21.5
−43.0
−17.6
−0.6
−9.0
6.0
29.4
15.4
−24.9
−50.1
−44.5
0.6
0.4
0.2
−20.9
21.8
−42.8
−10.7
−3.3
DTT + 2 •NO
6.8
DTT + NODTTNO
DTTox + HNO
3NODTT-S−
−2.8
−9.0
7.1
−2.5
−37.8
−9.4
−50.3
30.7
NODTT
−18.0
NODTT-S−
13.1
NODTT
3NODTT
26.9
3NODTT-S−
DTT + HNO
NODTT + DTT
DTTSONH2 + DTT
DTTox + 3NO−
DTTox + NH2OH
DTT-SONH2 + DTTox
2 DTTox + NH3
−22.4
−34.2
−100.4
−13.7
10
11
12
a Thermodynamic properties were calculated using the complete basis set (CBS-QB3) methodology. b Gas phase data. c Solvation corrections from
UHF/6-31+G(d)//CBS-QB3 single point calculations with the CPCM-UAHF solvation model for water.44 d DrGaq = DrGg + DrGsolv
.
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In conclusion, the spontaneous release of nitric oxide from
NODTTNO (and most likely of other dinitroso-dithiols) is a
highly efficient, thermodynamically feasible process because the
driving force of the reaction depends on a ring closure of a cyclic
disulfide.
10 (Fig. 5B). The pH dependence of Fig. 5B rather points to
the deprotonated, thiolate form of NODTT (NODTT-S−) as the
reacting species. This would be in agreement with typical acidity
constants of aliphatic thiols (compare, e.g. mercaptoethanol,
pKa = 9.5). Interestingly, our CBS-QB3 calculations predicted that
only the thiolate anion of NODTT has a rather low-lying triplet
state, about 13.1 kcal mol−1 above the singlet ground state (Table 4,
entry 7). It is therefore proposed that the reaction may take place
via a thermally accessible triplet state (reaction (11)), for which
CBS-QB3 calculation predicts a driving force of −9.3 kcal mol−1
in aqueous solution (Table 4, entries 7 and 9).
•
Now, the question arose why NO release is still fairly good
from nitroso-DTT solutions generated at [NO2−]/[DTT] ratios ≤
1, i.e. where mainly NODTT is formed and thus the release of
HNO has to be expected (Fig. 6B–D). In this case, two processes
•
for NO production can be imagined: First, reaction of HNO
with the parent S-nitrosothiol (reaction (4)) as proposed by Wong
et al.13 must be taken into account. In fact, the reaction of HNO
with NODTT is predicted to be exergonic by 10.7 kcal mol−1
(Table 4, entry 4). Second, since trans-nitrosation between thiols
and nitrosothiols is a well-known process,42 NODTT might be in
•
equilibrium with NODTTNO (reaction (5)), from which NO is
(11)
then released according to reaction (7) (Table 4, entry 1).
1
The possibility that NODTT-S− released alternatively NO−
The fact that NODTT ([NO2−]/[DTT] = 1) did not decay by
a clean first-order process (Fig. 5) would be in correspondence
with reaction (5) because CBS-QB3 calculations predict that this
reaction is slightly exergonic by 3.3 kcal mol−1 (Table 4, entry 5).
However, in case of the physiological dithiol-thioredoxin, trans-
nitrosation of its S-mononitrosated form (NOTrxn) analogous to
reaction (5) appears unlikely due to high steric hindrance by the
protein which would hamper exchange of the nitroso function
is unlikely because the experimentally determined singlet–triplet
energy gap of the nitroxyl anion is −16.1 kcal mol−1,46 so that
reaction (11) would lose its negative driving force. The 3NO−
anion comprises a unique conjugate acid–base couple (reaction
(12)) with different ground-state multiplicities, preferring HNO
formation at pH values lower than 11.4, the pKa value of HNO.47
3NO− + H3O+ ꢁ HNO + H2O
(12)
•
to give S-dinitroso-Trxn. Since, however, ample NO production
from NOTrxn has been found, we conclude that •NO is primarily
liberated from S-mononitrosothiols (in the absence of copper ions)
via the HNO pathway (reaction (4)). Nevertheless, for sterically
unhindered dithiols like DTT a contribution by reaction (5) cannot
be ruled out.
The fact that the half-life of NODTT again increased at pH > 10
(Fig. 5B) thus can be explained by the pKa value of HNO and the
reversibility of reaction (11). Since the spin inversion of NODTT-
S− (reaction (11)) possesses a sizeable barrier of ca. 13 kcal mol−1,
one had to expect that bimolecular reactions (reactions (4) and (5))
could compete with reaction (11). The concentration of NODTT
is governed by its self-reaction (reaction (5)) as well as by the
Since reaction of HNO with the parent S-nitrosothiol is
obviously the dominating pathway of •NO generation from
mononitroso-dithiols, the question remained how HNO is released
from NODTT. Although any details about the mechanism are
missing in the literature, the reaction schemes given in those
publications14,15 suggest that generation of HNO from NODTT
proceeds by direct, concerted elimination (reaction (9)).
•
HNO-mediated release of NO (reaction (4)). At excess dithiol
concentration, HNO is trapped by DTT and this reaction—as
first discussed by Wong et al.13 for the GSNO/GSH system—is
the source of the observed products hydroxyl amine, sulfinamide,
and ammonium ion. The formation of these compounds via the
Wong et al. mechanism is also strongly supported by our CBS-QB3
calculations (Table 4, entries 10–12).
According to Singh et al.12, the HNO-independent formation
(9)
+
of NH4 from GSNO via a stepwise mechanism with GSNH2 as
intermediate requires four equivalents of GSH. Such a mechanism
cannot be ruled out with certainty. Nevertheless, the intermediacy
of both HNO and the DTT-derived sulfinamide strongly sup-
We were unable to locate a transition structure for reaction (9), so
that the barrier height of the suggested elimination could not be
calculated. The CBS-QB3 calculations predicted that the NODTT-
dependent formation of HNO (reaction (10)) may be a proton-
catalyzed process:
+
ported the view that formation of NH4 from decay of NODTT
should primarily follow the Wong et al. mechanism. In conclusion,
formation of all observed products is proposed to occur via the
reactions summarized in Scheme 3.
Since the chemical properties of nitroso-DTT and nitroso-
dihydrolipoic acid3 on the one hand and of nitroso-DHLA and
nitroso-Trxn48 on the other hand are similar, we believe that the
reactions outlined in Scheme 3 are quite general ones for the
decay of nitroso-dithiols at physiological pH values. It has been
(10)
•
suggested that Trxn reacts in the cytosol to yield either NO19
However neither direct elimination of HNO from the ni-
trosodithiol (reaction (9)) nor a proton-catalyzed process (reaction
(10)) would explain the observed pH dependence of the decay
rate of NODTT with a maximum (lowest half-life) at pH 9–
or NH2OH.48 In this work, experimental evidence is presented
that the formation of the products depends on the degree of
nitrosation of the dithiol. A low level of nitrosation means
that the concentration of dinitroso-dithiol is negligibly low since
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(Michigan, US). All other chemicals were of the highest purity
commercially available.
Experimental conditions
All experiments were carried out at 25 ◦C in 50 mM phosphate
buffer (PB) if not described otherwise. Because nitrosation reac-
tions are sensitive to the presence of metal ions,42 solutions were
treated with the chelating resin Chelex 100 prior to use as described
previously,52 and further supplemented with 100 lM EDTA.
Synthesis of nitrosated dithiothreitol
As previously described for other thiols,53 154.25 mg DTT were
diluted in 50 mM PB containing 100 lM EDTA, and 69.0 mg
NaNO2 or 70.0 mg Na[N-15]NO2 were diluted in doubly distilled
water. Both solutions were mixed at the required ratio, and the
synthesis was started by adding 1 M hydrochloric acid until a pH
of 2 was attained. The reddish solutions were diluted immediately
with 50 mM PB and 100 lM EDTA to give a 10-fold stock solution.
The concentration was photometrically controlled (see Fig. 1).
Samples were prepared as fast as possible because of the low half-
life of nitroso-DTT15 (see Results), and the pH was immediately
adjusted to 7.4 with 1 M NaOH.
Synthesis of S-nitroso-glutathione
As described previously,33 3.07 g GSH and 0.69 mg NaNO2 were
allowed to react in a mixture of 20 ml purified water and 10 ml 1 M
HCl for 40 min at 4 ◦C. Then, 20 ml acetone were added and the
solution was stirred for another 10 min. The formed precipitate
was filtered off, washed with 10 ml water, 3 times with 20 ml
diethyl ether, dried in a desiccator and stored at −20 ◦C. The
concentration of the prepared GSNO was quantified by reading
Scheme 3
reaction (5) is shifted to the left side. In intracellular milieu,
the concentrations of RSNOs have been determined to be in
the submicromolar range,49 whereas that of Trxn and GSH are
in the range of 5–50 lM48 and 3–5 mM,50 respectively. Under
such conditions, the formation of the nitric oxide-yielding entity
dinitroso-Trxn would be extremely low and only NH2OH and
−1
the optical density at kmax = 545 nm (e545 = 20 M cm−1).54
Synthesis of 1-nitroso-melatonin
+
+
1-Nitroso-melatonin (NOMela) was prepared as described by
Kirsch and de Groot.53 In brief, melatonin (232 mg) was dissolved
in 4 ml acetone,10 ml water was added and the mixture stirred for
several minutes. Afterwards 207 mg NaNO2 and another 2 ml of
water were added. 1.5 ml of 1 M HCl were added to the yellow
liquid, shaken well and cooled to 1 ◦C. The mixture was extracted
with 20 ml cold (1 ◦C) dichloromethane, and the separated organic
phase dried with 750 mg Na2SO4. The solvent was evaporated
at −20 ◦C, and the yellow solid was stored at −20 ◦C. The
purity was determined photometrically at 345 nm (e345 = 7070 M−1
cm−1).53 To a 100-fold stock solution used for the experiments
20 vol% DMSO were added to prepare an aqueous solution of the
lipophilic substance.
NH4 would be formed. The generation of NH4 is favourable
for biological systems as ammonium can be rapidly detoxified by
many cell types via the urea cycle.51 Thus, Trxn is expected to
additionally act in vivo as a GSNO reductase but not as a GSNO-
dependent nitric oxide synthase.
Experimental
Chemicals
Thioredoxin (Escherichia coli, purity ≥ 90%), N-acetyltryptophan
(NAT), dithiothreitol (DTT), glutathione (GSH), Chelex 100,
5,5ꢀ-dithio-bis(2-nitrobenzoic acid) (DTNB) and L-glutamic de-
hydrogenase (GlDH) from bovine liver were obtained from
Sigma (Hamburg, Germany). Na[N-15]NO2 and [N-15]NH2OH
labled with 99.3% N-15 were purchased from Aldrich/Isotech
Inc (Hamburg, Germany). NaNO2 and (NH4)2SO4 were pro-
duced by Merck (Darmstadt, Germany). 4,5-Diaminofluorescein
(DAF-2), diaminofluorescein-2T (DAF-2T), hydroxyphenyl flu-
orescein (HPF) and aminophenyl fluorescein (APF) were from
Alexis (Gru¨nberg, Germany). Angeli’s salt (AS; purity ≥ 99%)
and Mn(III)-tetrakis(1-methyl-4-pyridyl)porphyrin pentachlo-
ride (Mn(III)TMPyp) were obtained from Cayman Chemical
Synthesis of N-nitroso-N-acetyltryptophan (NANT)
The preparation of NANT as described by Bonnett and
Holleyhead55 was improved by Sonnenschein et al.33 In brief,
526 mg NAT and 162 mg NaNO2 were stirred in 20 ml purified
water for 2 h at room temperature in the dark. The yellow mixture
was cooled to 1 ◦C, and 10 ml of cold (1 ◦C) 1 M HCl were
added. The yellow precipitate was immediately extracted with
60 ml ethyl acetate (1 ◦C). The organic layer was separated, and the
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solvent evaporated at room temperature under reduced pressure
(18 Torr) to yield 500 mg of NANT. The concentration of NANT
was photometrically controlled with a SPECTROCORD S 100
nicotinamide-adenine dinucleotide phosphate (NADPH) to nitrite
in the presence of the enzyme nitrate reductase. To the samples,
which were incubated for 20 min at room temperature, pyruvate
and LDH were added, and the samples were incubated for another
20 min at room temperature. The total nitrite-concentration was
determined by using the Griess-reagent as described above. The
base level of nitrite was directly quantified from a similarly treated
sample in the absence of nitrite reductase. The test was calibrated
with a solution of neat NaNO3, whose purity was controlled
−1
spetrometer from Analytic Jena (e335 = 6100 M cm−1).55
H-1 NMR spectrometric measurements
Solutions of 10 mM NODTTNO plus 10 mM NAT, 10 mM
NODTTNO, 10 mM NAT, and 5 mM NANT, respectively, were
prepared in 50 mM PB with 10% D2O and 100 lM EDTA at
pH 7.4. Spectra were recorded by 500 MHz H-1 NMR on a Bruker
AVANCE DRX 500 instrument 90 min after sample preparation.
Chemical shifts (d) are given in parts per million (ppm) relative to
tetramethyl silane (d = 0) as an external standard.
−1
photometrically (e302 = 10 M cm−1).58
Quantification of dihydroperoxide
Hydrogen peroxide was determined by the horseradish peroxidase-
catalysed reaction of H2O2 with 4-amino-antipyrine and 3,5-
dichloro-2-hydroxyl-benzenesulfonic acid. The pink-coloured
product was measured photometrically at 513 nm.59 Samples of
nitroso-DTT having reacted 10 min in the absence and presence
of SOD (100 units/ml) were measured after another 10 min of
incubation at room temperature. Standard buffer solutuons of
H2O2 were used for calibration.
N-15 NMR identification of adducts
After incubation for 6 h at room temperature, sample aliquots
were supplemented with 10% D2O and analyzed by N-15 NMR
spectrometry. The products were identified by 50.67 MHz N-15
NMR on a Bruker AVANCE DRX 500 instrument. Chemical
shifts (d) are given in parts per million (ppm) relative to neat
nitromethane (d = 0) as an external standard.
Quantification of ammonium
+
The concentration of NH4 was determined as described by
Bergmeyer et al.58 Samples were incubated at room temperature for
3 h, then, aliquots were diluted 2.48-fold with 1 M triethanolamine
buffer (TEA, pH = 8.0). To this mixture were added 2.8 mM
a-ketoglutarate, 177 lM NADH, and 0.8% glycerine. After incu-
bation for 20 min at room temperature, optical density of the
samples was read photometrically at 340 nm. Then, 7.2 units ml−1
L-glutamic dehydrogenase (GlDH) were added and the samples
were incubated for another 20 min at room temperature. Finally,
the optical density at 340 nm was read to calculate the decay of
NADH (e340 = 6200 M−1 cm−1),58 which is directly proportional to
Quantification of •NO
Release of nitric oxide was detected polarographically with a
graphite nitric oxide-sensing electrode (World Precision Instru-
ments, Berlin, Germany) immediately after sample preparation.
Quantification of dithiothreitol
The concentration of thiol functions of DTT was quantified as
described by Ellmann and improved by us.56 DTNB (39.6 mg) was
dissolved in 50 mM PB (10 ml, pH = 7.0). The samples were diluted
10-fold with 100 mM PB (pH = 7.0) and to 1 ml of the diluted
sample 6.67 ll stock solution of DTNB were added. The optical
density of the formed p-nitrothiophenol anion (e412 = 13 600 M−1
cm−1)57 was read photometrically after incubation for 20 min at
room temperature. The test was calibrated with a solution of neat
DTT.
+
the concentration of NH4 . The test was calibrated with a solution
of neat (NH4)2SO4.
Qualitative detection of nitroxyl
HNO was determined as described by Marti et al.34 for a related
Mn(III)–porphyrin complex. To the prepared samples were added
10 lM of Mn(III)TMPyp. Aliquots were immediately photo-
metrically measured by a SPECTROCORD S 100 spetrometer
from Analytic Jena. On reaction with HNO, the Soret-band of
Mn(III)TMPyp at 463 nm (e463 = 9.2 × 104 M−1 cm−1;35) decreased
and that of Mn(II)TMPyp-NO at 435 nm (e435 = 1.1 × 105 M−1
cm−1;36) increased.
Quantification of nitrite
Nitrite was quantified by using the Griess-reagent (0.1%
naphthylethylendiamine-dichloride [Sigma] + 1% sulfanil-amide
[Sigma] in 5% H3PO4).58 At the given time of reaction, the samples
were diluted 30-fold with 50 mM PB (pH = 7.5), afterwards
a 2.5-fold volume of Griess-reagent was added. After 10 min
of incubation at room temperature, the optical density was
photometrically read at 542 nm. Calibration was carried out
Determination of reactive intermediates from NODTTNO
NAT or melatonin (10 mM each, 50 mM PB, 100 lM EDTA, 20%
DMSO, pH = 7.4, room temperature) were allowed to react with
preformed NODTTNO (10 mM) for 90 min and the concentration
with a photometrically controlled solution of neat NaNO2 (e354
22.9 M−1 cm−1).58
=
of either NANT (e335 = 6100 M−1 cm−1) or nitroso-melatonin (e345
=
7070 M−1 cm−1) was calculated.
Quantification of nitrate
DHR-123. NODTTNO (50 lM) was allowed to react with
DHR-123 (50 lM) for 180 min at 37 ◦C, the concentration of
rhodamine-123 was quantified photometrically at kmax = 500 nm
(e500 = 78 000 M−1 cm−1).
The concentration of nitrate was determined by a commercially
available Nitrate-Test-Kit (Boeringer Mannheim/R-Biopharm,
Germany) improved by us. In brief, nitrate is reduced by
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DAF-2, APF, HPF. 15 lM NODTTNO were mixed immedi-
ately with DAF-2, APF or HPF (10 lM each), and the samples were
incubated for 180 min at 37 ◦C in the dark room. Afterwards, the
concentration of DAF-2T or fluorescein was read fluorometrically.
The APF- and HPF-derived formation of fluorescein or the DAF-
2-mediated formation of DAF-2T were quantified by reading
their fluorescence with excitation at 495 nm and emission at
515 nm, respectively. Standard calibration curves were prepared
from known amounts of fluorescein and DAF-2T.
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Sonja Liebeskind was supported by an IFORES grant of the
Universita¨t Duisburg-Essen.
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