Hydroxyl radical induced decomposition of S-nitrosoglutathione

Article abstract of DOI:10.1039/b004706f

S-Nitrosoglutathione (GSNO) undergoes decomposition induced by hydroxyl radicals (OH) in aqueous medium at neutral pH forming nitrite (NO₂-) and glutathione disulfide (GSSG) and therefore it is proposed that OH could interfere in the GSNO metab

Full text of DOI:10.1039/b004706f

Hydroxyl radical induced decomposition of S-nitrosoglutathione
Veleeparambil M. Manoj and Charuvila T. Aravindakumar*
School of Chemical Sciences, Mahatma Gandhi University, Kottayam 686 560, Kerala, India.
E-mail: mgu@md2.vsnl.net.in
Received (in Cambridge, UK) 13th June 2000, Accepted 16th October 2000
First published as an Advance Article on the web
S-Nitrosoglutathione (GSNO) undergoes decomposition in-
duced by hydroxyl radicals (·OH) in aqueous medium at
neutral pH forming nitrite (NO22) and glutathione disulfide
(GSSG) and therefore it is proposed that ·OH could interfere
in the GSNO metabolism.
presence of N2O the G(·OH) = 0.56 and G(OH 2) = 0.28 mmol
J21 as per reaction (2).
GSNO was synthesised from NaNO2 in presence of HCl.16
N2O saturated solutions containing GSNO (1023 M) and EDTA
(1024 M) at pH 7.3 were irradiated at different doses in a 60Co-
g-source and the decay of GSNO was monitored by both UV–
VIS spectrophotometry and HPLC. The unirradiated solution of
GSNO was found to be stable for many hours when protected
from light. The G(2GSNO) values obtained in both cases were
0.53 and 0.54 mmol J21 respectively. GSSG and nitrite were
found to be the major products of radiolysis from the HPLC
analysis. The decay of GSNO and the corresponding products
build up are shown in Fig. 1. The calculated G(GSSG) and
G(NO22) are 0.13 and 0.41 mmol J21, respectively. The pH of
the solutions were determined before and after irradiation and a
dose dependent reduction in pH was observed which is
tabulated in Table 1. A blank solution without GSNO under
similar conditions was also irradiated and obtained no major pH
changes.
S-Nitrosothiols (RSNO) are important class of compounds
which are now believed to play a major role in vivo in
connection with the storage and transport of nitric oxide (·NO)
within the body.1,2 The mechanism of the formation of RSNO
from the reaction of ·NO with protein thiols in the presence and
in the absence of oxygen is reasonably well understood.3–6 The
·
involvement of RSNOs in the storage and transport of NO
within the body makes them potential candidates for medical
applications. For example S-nitrosoglutathione (GSNO) is
currently used to inhibit platelet aggregation during some
operations.7,8 Therefore, the kinetics and mechanism of the
release of ·NO by RSNO is very important. Excellent reports on
the kinetics and mechanism of the degradation of RSNO leading
·
to the release of NO by metal ions and some nucleophiles are
A good material balance can be observed from the G(GSSG)
and G(NO22) values as G(2GSNO) ≈ G(GSSG) + G(NO22).
A minor contribution of NO22 is anticipated from the decay of
peroxynitrite which could be formed as a result of the reaction
of GSNO with H2O2 formed during radiolysis, as reported
earlier.11 However, this contribution is expected to be @ 0.072
mmol J21 [this value corresponds to G(H2O2) = 0.072 mmol
now available.9,10 Reaction of S-nitrosocysteine (SNCys) with
hydrogen peroxide yields peroxynitrite anion.11 It is also
·2
reported that GSNO reacts with superoxide radicals (O2
)
generating glutathione disulfide (GSSG) and equimolar quan-
tities of nitrite and nitrate.12
Glutathione (GSH) is the most abundant sulfur-containing
intracellular entity (cellular concentration ca. 5 mM) and
therefore the endothelial nitric oxide has to diffuse through the
cells in presence of GSH. This leads to the assumption that in
vivo conditions, the most likely S-nitrosation product could be
the GSNO.13 Hydroxyl radicals (·OH) are the main DNA
damaging agent which can be produced in vivo during oxidative
stress and on exposure to ionizing radiations.14 Understanding
of the reaction between ·OH and GSNO is, therefore, a matter of
utmost importance in a biological perspective. The present
communication describes a novel reaction pathway for the
decomposition of GSNO in presence of ·OH at neutral pH. To
our knowledge, this is the first report on the reaction of ·OH with
a possible reservoir for ·NO in biological systems.
·
J21]. Therefore, the major reaction is definitely between OH
and GSNO. ·OH generally reacts with thiols including GSH by
·
One of the major difficulties involved in the OH reaction
·
with RSNOs is that the components of most of the OH
generating systems such as H2O2 photolysis, Fenton reaction,
etc., themselves can induce degradation of RSNOs. In this
context, radiation chemical method is an ideal choice where
ionizing radiations such as g-rays can radiolyze water and
produce both oxidising and reducing radicals. Therefore, in the
present work we have used radiation chemical technique to
produce ·OH as shown in reactions (1) and (2).
Fig. 1 Dose dependent decay of GSNO (5) at pH 7.3 and the corresponding
2
formation of NO2 (8) and GSSG (D) determined by using HPLC
(Column: 25 cm, Nucleosil, 5C-18; eluent: mixture of sodium phosphate
(1023 M) and sodium sulfate (1022 M) in water; flow rate: 1 ml min21; l
= 210 nm).
Table 1 The observed pH changes in a g-irradiated N2O saturated solution
H2O À eaq2, H·, ·OH, H2, H2O2, H3O+
(1)
(2)
containing GSNO (1023 M) and EDTA (1024 M) at different dose values
·
N2O + eaq2 + H2O ? OH + OH 2 + N2
Dose/Gy
pH
0
525
1049
1574
2098
2728
7.3
5.9
5.1
4.8
4.6
4.1
The yields of various radicals and molecular products are
normally expressed as G-values which are defined as the
number of molecules formed or destroyed per 100 eV
absorption of radiation energy, in SI units, the yields are,
G(·OH) ≈ G(eaq2) ≈ G(H3O+) = 0.28, G(·H) = 0.062,
G(H2O2) = 0.072 and G(H2) = 0.047 mmol J21 15
In the
.
DOI: 10.1039/b004706f
Chem. Commun., 2000, 2361–2362
This journal is © The Royal Society of Chemistry 2000
2361

H-abstraction (from –SH) forming thiyl radicals (RS·) as the
main intermediate as reported earlier.17,18 Although the sulfur is
bonded to NO in GSNO, the most potential site for ·OH attack
is expected at the sulfur centre. On the other hand, the H-
abstraction reaction which is reported in the case of GSH will
not be possible in the present case. Therefore, based on the
above observations we propose a reaction mechanism involving
protective role against oxidative stress? However, such a role
can be established only after a clear understanding of the
concentration of GSNO in vivo and of the exact rate constant of
·OH with GSNO, which are yet to be investigated. Our work is
currently being concentrated in these directions.
We thank the Rubber Research Institute of India, Kottayam,
for the g-irradiation experiments. V. M. M. thanks the Nuclear
Science Centre, New Delhi, for a fellowship. Part of the
financial support for this work is from the Board of Research in
Nuclear Sciences (BRNS), Govt. of India.
·
the attack of OH at the electron rich sulfur centre of GSNO
[reactions (3)–(6)]. The initial attack of ·OH in GSNO (electron
transfer) would produce a highly unstable cationic species as
shown in reaction (3), which may lead to the breakage of the S–
N bond and result in the formation of GS· and NO+. However,
such a cationic intermediate (GS+NO) is expected to be very
short lived and no experimental evidence for its exact identity as
well as its transnitrosation reaction [reaction (4)] is available at
this moment. The subsequent reactions of NO+ with OH2 which
is formed as shown in reaction (3), can lead to nitrite and H+
formation. The combination of two sulfur centered radicals
(RS·) and the corresponding formation of disulfide (RSSR) is a
well known reaction reported in the case of low molecular
weight thiols.19 Therefore, a similar radical–radical reaction of
GS· is proposed for the formation of GSSG.
Notes and references
1 J. S. Stamler, O. Jaraki, J. Osborne, D. I. Simon, J. Keaney, J. Vita, D.
Singel, C. R. Valeri and J. Loscalzo, Proc. Natl. Acad. Sci. USA, 1992,
89, 76746.
2 B. Gaston, Biochim. Biophys. Acta, 1999, 1411, 323.
3 V. G. Kharitonov, A. R. Sundquist and V. S. Sharma, J. Biol. Chem.,
1995, 270, 28 158.
4 A. J. Gow, D. G. Buerk and H. Ischiropoulos, J. Biol. Chem., 1997, 272,
2841.
5 C. T. Aravindakumar, J. Ceulemans and M. De Ley, Biochem. J., 1999,
344, 253.
6 C. T. Aravindakumar, J. Ceulemans and M. De Ley, Biophys. Chem.,
2000, 85, 1.
7 E. J. Langford, A. S. Brown, R. J. Wainwright, A. J. de Belder, M. R.
Thomas, R. E. A. Smith, M. W. Radomski, J. F. Martin and S. Moncada,
Lancet, 1994, 344, 1458.
8 A. de Belder, C. Lees, J. Martin, S. Moncada and S. Campbell, Lancet,
1995, 345, 124.
GSNO + ·OH ? [GS+NO] + OH2
(3)
(4)
(5)
(6)
[GS+NO] ? GS· + NO+
OH2 + NO+ ? H+ + NO2
2
9 H. R. Swift and D. L. H. Williams, J. Chem. Soc., Perkin Trans. 2, 1997,
1933.
10 A. P. Munro and D. L. H. Williams, J. Chem. Soc., Perkin Trans. 2,
1999, 1989.
11 P. J. Coupe and D. L. H. Williams, J. Chem. Soc., Perkin Trans. 2, 1999,
1057.
1
GS· ? 2 GSSG
Therefore, the overall reaction mechanism can be written as
1
GSNO + ·OH ? 2 GSSG + NO22 + H+
(7)
12 J. David, T. M. Christie, F. L. Stephen, A. W. David and B. G. Matthew,
Biochem. Biophys. Res. Commun., 1998, 244, 525.
13 J. S. Stamler, Curr. Top. Microbiol. Immunol., 1995, 196, 19.
14 C. von Sonntag, The Chemical Basis of Radiation Biology, Taylor &
Francis, London, 1987.
15 J. W. Spinks and R. S. Wood, An Introduction to Radiation Chemistry,
John Wiley & Sons, Inc., New York, 3rd edn., 1990.
16 T. W. Hart, Tetrahedron Lett., 1985, 26, 2013.
17 W. A. Armstrong and W. G. Humphries, Can. J. Chem., 1967, 45,
2589.
18 A. B. Ross, W. G. Mallard, W. P. Helman, G.V. Buxton, R. E. Hui and
P. Neta, NDRL-NIST Solution Kinetics Database: ver.2.0, National
Institute of Standards and Technology, Gaithersburg, MD, 1994.
19 W. A. Pryor, D. F. Church, C. K. Govindan and G. Gank, J. Org. Chem.,
1982, 47, 159.
The dose dependent reduction in the pH values (Table 1)
provides clear support to the above mechanism. Further, we
exclude the possibility of the formation of nitrate as the reaction
is carried out in N2O saturated solutions where the presence of
oxygen is insignificant.
In conclusion, the mechanistic aspects of the reaction of ·OH
with GSNO, one among the biologically important S-ni-
trosothiols, have been proposed for the first time. The fast decay
·
of GSNO in the presence of OH and the corresponding
formation of nitrite and glutathione disulfide provide evidence
·
for the possible interference of OH in the GSNO metabolism.
One more question to be asked from these findings is, does
GSNO have any sacrificial role like glutathione in terms of its
2362
Chem. Commun., 2000, 2361–2362