Selective scavenging property of the indole moiety for the nitrating species of peroxynitrite

Article abstract of DOI:10.1248/cpb.52.146

The inhibitory effect on tyrosine nitration and oxidation of peroxynitrite was evaluated for more than 40 reagents including natural and synthetic compounds, and the inhibiting efficiency of each compound for nitration was compared with that for oxidation, to characterize its property as a peroxynitrite scavenger. In the presence of various concentrations of testing compounds, the nitrating and oxidizing activities were measured by monitoring the formation of 3-nitrotyrosine and dityrosine with an HPLC-UV-fluorescence detector. The IC₅₀ values for nitration and oxidation were determined, and the ratio of these two IC₅₀ values was calculated for each compound. Although the IC₅₀ values varied from compound to compound, it was revealed that the ratio of two IC₅₀ values (IC ₅₀ for oxidation/IC₅₀ for nitration) was 1 in almost all the compounds tested, except five indole derivatives (L-tryptophan, melatonin, 5-methoxytryptamine, tryptamine, and tetrahydro-beta-carboline) and one synthetic selenium-containing compound ((2R,3R,4S)-2-amino-3,4-dihydroxy-5- phenylselenopentan-1-ol, ADPP). The indole derivatives showed a specific inhibitory effect on tyrosine nitration without affecting the oxidation. ADPP was confirmed to have a preferable inhibitory activity for tyrosine oxidation. It was suggested that compounds showing an IC₅₀ value ratio of 1 scavenged the common species for nitration and oxidation, while the indole derivatives and ADPP preferably scavenged the nitrating and oxidizing species, respectively. From a stopped flow study, it was also revealed that the nitrotyrosine formation was relatively slow, unlike an OH radical reaction. These results imply that the peroxynirite reaction at least partly proceeds through specific species for nitration.

Full text of DOI:10.1248/cpb.52.146

1
46
Notes
Chem. Pharm. Bull. 52(1) 146—149 (2004)
Vol. 52, No. 1
Selective Scavenging Property of the Indole Moiety for the Nitrating
Species of Peroxynitrite
a
a
a
a
Hidehiko NAKAGAWA, Mitsuko TAKUSAGAWA, Hiromi ARIMA, Kumiko FURUKAWA,
b
a
,a
Takeshi KINOSHITA, Toshihiko OZAWA, and Nobuo IKOTA*
a
Redox Regulation Research Group, National Institute of Radiological Sciences; 4–9–1 Anagawa, Inage-ku, Chiba
63–8555, Japan: and Faculty of Pharmaceutical Sciences, Teikyo University; 1091–1 Suarasi, Sagamikomachi, Tsukui-
b
2
gun, Kanagawa 199–0195, Japan.
Received August 5, 2003; accepted October 23, 2003; published online October 28, 2003
The inhibitory effect on tyrosine nitration and oxidation of peroxynitrite was evaluated for more than 40
reagents including natural and synthetic compounds, and the inhibiting efficiency of each compound for nitra-
tion was compared with that for oxidation, to characterize its property as a peroxynitrite scavenger. In the pres-
ence of various concentrations of testing compounds, the nitrating and oxidizing activities were measured by
monitoring the formation of 3-nitrotyrosine and dityrosine with an HPLC-UV-fluorescence detector. The IC50
values for nitration and oxidation were determined, and the ratio of these two IC50 values was calculated for each
compound. Although the IC50 values varied from compound to compound, it was revealed that the ratio of two
IC50 values (IC50 for oxidation/IC50 for nitration) was 1 in almost all the compounds tested, except five indole de-
rivatives (L-tryptophan, melatonin, 5-methoxytryptamine, tryptamine, and tetrahydro-beta-carboline) and one
synthetic selenium-containing compound ((2R,3R,4S)-2-amino-3,4-dihydroxy-5-phenylselenopentan-1-ol, ADPP).
The indole derivatives showed a specific inhibitory effect on tyrosine nitration without affecting the oxidation.
ADPP was confirmed to have a preferable inhibitory activity for tyrosine oxidation. It was suggested that com-
pounds showing an IC50 value ratio of 1 scavenged the common species for nitration and oxidation, while the in-
dole derivatives and ADPP preferably scavenged the nitrating and oxidizing species, respectively. From a stopped
flow study, it was also revealed that the nitrotyrosine formation was relatively slow, unlike an OH radical reac-
tion. These results imply that the peroxynirite reaction at least partly proceeds through specific species for nitra-
tion.
Key words peroxynitrite; nitrotyrosine; tryptamine; scavenger; selenium; dityrosine
Reactive oxygen species are now well-known and impor- inhibitory effects of a certain compound have not been quan-
1
9)
tant components of oxidative stress in several diseases, such titatively discussed. We have briefly communicated that 5-
as inflammation, Parkinson’s disease, Alzheimer’s disease, methoxytryptamine (5MT) and a-lipoic acid (LA) are selec-
etc. It has also been suggested that nitric oxide (NO) and its tive inhibitors for tyrosine nitration by PN, but not for oxida-
activated species, reactive nitrogen species, are involved in tive dityrosine formation. In this paper, we report the evalua-
the oxidative damage in such diseases. Peroxynitrite (PN) is tion and comparison of the inhibiting activity for nitration
known as a reactive nitrogen species, and is thought to be and oxidation of tyrosine by PN of more than 40 reagents in-
Ϫ
2
1,2)
formed from NO and superoxide (O ·) in vivo. PN is a cluding natural and synthetic compounds, and the elucidation
highly reactive oxidant, and causes nitration on the aromatic of a unique property of five indole derivatives and one syn-
3
)
ring of free tyrosine and protein tyrosine residues. Since the thetic selenium-containing compound tested.
nitration of tyrosine residues is a characteristic reaction of
Experimental
PN, the presence of nitrotyrosine in tissues or cell cultures is
Chemicals L-Tyrosine and p-fluoro-L-phenylalanine were purchased
from Wako Pure Chemical Ind. (Osaka, Japan). 3-Nitro-L-tyrosine, 5-
often used as a marker of PN production, although it was re-
cently reported that myeloperoxidase and nitrite can induce
methoxytryptamine (5MT), melatonin, and tetrahydro-b-carboline were
4
)
tyrosine nitration in the presence of hydrogen peroxide. It from Aldrich (Milwaukee, WI, U.S.A.). All reagents purchased were of ana-
was also reported that PN induced various oxidative damage lytical grade.
Synthetic Compounds (2S,3R,4S)-N-3-[4-Hydroxy-2,3,5-trimethylphe-
noxy]propyl]-3,4-dihydroxy-5-hydroxymethylpyrrolidine (TMP-SRS-
DHHMP) hydrochloride was prepared from 3-[4-[(tert-butyldimethylsi-
lyl)oxy]-2,3,5-trimethyl-phenoxy]propyl bromide and (2S,3R,4S)-3,4-iso-
in vitro, for example low density lipoprotein oxidation, lipid
5
—14)
peroxidation, DNA strand breakage and so on.
Addition-
2
0)
ally, tyrosine nitration is thought to affect the phosphoryla-
21)
2
2)
tion of tyrosine residues in substrate proteins of tyrosine ki- propylidenedioxy-5-methoxymethoxymethylpyrrolidine in the presence of
5
—17)
nase in cellular signal transduction.
These data imply potassium carbonate in acetone followed by the removal of tert-butyl-
dimethylsilyl and methoxymethoxy protecting groups with tetrabutylammo-
that the oxidizing and nitrating reactions of PN may play dif-
ferent pathological roles in the oxidative stress process. From
this point of view, it is very useful and important to discrimi-
nium fluoride in tetrahydrofuran and with aqueous hydrochloric acid in
methanol at 60 °C, respectively.
1
mp 163—165 °C; H-NMR (D O) d: 1.90—2.30 (2H, m, CH ), 2.00,
2
2
nate the reaction of nitrating (nitrative) damage from that of 2.04, 2.09 (3H each, s each, 3ϫCH ), 3.05—3.80 (6H, m, 2ϫCH ), 3.85—
3
2
the oxidative damage in PN-induced oxidative stress reac- 4.05 (3H, m, 3ϫCH), 4.33—4.55 (2H, m, CH
), 6.62 (1H, s, aromatic pro-
2
1
3
ton); C-NMR (D O) d: 12.28 (q), 12.86 (q), 16.71 (q), 25.78 (t), 55.07 (t),
tions.
2
5
1
7.06 (t), 58.38 (d), 67.83 (t), 69.83 (t), 70.80 (d), 71.44 (d), 114.56 (d),
24.36 (s), 125.38 (s), 126.99 (s), 146.82 (s), 150.57 (s); [a] ϩ19.3° (cϭ1,
Various antioxidants have been reported to have an in-
20
D
3
,18)
hibitory effect on the nitration of tyrosine,
as well as oxi-
H O).
2
dation by PN. However, the relationships between these two
All other synthetic compounds were prepared as previously described or
∗
To whom correspondence should be addressed. e-mail: ikota@nirs.go.jp
© 2004 Pharmaceutical Society of Japan

January 2004
147
were generous gifts. (See Table 2 and References and Notes).
fective compounds, they were each plotted on the graph on
Peroxynitrite Preparation PN was synthesized as an alkaline solution
which the X-axis represented the IC value for the 3-nitroty-
2
3)
50
based on the method of Pryor et al. Briefly, NaN (138 mg) was dissolved
3
rosine formation and the Y-axis the dityrosine formation. It
was found that 20 compounds of 26 were plotted on the line
in 10 ml of H O, and the solution was then adjusted to pH 12 with 1.5 N
2
NaOH. The solution was bubbled with an oxygen stream containing ozone
(
generated at 1 g/h by an ozone generator) for 15 min while in an ice bath, so of yϭx, meaning that these compounds have the same in-
that the solution gradually turned yellow. The resulting solution was frozen
in a dry-ice/acetone bath, and then slightly defrosted at room temperature to
collect a concentrated yellow alkaline solution of PN. The obtained PN solu-
tion was stored at Ϫ80 °C until use. The concentration of PN was deter-
mined photometrically by measuring the absorbance at 302 nm (eϭ
hibiting potency for nitration as for oxidation. It was sug-
gested that these compounds scavenged the common species
for both the nitration and oxidation reaction of PN. On the
other hand, it was also found that the other 6 compounds,
Ϫ1
Ϫ1
1
670 M cm ). Using this method, up to a 240 mM solution of PN was ob- melatonin, 5-methoxytryptamine, tryptamine, tetrahydro-
tained. The concentration of the stock solution was determined again before
use, and then the stock solution was diluted to the desired concentration with
0
.01 N NaOH.
Inhibitory Effect of Various Compounds on the Reaction of Peroxyni- oxynitrite
trite with L-Tyrosine PN (0.2 mM) was added at 37 °C to the L-tyrosine
1 mM) solution in 0.1 M sodium phosphate buffer (pH 7.4) containing vari-
Table 1. Inhibitory Activity for Tyrosine Nitration and Oxidation by Per-
(
3-Nitrotyrosine
2,2Ј-Dityrosine
ous concentrations of testing compounds. After 10-min incubation at 37 °C,
p-fluorotyrosine (final 0.91 mM) was added to the reaction mixture as an in-
ternal standard. Due to the short lifetime of PN in the neutral solution, the
remaining PN was thought to decompose during the 10-min incubation. An
aliquot of the reaction mixture was analyzed with an HPLC-UV-fluorescence
detector system (TOSOH Corp., Tokyo, Japan). The HPLC conditions were
as follows; column: TSK-GEL ODS 80-Ts 4.6ϫ150 mm (TOSOH Corp.,
Tokyo, Japan), mobile phase: 0.1 M potassium phosphate (pH 3.5), flow rate:
Compounds
Inhibition IC50 (mM) Inhibition IC50 (mM)
Glutathione
ϩ
ϩ
ϩ
ϩ
ϩ
ϩ
ϩ
ϩ
ϩ
ϩ
ϩ
(ϩ)
ϩ
ϩ
ϩ
ϩ
ϩ
ϩ
ϩ
ϩ
ϩ
ϩ
ϩ
ϩ
(ϩ)
ϩ
0.04
0.289
0.438
0.032
1.53
0.038
0.5
ϩ
Ϫ
Ϫ
ϩ
ϩ
ϩ
ϩ
Ϫ
ϩ
ϩ
ϩ
(ϩ)
ϩ
ϩ
ϩ
ϩ
ϩ
ϩ
ϩ
ϩ
ϩ
Ϫ
Ϫ
ϩ
(ϩ)
ϩ
0.042
Ϫ
Ϫ
Melatonin
5MT
2
2
6)
7)
DTCP
ADPP
0.035
0.154
0.038
0.372
Ϫ
0.06
0.05
0.017
2
7)
1
.0 ml/min. The 3-nitro-L-tyrosine and 2,2Ј-dityrosine formed were detected
by monitoring the absorbance at 274 nm and the fluorescence at 410 nm (ex.
95 nm), respectively. There was no disturbance in the detection of nitroty-
NEMP
2
7)
APCB
2
Tryptamine
3Ј4Ј-DHBnTIQ
1.8
2
8)
rosine and dityrosine by the testing compounds used in this paper under
these analytical conditions. The detected 3-nitro-L-tyrosine was quantified
using a standard curve. The formation of dityrosine was confirmed by com-
paring the retention time and the excitation/fluorescence spectra of a de-
tected peak with the enzymatically synthesized authentic sample, as previ-
0.07
0.05
0.020
Ͼ2
0.025
0.01
0.0178
0.016
0.039
0.017
0.007
0.023
0.015
1.0
2
8)
67-DHBnTIQ
Curcumin
PBN
Uric acid
Catechin
Ͼ2
0.025
0.005
0.014
0.016
0.023
0.02
0.006
0.019
0.018
Ϫ
19)
ously described. The percent changes of the produced dityrosine were cal-
culated from the fluorescence peak area. When a testing compound showed
inhibitory activity, its IC50 value was determined for 3-nitro-L-tyrosine and
2
9)
RKO2B
3
0)
RK18R
RK16O
Trolox
2
5)
31)
for 2,2Ј-dityrosine formation.
Stopped Flow Measurement of the Nitration of Tyrosine A product
of L-tyrosine and PN, 3-nitriotyrosine has a characteristic local absorption
maximum (lmax) at 274 nm. By monitoring the absorbance change at this
wavelength, the production rate of 3-nitrotyrosine was measured. L-tyrosine
solution in 0.1 N HCl and PN solution in 0.01 N NaOH were mixed, and the
absorbance change at 274 nm was recorded with a stopped flow photometer
3
2)
THCL
HTP
3
3)
3
4)
DDFC
Tetrahydro-b-carboline
L-Tryptophan
2.0
Ϫ
3
5)
(
Otsuka Electronics Co., LTD, Osaka, Japan). From the observed reaction
TMG
0.022
Ͼ2 (5.0)
0.018
0.022
Ͼ2 (4.2)
0.020
3
6)
rate at various concentrations of L-tyrosine and PN, the reaction rate con-
stant was calculated as a second order reaction.
MS-818
HTPP
IC50 values are calculated from the dose-dependent curve determined from at least
three independent experiments for 1 mM tyrosine and 0.2 mM peroxynitrite. ϩ: in-
hibitory effect was observed, (ϩ): inhibitory effect was observed but very weak, Ϫ: in-
hibitory effect was not observed at all in the range of this work.
Results and Discussion
Various compounds, including natural and synthetic com-
pounds, were subjected to the assay for the inhibition of tyro-
sine nitration and oxidation by PN. In the presence of the
testing compound, 0.2 mM of freshly prepared PN solution
was mixed with 1 mM of L-tyrosine in sodium phosphate
buffer at physiological pH. The products were analyzed by
HPLC with UV and fluorescence detectors. It was confirmed
that 3-nitrotyrosine was formed as a major product of the re-
action. The formation of 3-nitrotyrosine was dependent on
the concentration of PN and L-tyrosine, as previously re-
1
9)
ported. The formation of 2,2Ј-dityrosine was also detected
1
4)
by fluorescence at 410 nm (ex. 295 nm). The production of
-nitrityrosine and 2,2Ј-dityrosine was measured as the ni-
3
trated and oxidized products, respectively, and the percentage
of production was calculated for the control reaction (con-
taining no testing reagents). Among the more than 40 tested
reagents showing an inhibitory effect on either nitration or
oxidation, the IC50 values were determined for 26 com-
pounds having sufficient efficacy (Table 1).
Fig. 1. Comparison of IC50 Values for Nitration and Oxidation of Tyrosine
with Peroxynitrite
IC50 values of the compounds for nitrotyrosine and dityrosine formation were deter-
To compare and characterize the inhibitory effect of 26 ef- mined for 1 mM of tyrosine and 0.2 mM peroxynitrite as described in Experimental.

1
48
Vol. 52, No. 1
beta-carboline, tryptophan, and ADPP were plotted out of the ond order rate constant (Fig. 2). This value was comparable
2
4)
yϭx line (Fig. 1). Melatonin, 5-methoxytryptamine, trypta- to the rate for tryptophan as previously reported. It is
mine, tetrahydro-beta-carboline, and tryptophan, which con- known that the reactivity of PN for the oxidation is similar to
tain indole moiety, showed an inhibitory effect only on the OH radicals, which is a diffusion rate-determining reaction,
nitration of tyrosine, and were plotted far above the line. This whereas the rate constant for the nitration calculated in this
result indicates that these compounds apparently scavenge experiment was much smaller than that for the OH radical re-
the specific species promoting the nitrating reaction. In con- action. This result suggests that the nitrating reaction has a
trast, ADPP, a selenium-containing synthetic compound, was different rate-limiting step. In the light of this result, the ni-
plotted below the line, indicating that this compound scav- trating reaction is not likely to be a direct rebound reaction of
enged the species for oxidation more effectively than that for caged NO radicals after electron subtraction by caged OH
2
nitration.
From the results of the reaction rate measurements using a
radicals from the aromatic ring of tyrosine.
It is known that PN couples with carbon dioxide to form
stopped-flow method, the reaction rate constant for the tyro- more reactive species. In our system, all the experiments
Ϫ1 Ϫ1
sine nitration was found to be 3.63ϫ102 M
s
as the sec- were conducted without additional carbon dioxide, so our re-
sults may not be directly applicable to the carbonate contain-
ing conditions such as in cell culture with carbonate ions, or
in serum in the veins. However, when the PN reaction with
tyrosine was carried out in Earle’s buffered salt solution con-
taining bicarbonate ions, 5-methoxytryptamine actually in-
hibited the nitration of tyrosine selectively without inhibiting
the dityrosine formation. Therefore, it is still likely that there
are different species or steps for tyrosine nitration by PN
even in the presence of carbonate ions.
In conclusion, almost all the compounds tested in this ex-
periment, including antioxidants, showed equal inhibitory ac-
tivity for both nitration and oxidation, suggesting that these
compounds scavenged the common species for nitration and
oxidation. However, five indole derivatives, tested selectively,
inhibited tyrosine nitration, suggesting that they scavenged
specific species for nitration. In contrast, ADPP, a selenium-
Fig. 2. Kinetics of the Reaction between Tyrosine and Peroxynitrite
Pseudo first order rates were determined by the stopped-flow method using indicated
containing compound, preferentially inhibited dityrosine for-
concentrations of L-tyrosine and synthetic peroxynitrite. The second order rate constant mation. Together with our results, it was suggested that there
was calculated from the pseudo first order rates.
may be specific species for tyrosine nitration by PN different
Fig. 3. Structures of Synthetic and Natural Compounds Used

January 2004
149
to the specie for the oxidation reaction. The rate-determining
step for nitration was assumed to be very slow compared
with the OH radical-like reaction of PN. It was also demon-
strated that the nitrating reaction of PN could be distin-
Redox Signaling, 1, 239—244 (1999).
2
0) Ikota N., Ueda J.-I., Gamage R., Shimazu Y., Hama-Inaba H.,
Takusakawa M., Ozawa T., Satoh T., “Biodefence Mechanisms against
Environmental Stress,” eds. by Ozawa T., Tatsumi K., Hori T., Kodan-
sha, Tokyo, 1998, pp. 1—12.
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K., Horikoshi H., J. Med. Chem., 32, 421—428 (1989).
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2
2
3) Pryor W. A., Cueto R., Jin X., Koppenol W. H., Ngu-Schwemlein M.,
Squadrito G. L., Uppu P. L., Uppu R. M., Free Radic. Biol. Med., 18,
Acknowledgments The authors acknowledge the late Professor Toshio
Satoh and Dr. Hideki Miyataka, Tokushima Bunri University, Professor Tsu-
tomu V. Kagiya, Kinki Research Foundation, Professor Shigeru Ohta and Dr.
Yaichiro Kotake, Hiroshima University, and Dr. Akira Awaya, for kindly
providing the valuable compounds. Partial financial support by a Grant-in-
Aid for Scientific Research (No. 10357021, 11771475) from Ministry of Ed-
ucation, Culture, Science, Sports and Technology Japan, and a grant from
the Cosmetology Research Foundation are gratefully acknowledged. This
study was performed using Special Coordination Funds of the Ministry of
Education, Culture, Sports, Science and Technology, Japan.
7
5—83 (1995).
2
2
4) Radi R., Chem. Res. Toxicol., 11, 720—721 (1998).
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6) N-Dithiocarboxy-L-proline (DTCP) was prepared as previously de-
scribed: Nakagawa H., Ikota N., Ozawa T., Masumizu T., Kohno M.,
Biochem. Mol. Biol. Int., 45, 1129—1138 (1998).
2
2
7) (2S,3R,4S)-2-Amino-3,4-dihydroxy-5-phenylselenopentan-1-ol
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pyrrolidine (NEMP), and (2S,4RS)-2-amino-4-phenylseleno-5-car-
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6) 2-Piperadino-6-methyl-5-oxo-5,6-dihydro(7H)-pyrrolo[3,4-d]pyrimi-
dine maleate (MS-818) was a generous gift from Dr. Akira Awaya:
Awaya A., Kobayashi H., Horikomi K., Tanaka S., Kabir A. M.,
Yokoyama K., Ohno H., Kato K., Kitahara T., Tomino I., Isayama S.,
Nakamura S., Biol. Pharm. Bull., 16, 248—253 (1993).
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3—66 (1996).
7) Li X., DeSarno P., Song L., Beckman J. S., Jope R. S., Biochem. J.,
31, 599—606 (1998).
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8) Whiteman M., Halliwell B., Free Radic. Res., 25, 275—283 (1996a).
9) Nakagawa H., Sumiki E., Ikota N., Matsushima Y., Ozawa T., Antioxi.