January 2004
Biol. Pharm. Bull. 27(1) 17—23 (2004)
17
Effects of Oxygen on the Reactivity of Nitrogen Oxide Species Including
Peroxynitrite
Kiyomi KIKUGAWA,* Kazuyuki HIRAMOTO, and Takumi OHKAWA
Tokyo University of Pharmacy and Life Science School of Pharmacy; 1432–1 Horinouchi, Hachioji, Tokyo 192–0392,
Japan. Received June 9, 2003; accepted September 30, 2003
This paper describes the O2-dependent control of the reactivity of nitrogen oxide species for the production
of biologically important nitrated and nitrosated compounds. In this study, the effects of O2 on the reactivity of
NO, NO2, and ONOO2/ONOOH for nitration of tyrosine (Tyr) and nitrosation of glutathione (GSH) and morpholine (MOR) were examined. NO produced S-nitrosoglutathione (GSNO) and N-nitrosomorpholine (NMOR)
through the formation of N2O3 under aerobic conditions, and NO2 produced 3-nitrotyrosine (3-NO2Tyr), GSNO,
and NMOR. Transnitrosation from GSNO to MOR was observed only in the presence of O2. Although preformed ONOO2/ONOOH produced all the products under aerobic conditions, the formation of 3-NO2Tyr and
GSNO was markedly reduced and the formation of NMOR was enhanced under anaerobic conditions. The reactivity of the CO2 adduct of ONOO2 was similarly dependent on O2. 3-NO2Tyr was produced effectively by reaction with ONOO2/ONOOH at the O2 concentration of 270 m M and by reaction with its CO2 adduct at O2 concentrations greater than 5 m M. Generation of · OH from ONOO2/ONOOH was suppressed under anaerobic conditions. The reactivity of ONOO2/ONOOH and · OH generation from ONOO2 were reversibly controlled by the O2
concentration.
Key words nitric oxide; peroxynitrite; tyrosine; glutathione; morpholine; oxygen
Nitric oxide (NO) has many important biological functions,1,2) but related nitrogen oxide species including dinitrogen trioxide (N2O3), nitrogen dioxide (NO2), and peroxynitrite/peroxynitrous acid (ONOO2/ONOOH) are known to
cause damage to biomolecules such as lipids, proteins, and
DNA. There are many reports showing the reactivitiy and decomposition of these nitrogen oxide species in relation to the
biologically toxic functions of NO. The reactivity of NO is
enhanced by oxygen (O2) through its conversion into the reactive intermediates NO2 and N2O3 as in Eqs. 1 and 2, and finally into nitrite (NO22) as in Eqs. 3 and 4.3,4) NO2 is rapidly
hydrolyzed into nitrate (NO32) and NO22 as in Eq. 5. The reactivity and decomposition of ONOO2, which is produced
rapidly by reaction of NO with superoxide (O22),5) are complex. ONOO2 is converted into reactive ONOOH by protonation at pKa56.8 as in Eq. 6,6) which undergoes homolytic
cleavage to germinate a radical pair [NO2/ · OH]cage or alternative decomposition of the activated form *ONOOH into
HNO3 as shown by Eq. 7.5—11) There is another decomposition pathway of ONOO2/ONOOH above pH 7.5 affording
NO22 and O2 as shown by Eq. 8.12) The reactivity and decomposition of ONOO2 are regulated by rapid formation of the
CO2 adduct shown by Eq. 9,13—17) and the reactivity of this
adduct is considered to be important because biological fluids contain a high concentration of CO2.
net
2NO1O2 →2NO2
(1)
2NO212NO→2N2O3
(2)
2N2O312H2O→4NO2214H1
(3)
4NO1O212H2O→4NO2214H1
(4)
2NO21H2O→NO221NO3212H1
(5)
1
2 H
NO1O2
2 →ONOO →ONOOH
∗ To whom correspondence should be addressed.
(6)
[NO2/ · OH]cage
ONOOH
(7)
*ONOOH→HNO3
ONOO21ONOOH→HNO21NO221O2
(8)
ONOO21CO2→ONOOCO22→[NO2/CO3 · 2]cage
(9)
Biologically important nitrated and nitrosated compounds
are produced by reaction with the nitrogen oxide species. 3Nitrotyrosine (3-NO2Tyr) is produced by reaction of tyrosine
(Tyr) with NO2,18,19) ONOO2/ONOOH20,21) and other nitrogen oxide species.22) S-Nitrosoglutathione (GSNO)23,24) and
N-nitrosomorpholine (NMOR)23,25) are formed by reaction of
glutathione (GSH) and morpholine (MOR), respectively,
with NO/O2. The reaction of GSH with ONOO2/ONOOH
gives small amounts of GSNO,26,27) whereas the reaction of
MOR gives NMOR and N-nitroMOR.28) GSNO is considered
to be a stable NO pool in biological systems,27,29) and thiol-30)
and metal ion31)-induced release of NO from nitrosothiols
and transnitrosation are known. N-Nitrosamines can be metabolized to form strongly alkylating electrophiles that react
with DNA.32) It is conceivable that the presence or absence of
O2 at the locus of generation of nitrogen oxide species can
greatly affect the reactivity of the species for the production
of nitrated and nitrosated products.
The aim of the present study was to compare the reactivity
of these nitrogen oxide species for the formation of 3NO2Tyr, GSNO, and NMOR in the presence and absence of
O2 and to determine the effects of O2 on their reactivity.
MATERIALS AND METHODS
Materials Purified air, NO gas (purity 99.9%), and NO2
gas (5% in nitrogen gas) were obtained from Nihonsanso
Ltd. (Tochigi, Japan), and highly purified nitrogen gas (purity
more than 99.9%) was obtained from Taiyo-Toyosanso Ltd.
e-mail: [email protected]
© 2004 Pharmaceutical Society of Japan
18
(Kanagawa, Japan). Glutathione reduced form (GSH), 3NO2Tyr, MOR, and NMOR were obtained from Sigma
Chemical Company (St. Louis, MO, U.S.A.). GSNO and 3(4-morpholinyl)sydnonimine hydrochloride (SIN-1) were obtained from Dojindo Laboratories (Kumamoto, Japan). 5,5Dimethyl-1-pyrroline-N oxide (DMPO) was obtained from
Labotec (Tokyo, Japan).
All the aqueous solutions were prepared with deionized,
distilled, purified water using a Milli-Q water purification
system (Simpli Lab, Nihon Millipore Ltd., Tokyo, Japan),
and finally by passing through a column of Chelex 100 resin
(sodium form, 100—200 mesh) (Bio-Rad Laboratories, CA,
U.S.A.).
Nitrogen Oxide Species. NO Solution One hundred
milliliters of NO solution in deaerated 0.1 M phosphate buffer
(pH 7.4) was prepared by introducing pure NO gas as described elsewhere.33) Three precautions were taken to minimize the contamination of NO2. NO gas was purified by a
column of KOH pellets to remove NO2 in the NO gas tank
generated by dismutation of NO before introduction into the
deaerated buffer in the flask. A column of sodium hydrosulfite on glass wool was attached to the flask to avoid exposure
of the flask contents to atmospheric O2. Nitrogen gas was
purged to remove NO in the headspace of the flask before
opening the flask to avoid conversion of gaseous NO into
NO2 upon contact with atmospheric O2.
Nitrogen oxide species in the solution in the absence of O2
were determined by the modified Saltzman method33) to be
1.8 mM NO/0.01 mM NO2/0.1 mM NO22/0.1 mM NO32. Nitrogen oxide species in the solution after exposure to atmospheric O2 were determined to be 0.05 mM NO/0.01 mM
NO2/1.7 mM NO22/0.1 mM NO32.
NO2 Solution Deaerated 0.1 M phosphate buffer (pH 7.4)
(100 ml) was saturated with NO2 gas. Nitrogen oxide species
in the solution were determined to be 0.04 mM NO/0.014 mM
NO2/3.21 mM NO22/2.12 mM NO32.
ONOO2 Stock Solution A ONOO2 alkaline stock solution was prepared by reaction of NaNO2 and H2O2 according
to the method of Beckman et al.34) The concentration was determined by molecular extinction coefficient 1620 at 302 nm.
The solution may have contained NaNO2 and H2O2 as contaminants. On neutralization ONOO2 was converted into
ONOO2/ONOOH.
SIN-1 Stock Solution A solution of 10 mM SIN-1 in
15 mM hydrochloric acid was used as a stock solution. On
neutralization SIN-1 may generate ONOOH together with
SIN-1C.21,35)
Measurement of Dissolved O2 Dissolved O2 was measured with the galvanic-type oxygen electrode Able DO indicator model 1032 (Tokyo, Japan). The partial O2 pressure of
the meter was set at 0 mmHg with buffer containing 2%
sodium sulfite and set at 160 mmHg with buffer saturated
with air. The partial O2 pressure at 160 mmHg corresponded
to the concentration of dissolved O2 at 270 m M. The sample
solution (2.0 ml) was placed in a 3.0 ml-sealed cuvette attached to the electrode. Air in the headspace of the cuvette
was replaced with nitrogen gas. The concentration of dissolved O2 was controlled by introducing nitrogen gas.
Electron Spin Resonance (ESR) Studies The ESR
spectrum was obtained on an X-band JES-REIX spectrometer (JEOL, Tokyo, Japan) with a Mn21 marker at room tem-
Vol. 27, No. 1
perature with a quartz flat cell. The instrumental conditions
were: field setting at 336.5 mT, scan range of 10 mT, modulation frequency of 100 kHz, microwave power of 4 mW, and
modulation amplitude of 0.1 mT.
Reactions with NO or NO2 Gas For the reaction under
anaerobic conditions, a 10-ml solution of 1.0 mM (10 m mol)
Tyr, GSH or MOR in 0.1 M phosphate buffer (pH 7.4) was
deaerated exhaustively by purging with nitrogen gas for
30 min. NO gas (446 m mol) or NO2 gas (22.3 m mol) was introduced for 1 min at a flow rate of 10 ml/min in a sealed
tube. The solution was kept at 20 °C for 30 min. For the reaction under aerobic conditions, NO gas together with purified
air, or NO2 gas together with purified air was introduced to
the substrate solution, and the solution was kept at 20 °C for
30 min.
With NO Solution or NO2 Solution For the reaction
under anaerobic conditions, 9.6 ml of NO saturated solution
(NO 16—17 m mol) or NO2 saturated solution (31 m mol
NO22/20 m mol NO32) was added to 0.40 ml of deaerated solution of 25 mM (10 m mol) Tyr, GSH, or MOR in 0.1 M phosphate buffer (pH 7.4), and the solutions were kept at 20 °C
for 30 min. For the reaction under aerobic conditions, the reaction was conducted in an atmosphere of air.
With ONOO2/ONOOH For the reaction under anaerobic conditions, several microliters containing 20 m mol of the
deaerated alkaline stock ONOO2 solution were added with
vigorous stirring, to 10 ml of the deaerated solution of
1.0 mM (10 m mol) Tyr, GSH, or MOR in 0.1 M phosphate
buffer (pH 7.4) with or without 20 mM NaHCO3 in a sealed
tube, and the solution was kept at 20 °C for 30 min. For the
reaction under aerobic conditions, the stock ONOO2 solution
was added to the substrate solution with vigorous stirring and
the mixture was kept in an atmosphere of air. As the reverseorder addition control,34) the stock ONOO2 solution and then
0.4 ml of 25 mM Tyr, GSH, or MOR solution in the buffer
was added to 9.6 ml of the buffer alone.
For the reaction under controlled O2 conditions, to 2.0 ml
of the substrate solution in a sealed cuvette of which the initial O2 concentration was regulated by purging with nitrogen
gas and by monitoring with an oxygen electrode, 4.0 m mol of
the ONOO2 stock solution was rapidly added at 20 °C with
stirring.
With SIN-1 Hydrolysate To a 10-ml solution of 1 mM
(10 m mol) Tyr, GSH, or MOR in 0.1 M phosphate buffer (pH
7.4), 1.0 ml (10 m mol) of SIN-1 stock solution was added.
The pH values of the solutions were kept at pH 7.4. The solutions were stored at 37 °C for 4 h under aerobic conditions.
Product Analysis Gaseous nitrogen oxide species in the
solutions were removed by introducing nitrogen gas before
analysis. Products were analyzed with high-performance liquid chromatography (HPLC) on a Hitachi 655-11 liquid
chromatograph equipped with a column of Inertsil ODS-2
(4.6 i.d.3250 mm) (YMC Company, Kyoto, Japan) and a Hitachi L-7420 UV-VIS detector.
For 3-NO2Tyr: The sample was diluted 10-fold in 0.1 M
Tris–HCl (pH 9.0) and 100 m l of the solution was injected
into the column. The column was eluted with a mobile phase
composed of 0.5% (v/v) acetic acid : methanol (29 : 1, v/v)19)
at a flow rate of 1.0 ml/min. The peaks were detected at
280 nm with a detector sensitivity of 0.05. The retention
times of Tyr and 3-NO2Tyr were 7 and 25 min, respectively.
January 2004
19
The concentration of the product in the sample solutions was
determined by comparing the peak area obtained from the
standard solution.
For GSNO: The sample was diluted 2-fold in the mobile
phase, and 100 m l of the solution was injected into the column. The column was eluted with a mobile phase composed
of 0.5% (v/v) acetic acid : methanol (199 : 1, v/v) at a flow
rate of 1.0 ml/min. The peaks were detected at 335 nm with a
detector sensitivity of 0.02. The retention times of GSNO
and NMOR were 20 and 12 min, respectively.
For NMOR: The sample (10 m l) was directly injected
into the column. The column was eluted with a mobile phase
composed of 0.5% (v/v) acetonitrile in 27 mM acetate/30 mM
citrate buffer (pH 3.2)29) at a flow rate of 1.0 ml/min. The
peaks were detected at 254 nm with a detector sensitivity of
0.002. The retention times of NMOR and GSNO were 19
and 17 min, respectively.
RESULTS
Reactivity of Nitrogen Oxide Species for Production of
3-NO2Tyr, GSNO and NMOR in the Presence and Absence of O2 Tyr, GSH, and MOR at concentration of 1 mM
were reacted with more than an equivalent amount of NO
gas, NO solution, NO2 gas, NO2 solution (NO22/NO32), or
ONOO2/ONOOH in 0.1 M phosphate buffer (pH 7.4) at
20 °C for 30 min under anaerobic and aerobic conditions.
Each substrate was also reacted with an equivalent amount of
SIN-1 hydrolysate in 0.1 M phosphate buffer (pH 7.4) at
37 °C for 4 h under aerobic conditions. The amounts of the
respective nitrated product (3-NO2Tyr) and nitrosated products (GSNO and NMOR) were determined by HPLC. Representative HPLC are shown in Figs. 1—3, and the results are
summarized in Table 1.
The reactions of Tyr with NO gas and NO solution under
anaerobic and aerobic conditions gave no detectable amount
of 3-NO2Tyr. Under aerobic conditions NO may be converted
into NO22 through the reactive intermediate N2O3, but 3NO2Tyr was not produced, indicating that the nitrogen oxide
species did not participate in 3-NO2Tyr production. The reaction of Tyr with NO2 gas produced 3-NO2Tyr in yields of 2—
4% under both anaerobic and aerobic conditions (Figs. 1A,
B), but the reaction of Tyr with NO2 solution gave no detectable amount of 3-NO2Tyr. The reaction of Tyr with
ONOO2/ONOOH solution conducted under anaerobic conditions gave no detectable amount of 3-NO2Tyr (Fig. 1C),
whereas the reaction under aerobic conditions gave 3-NO2Tyr
in a significantly high yield of 5.5% (Fig. 1D). Unexpectedly
high O2 dependency of the reactivity of ONOO2/ONOOH
for 3-NO2Tyr production was observed. Because the ONOO2
stock solution contains NO22 and H2O2 as contaminants, reverse-order addition control of ONOO2 solution to the neutral buffer before addition of the substrate did not produce 3
NO2Tyr under aerobic conditions. SIN-1 is known to be hydrolyzed at neutral pH and generate NO and O22 simultaneously and thus ONOO2/ONOOH under aerobic conditions,35)
but the reaction of Tyr with SIN-1 hydrolysate gave no detectable amount of 3-NO2Tyr (Fig. 1E).
The reactions of GSH with NO gas (Fig. 2A) and NO solution under anaerobic conditions gave no detectable amount
of GSNO. The reactions of GSH with NO gas (Fig. 2B) and
NO solution under aerobic conditions gave GSNO in significantly high yields of 16% and 26%, respectively. The reactions of GSH with NO2 gas gave GSNO in yields of 1%
under anaerobic and aerobic conditions (Fig. 2C). The reaction of GSH with ONOO2/ONOOH solution under anaerobic conditions did not give GSNO (Fig. 2D), whereas the reaction under aerobic conditions gave 0.9% GSNO (Fig. 2E).
The O2 dependency of the reactivity of ONOO2/ONOOH for
GSNO production was observed. The reverse-order addition
control of ONOO2 did not produce GSNO under aerobic
conditions. The reaction of GSH with SIN-1 hydrolysate did
not give GSNO.
While the reactions of MOR with NO gas (Fig. 3A) and
Table 1. Yields of 3-NO2Tyr, GSNO, and NMOR in the Reaction of Nitrogen Oxide Species under Anaerobic and Aerobic Conditions
Amount of product (nmol)
Nitrogen oxide species
O2
3-NO2Tyr
NO gas
NO solution
NO2 gas
NO2 solution
ONOOH solution
SIN-1 hydrolysate
2
1
2
1
2
1
2
1
2
1
1
nd
nd
nd
nd
263
350
nd
nd
nd
550
nd
GSNO
nd
1650
nd
2618
108
108
nd
nd
nd
90
nd
NMOR
nd
338
nd
399
142
243
nd
nd
795
100
49
Tyr, GSH or MOR (10 m mol) was reacted with NO gas (450 m mol), NO solution
(16—17 m mol), NO2 gas (23 m mol), NO2 solution (31 mmol NO22/20 m mol NO32) or
ONOO2/ONOOH (20 m mol) in 10 ml of 0.1 M phosphate buffer (pH 7.4) at 20 °C for
30 min. The substrate was reacted with SIN-1 hydrolysate (10 m mol) in the buffer at
37 °C for 4 h. The amount of the product was determined using HPLC. nd: not detected.
Fig. 1. HPLC of the Reaction Mixture of Tyr with Nitrogen Oxide Species
in the Presence and Absence of O2
HPLC of the reaction mixture of Tyr (10 m mol) with NO2 gas (23 m mol)2O2 (A),
NO2 gas (23 m mol)1O2 (B), ONOO2/ONOOH (20 m mol)2O2 (C) and ONOO2/
ONOOH (20 m mol)1O2 (D) in 0.1 M phosphate buffer (pH 7.4) at 20 °C. Tyr was reacted with SIN-1 hydrolysate (10 m mol) in 0.1 M phosphate buffer (pH 7.4) at 37 °C for
4 h (E).
20
Fig. 2. HPLC of the Reaction Mixture of GSH with Nitrogen Oxide
Species in the Presence and Absence of O2
HPLC of the reaction mixture of GSH (10 m mol) with NO gas (450 m mol)2O2 (A),
NO gas (450 m mol)1O2 (B), NO2 gas (23 m mol)2O2 (C), ONOO2/ONOOH
(20 m mol)2O2 (D) and ONOO2/ONOOH (20 m mol)1O2 (E) in 0.1 M phosphate buffer
(pH 7.4) at 20 °C.
Fig. 3. HPLC of the Reaction Mixture of MOR with Nitrogen Oxide
Species in the Presence and Absence of O2
HPLC of the reaction mixture of MOR (10 m mol) with NO gas (450 m mol)2O2 (A),
NO gas (450 m mol)1O2 (B), NO2 gas (23 m mol)2O2 (C), ONOO2/ONOOH
(20 m mol)2O2 (D) and ONOO2/ONOOH (20 m mol)1O2 (E) in 0.1 M phosphate buffer
(pH 7.4) at 20 °C. MOR was reacted with SIN-1 hydrolysate (10 m mol) in 0.1 M phosphate buffer (pH 7.4) at 37 °C for 4 h (F).
Vol. 27, No. 1
Fig. 4.
Transnitrosation from GSNO to MOR in the Presence of Cu21
A mixture of 1 mM GSNO, 1 mM MOR and 0.2 mM Cu21 in 0.1 M phosphate buffer
(pH 7.4) was incubated at 37 °C for the indicated periods under aerobic conditions. A:
HPLC pattern (NMOR analysis mode), and B: time course.
NO solution under anaerobic conditions did not give NMOR,
the reactions conducted under aerobic conditions gave
NMOR in yields of 3.4% (Fig. 3B) and 4.0%, respectively,
indicating participation of N2O3 in the NMOR formation.
The reaction of MOR with NO2 gas under anaerobic and aerobic conditions (Fig. 3C) gave NMOR in yields of 1—3%.
The reaction of MOR with ONOO2/ONOOH solution under
anaerobic conditions gave NMOR in a significantly high
yield of 7.6% (Fig. 3D), whereas the reaction conducted
under aerobic conditions markedly reduced the yield to 1.0%
(Fig. 3E). High O2 dependency of the reactivity of ONOO2/
ONOOH for NMOR production was thus observed. The reverse-order addition control of ONOO2 did not produce
NMOR under aerobic conditions. The reaction of MOR with
SIN 1 hydrolysate gave a small amount (0.5%) of NMOR
(Fig. 3F).
Transnitrosation of GSNO to MOR in the Presence and
Absence of O2 Transnitrosation from GSNO to MOR
under anaerobic and aerobic conditions was investigated.
When a mixture of GSNO and MOR in the presence of
0.2 mM Cu21 in 0.1 M phosphate buffer (pH 7.4) was incubated in the light under aerobic conditions at 37 °C, GSNO
was gradually decreased and NMOR was produced in a timedependent fashion (Fig. 4). After 6 h, 80% of GSNO was lost
and 2.2% NMOR was produced. When the mixture was incubated in the light under anaerobic conditions, GSNO decreased similarly but no detectable amount of NMOR was
produced (data not shown). When the mixture was incubated
in the light in the presence of 1 mM cysteine under aerobic
conditions, GSNO decreased more rapidly and NMOR was
produced in a time-dependent fashion (Fig. 5), although nitrosocysteine was not detected in the present analytical pro-
January 2004
21
Fig. 6. Yields of 3-NO2Tyr (A), GSNO (B), and NMOR (C) in the Presence and Absence of O2
Percent yields of 3-NO2Tyr (A), GSNO (B), and NMOR (C) in the reactions of Tyr,
GSH and MOR (10 m mol) with ONOO2/ONOOH (20 m mol) in the presence and absence of 20 mM NaHCO3 at pH 7.4 and 20 °C under aerobic and anaerobic conditions.
In the reactions under anaerobic conditions (O2 2), deaerated ONOO2 stock solution
was added to the deaerated neutral substrate solution. Deaerated ONOO2 stock solution
was reoxygenated and added to the aerated neutral solution (O2 2→1). Each product
was analyzed by HPLC.
Fig. 5.
Transnitrosation from GSNO to MOR in the Presence of Cysteine
A mixture of 1 mM GSNO, 1 mM MOR and 1 mM cysteine in 0.1 M phosphate buffer
(pH 7.4) was incubated at 37 °C for the indicated periods under aerobic conditions. A:
HPLC pattern (NMOR analysis mode), and B: time course.
cedure. When the mixture was incubated under anaerobic
conditions, GSNO decreased similarly but no detectable
amount of NMOR was produced (data not shown). The results indicate that the decomposition of GSNO was not O2
dependent, but nitrosation of MOR by released NO is O2 dependent.
O2-Dependent Control of the Reactivity of ONOO2/
ONOOH and Its CO2 Adduct The unexpected influence
of O2 on the reactivity of ONOO2/ONOOH observed above
was carefully examined. While the reactivity of 2 mM
ONOO2/ONOOH with 1 mM Tyr and GSH under aerobic
conditions (Figs. 6A, B: O21) was reduced under anaerobic
conditions (Figs. 6A, B: O22), the reactivity was recovered
when the solutions were reoxygenated with atmospheric O2
(Figs. 6A, B: O22→1), indicating that the reactivity was interconvertible depending on whether O2 was present or absent. The reactivity with Tyr under aerobic conditions was
enhanced by CO2 (Fig. 6A: O21, HCO321) as has been
shown,36—38) which was completely lost under anaerobic conditions (Fig. 6A: O22, HCO321). The reactivity for GSH
under aerobic conditions was suppressed by CO2 (Fig. 6B:
O21, HCO321) which was completely lost under anaerobic
conditions (Fig. 6B: O22, HCO321). The reactivity of 2 mM
ONOO2/ONOOH with 1 mM MOR under aerobic conditions
(Fig. 6C: O21) was enhanced under anaerobic conditions
(Fig. 6C: O22). The reactivity with MOR under aerobic conditions was slightly enhanced by CO2 (Fig. 6C: O21,
HCO321) as has been shown,29) which was highly enhanced
under anaerobic conditions (Fig. 6C; O22, HCO321). These
results were reproducible in several repeated experiments.
When 2 mM ONOO2/ONOOH was reacted with each of the
substrates at varying concentrations of 0.3—2 mM, a similar
O2 dependency was observed (data not shown).
Yields of 3-NO2Tyr in the reaction of Tyr with ONOO2/
Fig. 7.
O2 Concentration-Dependent Yield of 3-NO2Tyr
O2 concentration-dependent yield of 3-NO2Tyr produced in the reaction of Tyr
(2 m mol) with ONOO2/ONOOH (4 m mol) in the absence (s) and presence (d) of
20 mM NaHCO3 at pH 7.4 and 20 °C. O2 concentration was determined using an oxygen
electrode.
ONOOH and its CO2 adduct at different O2 concentrations
were compared (Fig. 7). The yields of 3-NO2Tyr in the reaction with ONOO2/ONOOH decreased gradually as the O2
concentration decreased, and the yield at an O2 concentration
of 20 m M was about half that at 270 m M. The yields in the reaction with the CO2 adduct decreased sharply at O2 concentrations less than 20 m M.
Adduct formation of ONOO2 with CO2 is due to the
chemical changes in ONOO2 accompanying spectral
changes.12) The interconvertible O2 dependent reactivity of
ONOO2 cannot be explained by the chemical alteration of
ONOO2. Changes in the ultraviolet absorption spectrum of
the ONOO2 alkaline stock solution through introduction of
nitrogen gas for 30 min and subsequently with purified air for
30 min were monitored (Fig. 8). No distinct changes in the
spectrum were observed throughout the gas exposures, although the absorbance at the maximum wavelength gradually
decreased with the treatments. The result indicates that deoxygenation and reoxygenation did not alter the chemical
structure of ONOO2. Changes in the · OH-generating potency of ONOO2/ONOOH solution by deoxygenation and
subsequent reoxygenation were examined. Incubation of
ONOO2/ONOOH solution with the spin-trapping agent
22
Fig. 8. Ultraviolet Absorption Spectra of ONOO2 Solution in the Presence and Absence of O2
ONOO2 solution 380 m M in 1 M NaOH was deoxygenated by introduction of nitrogen
gas for 30 min (O2 2) and the solution subsequently reoxygenated by introduction of
air for 30 min (O2 2→1).
Fig. 9. ESR Spectra of the Mixture of DMPO with ONOO2/ONOOH in
the Presence and Absence of O2
A: To 1.0 ml of 0.1 M DMPO in 0.1 M phosphate buffer (pH 7.4) 50 ml of 40 mM
ONOO2 solution in 0.1 M NaOH was added, and the mixture was incubated at 20 °C for
10 min under aerobic conditions. B: Before mixing, the DMPO solution and the
ONOO2 solution were deoxygenated by introducing nitrogen gas for 30 min, and each
solution was mixed in a sealed tube under anaerobic conditions. C: Before mixing, the
DMPO solution and the ONOO2 solution were deoxygenated and subsequently reoxygenated by gentle aeration, and each solution was mixed and incubated under aerobic
conditions.
DMPO at pH 7.4 under aerobic conditions gave characteristic four line ESR signals of the adduct of · OH, DMPOOH,39) with hyperfine splitting constants of aN5aH51.49 mT
(Fig. 9A). On careful deoxygenation of the solution the intensities of the signals were greatly reduced (Fig. 9B), and on
subsequent reoxygenation the intensities of the signals were
increased (Fig. 9C). The result indicates that the · OH-generating potency of ONOO2/ONOOH solution was reversibly
changed depending on the O2 concentration, which was in
concert with the reversible O2 dependent reactivity of
ONOO2/ONOOH solution for Tyr and GSH.
DISCUSSION
In the present study, the O2 dependency of the reactivity of
nitrogen oxide species for the formation of the biologically
important nitrated or nitrosated components was investigated. NO by itself was inactive for nitration of Tyr and nitrosation of GSH and MOR, but became reactive to nitrosate
GSH and MOR in the presence of O2, which is consistent
with the earlier observations.23—25) N2O3 generated during
NO oxidation3,4) must participate in GSNO and NMOR for-
Vol. 27, No. 1
mation. It is known that direct reaction of GSH with NO
does not give GSNO but disulfide and HNO.40,41) Decomposition of GSNO was promoted by Cu21 ion and cysteine in
the presence and absence of O2, but transnitrosation from
GSNO to MOR did not occur in the absence of O2. NO2 was
reactive to produce 3-NO2Tyr, GSNO, and NMOR only when
NO2 gas was used. Nitration of Tyr may proceed through abstraction of hydrogen by NO2 followed by addition of NO2.19)
Nitrosation of GSH and MOR by NO2 may proceed through
nitration followed by reduction.
It is known that in the reaction with ONOO2/ONOOH
under ambient conditions, Tyr gives 3-NO2Tyr,20,21) GSH
gives disulfide with formation of small amouts of GSNO,26,27)
and MOR gives NMOR and N-nitroMOR.28) The reactive
form of ONOO2/ONOOH has been considered to be complex: to germinate a radical pair [NO2/ · OH] in the solvent
cage generated by homolytic cleavage of the ONOOH molecules or to produce an alternative activated form *ONOOH
as shown by Eq. 7.5—11) It has been shown that Tyr nitration
occurs in the reaction of Tyr/NO/KO2 by the stopped flow reaction system under the anaerobic conditions.42) Simultaneous generation of NO and O22 in the pulse radiolysis of a
deaerated solution of Tyr/NaN3/KNO3/H2PO42 also produces
3-NO2Tyr.43) Hence it seems certain that Tyr nitration by
ONOO2/ONOOH occurs under anaerobic conditions. In the
present study, however, reversible O2 dependent reactivity of
ONOO2/ONOOH was observed. The reasons for this discrepancy are now obscure.
In the present study, 3-NO2Tyr and GSNO formation was
completely suppressed and NMOR formation was effectively
enhanced under anaerobic conditions. In the reactions of the
CO2 adduct, similar O2 dependency was observed. Moreover,
the reactivity was reversible depending on whether O2 was
present or absent. It is likely that certain interactions may
occur between ONOO2/ONOOH or its CO2 adduct with O2
to affect the reactivity and the generation of · OH, although a
plausible explanation of the reasons for the O2 dependency
cannot be given. Because the ultraviolet absorption spectrum
of ONOO2 was unchanged, the ONOO2 molecules may be
unchanged by deoxygenation. The · OH-generating potency
of ONOO2/ONOOH solution was lost by deoxygenation but
recovered by reoxygenation, which was in agreement with
the reversible O2 dependent reactivity.
Examination of the reaction of each substrate with SIN-1,
a known generator of ONOO2,21) revealed that the reaction
afforded NMOR but did not 3-NO2Tyr and GSNO. The low
reactivity of SIN-1 observed in the present study may be due
to the contaminated components such as SIN-1C. The result
is consistent with earlier observations showing slow reactivity of SIN-1 for Tyr.21)
O2 dependency of the reactivity of the nitrogen oxide
species NO and ONOO2/ONOOH may be important in understanding their reactions in biological fluids. The intracellular concentration of O2 is assumed to be 20—50 m M and the
mitochondria concentration to be 1—5 m M.44) NO is generated continuously at a particular locus at less than 0.1 m M45)
and diffuses to the site of action. Considering the rate constant,46) the rate of the reaction of NO with O2 may be too
small to give N2O3.47) Hence GSNO and NMOR formation
through the NO oxidation pathway is unlikely. A relatively
higher intracellular concentration of GSH (about 5 mM) may
January 2004
have a role in the formation of GSSG and HNO40,41) without
producing GSNO. Production of ONOO2 by the reaction of
NO and O22 (66 pM44)) as in Eq. 6 is considered to be sufficiently rapid,6—8) and ONOO2 thus produced may be protonated to form ONOOH at neutral pH or rapidly react with intracellular and interstitial concentrations of CO2 (12, 30 m M,
respectively46)) to form ONOOCO22. If the reactivity of
ONOO2 and ONOOCO22 to produce 3-NO2Tyr, GSNO and
NMOR are affected by O2 concentrations as observed here,
3-NO2Tyr, GSNO, and NMOR formation due to the nitrogen
oxide species in biological fluid may be highly regulated by
the O2 concentrations.
Acknowledgments This work was supported by Special
Coordination Funds of the Ministry of Education, Culture,
Sports, Science and Technology of the Japanese government.
REFERENCES
1)
2)
3)
4)
5)
6)
7)
8)
9)
10)
11)
12)
13)
14)
15)
16)
17)
18)
Moncada S., Palmer R. M. J., Higgs E. A., Pharmacol. Rev., 43, 109—
142 (1991).
Stamler J. S., Singel D. J., Loscalzo J., Science, 258, 1898—1902
(1992).
Ignarro L. J., Fukuto J. M., Griscavage J. M., Rogers N. E., Byrns R.
E., Proc. Natl. Acad. Sci. U.S.A., 90, 8103—8107 (1993).
Goldstein S., Czapski G., J. Am. Chem. Soc., 117, 12078—12084
(1995).
Beckman J. S., Beckman T. W., Chen J., Marshall P. A., Freeman B.
A., Proc. Natl. Acad. Sci. U.S.A., 87, 1620—1624 (1990).
Koppenol W. H., Moreno J. J., Pryor W. A., Ischiropoulos H., Beckman J. S., Chem. Res. Toxicol., 5, 834—842 (1992).
Crow J. P., Spruell C., Chen J., Gunn C., Ischiropoulos H., Tsai M.,
Smith C. D., Radi R., Koppenol W. H., Beckman J. S., Free Radic.
Biol. Med., 16, 131—138 (1994).
Yang G., Candy T. G. E., Boaro M., Wilkin H. E., Jones P., Nazht N.
B., Saadalla-Nazht R. A., Blake D. R., Free Radic. Biol. Med., 12,
327—330 (1992).
Merenyi G., Lind J., Chem. Res. Toxicol., 11, 243—246 (1998).
Merenyi G., Lind J., Goldstein S., Czapski G., Chem. Res. Toxicol., 11,
712—713 (1998).
Coddington J. W., Hurst J. K., Lymar S. V., J. Am. Chem. Soc., 121,
2438—2443 (1999).
Pfeiffer S., Gireb A. C. F., Schmidt K., Wrner E. R., Hansert R., Bohl
D. S., Mayer B., J. Biol. Chem., 272, 3465—3470 (1997).
Lymar S. V., Hurst J. K., J. Am. Chem. Soc., 117, 8867—8868 (1995).
Uppu R. M., Suadrito G. L., Pryor W. A., Arch. Biochem. Biophys.,
327, 335—343 (1996).
Denicola A., Freeman B. A., Trujillo M., Radi, R., Arch. Biochem.
Biophys., 333, 49—58 (1996).
Uppu R. M., Pryor W. A., J. Am. Chem. Soc., 121, 9738—9739 (1999).
Hodges G. H., Ingold K. U., J. Am. Chem. Soc., 121, 10695—10701
(1999).
Prutz W. A., Monig H., Buler J., Land E. J., Arch. Biochem. Biophys.,
243, 125—134 (1985).
23
19)
20)
21)
22)
23)
24)
25)
26)
27)
28)
29)
30)
31)
32)
33)
34)
35)
36)
37)
38)
39)
40)
41)
42)
43)
44)
45)
46)
47)
Kikugawa K., Kato T., Okamoto Y., Free Radic. Biol. Med., 16, 373—
382 (1994).
Ischiropoulos H., Zhu L., Chen J., Tsai M., Martin J. C., Smith C. D.,
Beckman J. S., Arch. Biochm. Biophys., 298, 431—437 (1992).
van der Vliet A., Eiserich J. P., O’Neill C. A., Halliwell B., Cross C.
E., Arch. Biochem Biophys., 319, 341—349 (1995).
Eiserich J. P., Hristova M., Cross C. E., Jones A. D., Freeman B. A.,
Halliwell B., van der Vliet A., Nature (London), 391, 393—397
(1998).
Goldstein S., Czapski G., J. Am. Chem. Soc., 118, 3419—3425 (1995).
Kharitonov V. G., Sundquist A. R., Sharma V. S., J. Biol. Chem., 270,
28158—28164 (1995).
Lewis R. S., Tannenbaum S. R., Deen W. M., J. Am. Chem. Soc., 117,
3933—3939 (1995).
Radi R., Beckman J. S., Bush K. M., Freeman B. A., J. Biol. Chem.,
266, 4244—4250 (1991).
Ouijano C., Alvarez B., Gatti M., Augusto O., Radi R., Biochem. J.,
322, 167—173 (1997).
Uppu R. O., Squadrito G. L., Bolzan R. M., Pryor W. A., J. Am. Chem.
Soc., 122, 6911—6916 (2000).
Hogg N., Free Radic. Biol. Med., 28, 1478—1486 (2000).
Hogg N., Singh R. J., Kalyanaraman R., FEBS Lett., 382, 223—228
(1996).
Williams D. L. H., Methods Enzymol., 268, 299—308 (1996).
Tannenbaum S. R., Tamir S., deRoojas-Walker T., Wishnok J. S., “Nitrosamines and Related N-Nitro Compounds,” ed. by Loeppky R. N.,
Michejda J. S., American Chemical Society, Washington DC, 1994,
pp. 120—135.
Ohkawa T., Hiramoto K., Kikugawa K., Nitric Oxide Biol. Chem., 5,
515—524 (2001).
Beckman J. S., Chen J., Ischiropoulos H., Crow J. P, Methods
Enzymol., 233, 229—240 (1994).
Hogg N., Usmar M. D., Wilson M. T., Moncada S., Biochem. J., 281,
419—474 (1992).
Lymar S. V., Jiang Q., Hurst J. K., Biochemistry, 35, 7855—7861
(1996).
Gow A., Duran D., Thom S. R., Ischirpoulos H., Arch. Biochem. Biophys., 333, 42—48 (1996).
Santos C. X. C., Bonini M. G., Augusto O., Arch. Biochem. Biophys.,
377, 146—152 (2000).
Saprin A. N., Piette L. H., Arch. Biochem. Biophys., 180, 480—492
(1977).
Pryor W. A., Church D. F., Govindn C. K., Crank G., J. Org. Chem.,
47, 156—159 (1982).
DeMaster E. G., Quast B. J., Redfern B., Nagasawa H. T., Biochemistry, 34, 11494—11499 (1995).
Reiter C. D., Teng R.-J., Beckman J. S., J. Biol. Chem., 275, 32460—
32466 (2000).
Goldstein S., Czapski G., Lind J., Merenyi G., J. Biol. Chem., 275,
3031—3036 (2000).
Tyler D. D., Biochem. J., 147, 493—504 (1975).
Wink D. A., Darbyshire J. F., Nima R. W., Saavedra J. F., Ford P. C.,
Chem. Res. Toxicol., 6, 23—27 (1993).
Carola R., Harely J. P., Noback C. R., “Human Anatomy and Physiology,” McGraw-Hill, New York, 1990, p. 803.
Fukuto J. M., Hibbs A. J., Ignaro L. J., Biochem. Biophys. Res. Commun., 196, 707—713 (1993).