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INTRODUCTION |
Protein nitration has been detected in several inflammatory
disease states (1-3). Nitration of tyrosine or tyrosyl residues associated with peptides and proteins is perceived to be a diagnostic marker of reactive nitrogen species (e.g. peroxynitrite,
OONO
/ONOOH; nitrogen dioxide radical,
·NO2) (3-5). Nitration of protein tyrosyl has been
postulated to modulate protein metabolism, function, and signal
transduction pathways (1). Recent reports indicate that hydrophobic
protein tyrosyl residues are preferentially nitrated by reactive
nitrogen species (1, 6, 7). Thus, in vitro mechanistic
studies using "free" tyrosine in the aqueous phase may not
adequately reflect the mechanisms of nitration of protein tyrosyl
residues in vivo (8-10). In addition, the efficiency of
nitration of tyrosine induced by peroxynitrite in the aqueous phase has
been reported to be remarkably low (1-4%) (11-13). In contrast,
nitrations of membrane-incorporated
-tocopherol (a
membrane-associated chromanol) and N-t-BOC
l-tyrosine tert-butyl ester were increased by 3- to 5-fold (12, 14, 15). A plausible reason for the increased efficiency
of lipid-phase nitration is the greater membrane permeability of
reactive nitrogen species coupled with decreased lateral diffusion of
membrane-bound tyrosyl free radical, making radical-radical dimerization unlikely (15).
These previous studies demonstrated preferential nitration of
-tocopherol and tyrosyl analogs that are distributed throughout the
hydrophobic phase of the bilayer (12, 15). The objective of this study
was to test the hypothesis that the extent of nitration of
membrane-incorporated tyrosyl probe is dependent on its intramembrane location. To test this hypothesis, we have synthesized membrane spanning 23-mer peptides containing a single tyrosyl residue at positions 4, 8, and 12 and monitored their nitration reactions in the
membrane bilayer.
In this study, we have used
HPLC,1 electron spin
resonance (ESR), and fluorescence techniques for detection and
characterization of transmembrane nitration reactions of tyrosyl
probes induced by peroxynitrite and ·NO2.
Nitrating agents employed include slow infusion of peroxynitrite, generation of superoxide and nitric oxide (using SIN-1) that
rapidly combines to form peroxynitrite in situ, and a
myoperoxidase/H2O2/ NO
system that slowly generates ·NO2 free radical gas
(16-21). The present findings point to an intermediacy of
·NO2 in transmembrane nitration reactions.
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EXPERIMENTAL PROCEDURES |
The following chemicals and enzymes were purchased from various
sources as indicated: tyrosine, hydrogen peroxide, sodium nitrite,
sodium bicarbonate, 3-nitrotyrosine, and thiobarbituric acid
(Sigma), tetranitromethane and tetramethoxypropane
(Aldrich), 3-morpholinosydnonimine HCl (SIN-1; Calbiochem),
1,2-dilauroyl-sn-glycero-3-phosphatidylcholine (DLPC) and
1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocoline (PLPC) (Avanti Polar
Lipids), and myeloperoxidase (Calbiochem). Rink amide
methylbenzhydrylamine resin and all Fmoc-protected amino acids were
purchased from Calbiochem-Novabiochem (La Jolla, CA).
Diisopropylcarbodiimide (DIC), 1-hydroxybezotriazole (HOBt), triisopropylsilane, piperidine, and N-methylpyrrolidione
(NMP) were purchased from Fisher Scientific and used without further purification. Spin-labeled fatty acids (5-doxyl)-stearic acid and
(12-doxyl)-stearic acid are from Avanti Chemicals. Peroxynitrite was
synthesized according to the published procedure (18, 19), and its
concentration in 0.01 M NaOH was determined using the extinction coefficient (
= 1,670 M
1
cm
1 at 302 nm). All reagents were prepared in
double-distilled deionized water.
Peptide Synthesis and Purification--
Sequences of the
peptides used in this study are given in Table I. The peptides were
acetylated and amidated to stabilize their
-helical secondary
structure. The peptides were chemically synthesized using standard Fmoc
solid phase peptide synthesis chemistry on an Advanced Chemtech Model
90 synthesizer (Louisville, KY). Rink amide methylbenzhydrylamine resin
(loading 0.72 mmol/g) was used as a solid support. Fmoc-protected amino
acids were coupled as HOBt esters. All amino acids were double coupled
using HOBt/DIC. The following steps were performed in the reaction
vessel for each double coupling: deprotection of the Fmoc group with
20% piperidine in NMP for 30 min (2 times), three NMP washes, two dichloromethane washes, first coupling for 1 h with 5-fold excess of Fmoc amino acid in 0.5 M HOBt and 0.5 M DIC,
second coupling using a fresh addition of the same reagent for another
hour, three NMP washes, and two dichloromethane washes. Final
acetylation was performed using acetic anhydride/HOBt/DIC for 30 min
(twice). The resin was washed twice with dichloromethane and three
times with methanol and then dried under vacuum prior to cleavage. The peptide was deprotected and cleaved from the resin with 90%
trifluoroacetic acid containing triisopropylsilane for 3 h
at room temperature. The resin was removed by filtration and washed
with trifluoroacetic acid, and the combined trifluoroacetic acid
filtrates were evaporated to dryness under a steam of dry
N2 gas. The oily residue was washed three times with cold
ether to remove scavengers, and the dry crude peptide was dissolved in
acetonitrile/H2O (1:1) and lyophilized. The crude peptides
were purified by semi-preparative reverse-phase HPLC on a Vydac RP-C8
(10 × 250 mm) column (Hesperia, CA) using a
CH3CN/water gradient (20 to 100% CH3CN over 50 min) containing 0.1% trifluoroacetic acid at a flow rate of 3 ml/min
with detection at 220 nm. Elution of the peptides generally occurred
between 50 and 70% CH3CN. Purity and homogeneity of the
peptides were cross-checked by analytical reverse-phase HPLC, and the
purified peptides (~95% pure) were characterized by liquid
chromatography/mass spectrometry on an Agilent 1100 series liquid
chromatography/mass spectrometer.
Synthesis of Nitro Peptides--
10 mg/ml peptide dissolved in
MeOH/1% trifluoroacetic acid was mixed with
NO2BF4 (5 mg/ml). After 20 min, the product was purified by a semi-preparative HPLC (C-18; 250 × 10 mm),
pre-equilibrated with 50% CH3CN in 0.1% trifluoroacetic
acid. The nitro peptide was eluted by a linear CH3CN
gradient (0.025%/min). Collected nitro peptide peak was dried under a
stream of nitrogen and redissolved in MeOH as standard for analysis.
Nitro peptides show a characteristic UV-visible spectrum. After adding
NaOH, the 350-nm absorption peak in MeOH was pH-sensitive-shifted to
430 nm with an extinction coefficient of 4100 M
1 cm
1.
Spin Labeling Depth Measurements--
HPLC-purified peptides in
methanol were mixed with a 5-fold molar excess of
(1-oxyl-2,2,5,5-tetramethylpyrroline)-3-methylmethanethiosulfonate (MTSL) (Toronto Research Chemicals) and incubated overnight at 4 °C.
Unreacted spin labeled and unlabeled peptides were removed by
semi-preparative HPLC as described above, and the MTSL-labeled peptides
were collected and lyophilized. To prepared liposomes, spin-labeled
peptides were dissolved in methanol and added to a thin film of
DLPC to give a lipid:peptide molar ratio of 200:1. The samples
were dried under a stream of N2 and placed under vacuum for
at least 1 h. The dried lipid:peptide films were then hydrated at
room temperature in 50 mM Tris, 100 mM NaCl, pH
7.5, with or without 200 mM nickel (II)
ethylenediaminediacetate (NiEDDA). ESR spectra were obtained on a
Varian E-9 spectrometer equipped with a loop-gap resonator using a
field modulation amplitude of 1.0 G. Samples were contained in a
TPX capillary to facilitate equilibration with either air or
high purity N2.
Incorporation of Peptides into DLPC Liposome--
Liposomes were
prepared according to a previous report (15). A methanolic solution of
peptide was added to DLPC dissolved in methanol. The mixture was then
dried under a stream of N2 gas and kept in a vacuum
dessicator overnight. Multilamellar liposomes were formed by thoroughly
mixing the dried lipid in phosphate buffer (100 mM, pH 7.4)
containing DTPA (100 µM). Unilamellar liposomes were
prepared by five cycles of freeze-thawing using liquid nitrogen
followed by five cycles of extrusion through a 0.2-µm polycarbonate
filter (Nucleopore, Pleasanton, CA) in an extrusion apparatus (Lipex
Biomembranes, Inc., Vancouver, BC). The nitration of peptides in
liposomes prepared by both methods showed no significant difference.
Nitration of Tyrosyl Peptides in Liposomes--
A typical
reaction was initiated by adding 1 µl of various concentration of
peroxynitrite diluted in 0.05 M NaOH into 29 µl of
liposome (30 mM DLPC)-containing peptide (0.3 mM peptide). Incubation mixtures were in 0.1 M
phosphate buffer containing 100 µM DTPA. After 20 min at
room temperature, the reaction mixture was diluted with 600 µl of
distilled water and centrifuged at 12800 rpm for 120 min, and the
pelleted liposome was dried by a speedvac. The dried liposome
was dissolved in MeOH containing 2.5% trifluoroacetic acid for HPLC
analysis. Compared with liposomes dried directly, the recovery of
peptide was greater than 95%. Neither decomposed peroxynitrite nor
addition of NO
/NO
(200 µM) gave any detectable nitration products.
Peroxynitrite Infusion--
The slow infusion of peroxynitrite
was performed using an infusion/withdraw pump from Harvard Apparatus
(model 966) under constant stirring of liposomes or aqueous solution
containing tyrosine probes. Peroxynitrite (15 mM diluted in
0.25 M NaOH) was infused at a constant rate (0.82 ± 0.05 µl per 10 min) into 0.25 ml of liposomes (30 mM DLPC
containing 300 µM peptide) until the final concentration
of peroxynitrite was measured to be 50 µM. The infusion
rates for peroxynitrite were 5.0 µM/min. The maximum pH
shift was less than 0.1 units after infusion. Peroxynitrite decomposition under these conditions was evaluated by following the
absorption change at 302 nm and its nitration efficiency before and
after infusion. After 45 min, the changes of optical absorption and
nitration efficiency after infusion were less than 5% of the starting
peroxynitrite stock solution.
Myeloperoxidase/H2O2/NO
System--
Peptides (0.3 mM) in 30 mM DLPC
liposomes were incubated with 0.5 mM NaNO2, 0.1 or 0.2 mM H2O2, and 50 nM MPO in 0.1 M phosphate buffer containing 100 µM DTPA at 37 °C for 1 h. Samples were extracted and analyzed by HPLC as described below.
HPLC Analysis of Nitration Products--
Typically, 2 µl of
sample was injected into a capHPLC system (HP1100) with a cap
C-18 column (150 × 2 mm) equilibrated with 50% CH3CN
in 0.5% trifluoroacetic acid. The peptide and its nitration product
were separated by a linear increase of CH3CN concentration (0.1%/min) at a flow rate of 15 µl/min. The elution was monitored by
a variable UV detector at 280 and 350 nm. Peptide and nitrated peptide
were eluted at 12 or 17.5 min, respectively.
Nitration of tyrosine in aqueous solution was performed under the same
conditions, and the product analysis of tyrosine reaction in aqueous
solution was according to previous reports (12, 19, 22, 23). Briefly, 2 µl of sample was injected into a capHPLC system (HP1100) with a cap
C-18 column (150 × 2 mm) equilibrated with 4% MeOH in 50 mM phosphate buffer, pH 3.0, and the tyrosine and
nitrotyrosine were eluted at 5 and 12 min at a flow rate of 15 µl/min. The elution was monitored by a variable UV detector at 280 and 350 nm.
Fluorescence Quenching Measurements--
Fluorescence spectra of
peptides incorporated into liposomes (0.3 mM peptide in 30 mM DLPC in 0.1 M phosphate buffer containing 100 µM DTPA) were monitored using an excitation
wavelength of 278 nm (slit width, 5 nm) and monitoring emission from
300-350 nm (slit width, 10 nm). The fluorescence quenching experiments were performed as follows: methanolic solutions of spin labels, 12-doxyl SA, or 5-doxyl SA in glass vials were dried under a stream of
nitrogen, and liposomes containing peptides (Y-4, Y-8, or Y-12) were pipetted into these vials and vortexed for 5 min, allowing incorporation of the doxyl SA into the liposomes.
TBA-MDA Assay--
We used a modified HPLC method to measure
TBA-MDA adduct (24). A mixture of 25 µl of sample (or standard), 75 µl of 0.15 M H3PO4, 25 µl of
0.44 mM TBA, and 25 µl of distilled H2O was heated in boiling water for 1 h and then cooled down on ice. An aliquot of 50 µl was neutralized with 50 µl of methanol-sodium hydroxide solution and analyzed by HPLC. Reverse-phase isocratic separation was applied on a Kromasil C-18 column (250 × 4.6 mm, 5 µm) with a methanol/water mobile phase (70/30%) (v/v) at
0.5 ml/min flow rate. The TBA-MDA adduct eluting at 4.7 min was
detected at 532 and 553 nm extinction and emission, respectively. The
injected amount was 10 µl.
ESR Measurements--
ESR spectra were recorded at room
temperature on a Bruker ER 200 D-SRC spectrometer operating at 9.8 GHz
and a cavity equipped with a Bruker Aquax liquid sample cell. Typical
spectrometer parameters were as follows: scan range, 100 G; field set,
3510 G; time constant, 0.64 ms; scan time, 20 s; modulation
amplitude, 5.0 G; modulation frequency, 100 kHz; receiver gain, 2 × 105; and microwave power, 20 mW. Reaction mixtures
consisting of Y-4 (0.4 mM) and 20 mM
2-methyl-2-nitrosopropane (MNP) spin trap incorporated into 40 mM DLPC liposome in a phosphate buffer (0.1 M,
pH 7.4) containing DTPA (0.1 mM) were rapidly mixed with 2 mM peroxynitrite. Samples were subsequently transferred to
a 100-µl capillary tube for ESR measurements.
Statistic Analysis--
The data are presented as average ± S.D. The significance was calculated using a Student's t test.
 |
RESULTS |
Characterization of Transmembrane Peptides in Liposomes--
Table
I shows the amino acid sequences and mass
spectral data for the various tyrosyl and cysteinyl peptides
used in the present study. These peptides were synthesized and purified
as described under "Experimental Procedures." To assess the
localization of the tyrosine residues in these peptides with respect to
the membrane bilayer, we examined their intrinsic fluorescence
properties and susceptibility to quenching by nitroxide-labeled fatty
acids. As shown in Fig. 1A,
fluorescence intensity for this series of peptides increases in the
order Y-12 > Y-8 > Y-4, indicating a progressively
increasing hydrophobic environment (membrane depth) for the tyrosine
residues. A similar order of sensitivity to fluorescence quenching by
nitroxides attached at the 5-position of stearic acid (5-doxyl SA) and
at the 12-position of stearic acid (12-doxyl SA) was observed (Fig.
1B). Fluorescence quenching by 5-doxyl SA, which localizes
near the glycerol backbone of phospholipid bilayers, was greater for
Y-4 than for Y-8 or Y-12 (Fig. 1B), further indicating that
the tyrosine residue of Y-4 resides just below the membrane surface. On
the other hand, fluorescence quenching by 12-doxyl SA was greater for
Y-12 than for Y-4 indicating that Y-8 and Y-12 are located deeper in
the bilayer. A schematic representation of the intramembrane position
of tyrosyl peptides and nitroxide-labeled fatty acids is shown in Fig.
1C. These data are all consistent with the expected
transmembrane orientation of these peptides (25-29) and indicate that
incorporation into a repeating leucine-alanine motif anchored at each
end by lysine residues is an effective strategy for positioning
tyrosine residues across the lipid bilayer.

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Fig. 1.
Fluorescence spectra of membrane-incorporated
tyrosyl peptides. A, fluorescence spectra of tyrosyl
peptides (0.3 mM) incorporated into liposomes (30 mM DLPC in 0.1 M phosphate buffer containing
100 µM DTPA) were obtained using an excitation wavelength
of 278 nm. B, the effect of 5-doxyl and 12-doxyl stearic
acid nitroxides (1 mM) on the ratio of the fluorescence
intensity of tyrosyl peptides in the absence
(F0) and in the presence of nitroxide
(F). C, schematic representation of relative
intramembrane position of tyrosyl peptides and doxyl probes.
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To further characterize bilayer depth we synthesized the analogous
peptides with cysteine replacing tyrosine (i.e. C-4, C-8, and C-12), modified the single cysteine in each peptide with a sulfhydryl-specific nitroxide spin label MTSL, and measured bilayer depth by established methods using O2 and NiEDDA as
paramagnetic relaxation agents (30, 31). The ESR spectra of
MTSL-labeled peptides in DLPC liposomes are shown in Fig.
2A. The ESR spectra of
spin-labeled peptides in Fig. 2 are significant in that they demonstrate that all of the peptide has been incorporated into the
membrane, giving homogeneous, single component spectra. Peptides with a
similar sequence may distribute between a transmembrane orientation and alignment along the surface of the bilayer, which would give rise to a two-component ESR spectrum. The spectra in Fig. 2
show that, at least for our methods of liposome preparation, this is
not the case. MTSL-C-12 shows a slightly greater degree of motional
restriction than the other two peptides. Isotropic hyperfine coupling
constants for the three peptides ranged between 13.45 and 13.9 G,
consistent with their hydrophobic location.

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Fig. 2.
ESR spectrum of MTSL-labeled cysteinyl
peptides in DLPC liposomes. A, the ESR spectrum of
MTSL-labeled C-4 peptide (top), C-8 peptide
(middle), and C-12 peptide (bottom). The
lipid:peptide ratio was 200:1. The scan width was 100 G. B,
continuous wave power saturation for MTSL-C8 in DLPC liposomes under
N2 (circles), air (squares), and in
the presence of 200 mM NiEDDA (triangles). The
greater shift to higher powers in the presence of O2
relative to NiEDDA is characteristic of a hydrophobic environment for
the spin label.
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Bilayer depths of the nitroxide side chains were determined by power
saturation ESR measurements (Fig. 2B) based on relative collision rates with O2 and NiEDDA (30, 31). In agreement with the fluorescence studies described above, the C-4 position was
nearest to the membrane surface at an estimated depth of 9 Å below the
lipid phosphates, whereas nitroxide side chains attached at C-8 and
C-12 were located further below the lipid phosphates (~ 12.5 Å).
Peroxynitrite-dependent Oxidation and Nitration of
Tyrosyl Peptides in Membranes: Comparison with Tyrosine in Aqueous
Solution--
Previous studies implicated that peroxynitrite-induced
nitration and oxidation reactions are favored in the hydrophobic phase of lipid membranes (12, 15). In this study, we sought to demonstrate this phenomenon with tyrosyl residues that are anchored at specific positions in the lipid bilayer. Peroxynitrite-dependent
nitration of tyrosyl-containing peptides in DLPC liposomes was
monitored by HPLC with UV-visible detection at both 280 and 350 nm
(Fig. 3, A and B).
The Y-8 peptide eluted with a retention time of 12 min, and
pre-synthesized NO2-Y-8 eluted after 17.5 min. A bolus addition of peroxynitrite (100 µM) to DLPC liposomes
containing Y-8 peptide (300 µM) resulted in the formation
of a new compound eluting at 17.5 min, along with the parent peptide
eluting at 12 min (Fig. 3, middle trace). The peak eluting
at 17.5 min was assigned to the nitration product, NO2-Y-8
peptide, by comparison with the authentic standard (Fig. 3, top
trace). Decomposed peroxynitrite did not induce any nitration of
peptide in DLPC liposomes (Fig. 3, bottom).

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Fig. 3.
HPLC traces of nitrated tyrosyl peptides in
liposome. Y-8 peptide (0.3 mM) in 30 mM
DLPC liposome was reacted with peroxynitrite in 0.1 M
phosphate buffer containing 100 µM DTPA and analyzed by
capillary HPLC. A, HPLC traces were monitored at 280 nm.
Top indicates the authentic 20 µM
NO2-Y-8 peptide, middle shows the nitrated
reaction product from the reaction between peroxynitrite and Y-8
peptide and bottom is the same as above but in the presence
of decomposed peroxynitrite (left) HPLC traces monitored at
280 nm that showed both original Y-8 peptide (12 min) and nitration
product (17.5 min). B, HPLC traces monitored at 350 nm for
nitration products. Experimental conditions were the same as in
A.
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Previous reports suggest that the rate at which peroxynitrite is added
to a solution containing tyrosine can drastically alter the nitration
yield (8, 9, 15, 22, 23, 32, 33). A bolus addition of increasing
concentrations of peroxynitrite showed a significant increase in
formation of NO2-Y-8 (Fig.
4A). Under identical
conditions, bolus addition of peroxynitrite to a phosphate buffer
containing tyrosine (300 µM) also yielded a dose-dependent increase in the formation of nitrotyrosine
as monitored by HPLC-UV. At high peroxynitrite concentrations
(~300 µM), the yields of NO2-Tyr
were about 2-fold higher than NO2-Y-8 peptide (Fig.
4A). However, this difference was diminished as
peroxynitrite concentration decreased; a bolus addition of 50 µM peroxynitrite induced greater nitration of Y-8 peptide
(6.8 µM NO2-Y-8 peptide) in DLPC liposomes as
compared with tyrosine (4.2 µM) in aqueous solution (Fig.
4A). The nitration profile of Y-8 peptide in DLPC membrane
was similar to that of N-t-BOC tyrosine
tert-butyl ester (BTBE), a hydrophobic tyrosine analog (15).
Fig. 4B shows the nitration yields of tyrosyl residues
anchored at different depths in DLPC liposomes. HPLC analysis showed
that the nitration yields of tyrosyl peptides in membranes were
significantly higher than that of tyrosine in aqueous phase. Results
showed that the formation of nitrated peptides slightly increased as
the depth of tyrosyl residues in the bilayer were increased
(NO2-Y-4, 5.8 µM; NO2-Y-8, 6.8 µM; and NO2-Y-12, 7.4 µM).

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Fig. 4.
Nitration of tyrosine in the aqueous phase
and tyrosyl peptides in membranes. A, tyrosine (0. 3 mM) or Y-8 (0.3 mM) in DLPC (30 mM)
liposomes was incubated with various concentrations of peroxynitrite
for 20 min in sodium phosphate buffer (0.1 M, pH 7.4)
containing DTPA (100 µM) and analyzed by HPLC
(n = 3 ± S.D.). B, tyrosine (0. 3 mM) or Y-4, Y-8, and Y-12 peptides (0. 3 mM
each) in DLPC (30 mM) liposomes were incubated with
peroxynitrite (50 µM) for 20 min in sodium phosphate
buffer (0.1 M, pH 7.4). C, tyrosine and tyrosyl
peptides (0.3 mM) in DLPC (30 mM) liposomes
were incubated with SIN-1 (1 mM) in sodium phosphate buffer
(0.1 M, pH 7.4) containing DTPA (100 µM)
overnight. Nitrated products were analyzed by HPLC. D,
tyrosine (0.3 mM) or tyrosyl peptides (0.3 mM)
in DLPC (30 mM) liposomes were incubated with
NaNO2 (0.5 mM), H2O2
(0.2 mM), and myeloperoxidase (50 nM) in a
phosphate buffer (0.1 M, pH 7.4) containing DTPA (100 µM) at 37 °C for 1 h. The sample was extracted
and analyzed by HPLC as described under "Experimental
Procedures."
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To investigate the nitrating ability of peroxynitrite that is generated
in situ (Fig. 4C), DLPC liposomes containing
tyrosyl peptides (300 µM) or tyrosine (300 µM) in phosphate buffer were incubated with SIN-1 (1 mM) (Fig. 4C). Nitration of tyrosine in aqueous
phase by SIN-1, which generates equal amounts of nitric oxide and
superoxide, gave very low yields of nitrotyrosine (~ 0.5 µM) (Fig. 4C) (9, 12). Under similar
conditions, the nitration yield of Y-12 peptide in membrane was about
18 times higher (9 µM) than that of tyrosine in aqueous
solution (Fig. 4C). Slow infusion of peroxynitrite (5 µM/min; final concentration, 50 µM) into
DLPC liposome containing Y-8 peptide also induced a 5-fold increase in
nitration (6.2 µM) compared with the nitration of
tyrosine (1.2 µM) in aqueous phase (Fig.
5C). Incubation of SIN-1 (1 mM) with DLPC liposome containing these peptides (Y-4, Y-8,
and Y-12) revealed that nitration of those peptides was enhanced with
increasing membrane depth for the tyrosine residues (Fig. 4C). Investigation of the nitration profile of Y-4, Y-8, and
Y-12 obtained in MPO/H2O2/NO
system produced an interesting yet unexpected result (Fig.
4D). In contrast to SIN-1-catalyzed nitration profile,
incubation of DLPC liposome containing Y-4, Y-8, and Y-12 in the
presence of MPO, H2O2, and NO
caused a decrease in the nitration of peptide
with increasing membrane depth (Fig. 4D).

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Fig. 5.
The effect of bicarbonate on
peroxynitrite-mediated nitration of tyrosine in aqueous solution and
tyrosyl peptides in membranes. A, tyrosine (0. 3 mM) or Y-4, Y-8, and Y-12 peptides (0. 3 mM
each) in DLPC (30 mM) liposomes were incubated with
peroxynitrite (50 µM) for 20 min in sodium phosphate
buffer (0.1 M, pH 7.4) containing DTPA (100 µM) with or without 25 mM bicarbonate.
B, tyrosine and tyrosyl peptides (0.3 mM) in
DLPC (30 mM) liposomes were incubated with SIN-1 (1 mM) and bicarbonate (25 mM) in sodium phosphate
buffer (0.1 M, pH 7.4) containing DTPA (100 µM) overnight. Nitrated products were analyzed by HPLC.
C, tyrosine (0.3 mM) and Y-8 peptide (0.3 mM) in DLPC (30 mM) liposomes were infused with
peroxynitrite at 5 µM/min (accumulating to a final
concentration of 50 µM) with and without bicarbonate (25 mM) in phosphate buffer (0. 1 M, pH 7.4)
containing 100 µM DTPA and analyzed by HPLC.
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To investigate the effect of tyrosine on the relative product yields,
we incubated SIN-1 or MPO in the reaction mixtures containing free
tyrosine at the same concentration as Y-8 peptide in DLPC liposomes.
Table II shows the relative yields of
nitrated peptide, nitrotyrosine, and dityrosine. Even in the presence
of free tyrosine, the nitration yield in the membrane is higher than
that of free tyrosine in the aqueous phase. However, the overall yield
of peroxynitrite from SIN-1 is considerably lower in the presence of
tyrosine. The effect of tyrosine on the mechanism of decomposition of
SIN-1 is not known. With SIN-1, there was no detectable change in the dityrosine concentration in the presence or absence of DLPC liposome containing Y-8 (Table II). With
MPO/H2O2/NO
-induced nitration, the relative nitration yield in the membrane was
significantly enhanced in the presence of tyrosine. Under these
conditions, the dityrosine yield in the aqueous phase was increased by
more than 2-fold (Table II). Clearly, the hydrophobic membranes
significantly affect the
MPO/H2O2/NO
-induced
dityrosine formation in the aqueous phase. We are actively
investigating the mechanism of this reaction in detail.
Effect of Bicarbonate on Peroxynitrite-mediated Nitration of
Tyrosyl Peptides--
Next we compared the effect of bicarbonate anion
(HCO
) on nitration of tyrosyl peptides in membrane with that of aqueous tyrosine, as HCO
had been shown
to enhance peroxynitrite-dependent oxidation and nitration
of tyrosine (34-39). Peroxynitrite reacts with CO2 to generate the nitrosoperoxycarbonate (ONOOCO
) intermediate that is responsible for increased nitration and oxidation of tyrosine in the aqueous phase (15). Fig. 5 shows the effect of
increasing concentrations of HCO
on the nitration of
tyrosine caused by a bolus and the slow addition of peroxynitrite.
Bicarbonate considerably enhanced the nitration of tyrosine in aqueous
solution as expected (15, 33, 39), whereas nitration of peptides in
DLPC liposomes by a bolus addition of peroxynitrite was inhibited by
bicarbonate (Fig. 5A). As shown in Fig. 5A, the
inhibitory effect of bicarbonate on the nitration of peptides in
membrane increased in the order Y-12 (60%) > Y-8 (30%) > Y-4 (20%). This indicates a lack of diffusion or slow diffusion of the
intermediate radical pair (·NO2 ...
CO
) into membranes (36). Although the degree to
which bicarbonate inhibited membrane nitration varied to some extent,
the overall effect of bicarbonate on membrane nitration was minimal
(Fig. 5, A-C). Based on these findings, we
conclude that bicarbonate does not affect nitration of tyrosyl peptides
in membrane exposed to co-generated O
and ·NO at the
same flux or to slowly infused peroxynitrite.
Effect of Unsaturated Fatty Acid on Tyrosine Nitration and Lipid
Oxidation--
Biological membranes are composed of unsaturated
lipids. Recently, several papers reported that peroxynitrite can
diffuse into membranes (15, 38, 40, 41) and oxidize unsaturated fatty
acids. To investigate the effect of unsaturated fatty acids on
transmembrane nitration of tyrosyl peptides, we used PLPC as a model
liposomal system. Peroxynitrite (50 µM) was added as a bolus or generated in situ from SIN-1 in incubations
containing Y-8 peptide (0.3 mM) incorporated into liposomes
(30 mM PC), composed of differing percentages of
PLPC. Nitration of tyrosyl-containing peptides was inhibited by
unsaturated fatty acid in a concentration-dependent manner
(Fig. 6A). The nitration yield
of Y-8 peptide was reduced from 6.5 µM in 100% DLPC to
about 1.6 µM in 100% PLPC liposome under otherwise
identical conditions. Concomitantly, an increase in MDA, an oxidation
product of polyunsaturated fatty acids, was observed (Fig.
6B). These results indicate that unsaturated fatty acid
effectively scavenges peroxynitrite or its reactive intermediate and
inhibits tyrosine nitration in membranes containing PLPC.

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Fig. 6.
The effect of unsaturation on peroxynitrite-
and MPO/H2O2/nitrite-induced nitration of
tyrosyl peptides. A, Y-8 peptide (0.3 mM)
in liposomes composed of various amounts of DLPC and PLPC (total, 30 mM) were incubated with 50 µM peroxynitrite
in sodium phosphate buffer (0.1 M, pH 7.4) containing 100 µM DTPA. Nitrated Y-8 was analyzed by HPLC
(n = 3 ± S.D.). The effect of unsaturation on
MPO/nitrite/H2O2-induced nitration of tyrosyl
peptide was determined. Conditions were essentially the same except
that incubations were performed with 0. 5 mM
NaNO2, 0.2 mM H2O2, and
50 nM MPO. Nitrated Y-8 was analyzed by HPLC
(n = 3 ± S.D.). B, formation of MDA
was measured by HPLC under the reaction conditions that were the same
as A.
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The effect of different proportions of PLPC on
MPO/H2O2/ NO
-dependent
transmembrane tyrosyl nitration was then investigated. As shown in Fig.
6B, nitropeptide formation was inhibited by unsaturated
fatty acid in a dose-dependent manner. With increasing
amount of unsaturated fatty acid,
MPO/H2O2/NO
caused an
increase in lipid oxidation (Fig. 6B) while decreasing tyrosine nitration. These data suggest that ·NO2
generated from MPO/H2O2/NO
is scavenged by PLPC resulting in increased MDA formation and decreased nitration.
The Effect of Nitric Oxide on Peroxynitrite- and
MPO/ H2O2/NO
-induced
Nitration of Tyrosyl Peptides--
Nitrogen dioxide radical is rapidly
scavenged by ·NO to form N2O3 that is
hydrolyzed to form NO
(42). Peroxynitrite also
reacts with ·NO to form N2O3 (43). As
both ·NO and ·NO2 can readily diffuse into
the membrane (44), this radical-radical recombination reaction should
rapidly occur in the hydrophobic domain of membranes. Fig.
7 shows that ·NO dramatically
inhibits transmembrane nitration. We investigated the effect of
·NO released slowly from spermine NONOate (SNN) (5 µM/min) on transmembrane nitration of tyrosyl peptides
induced by MPO/ H2O2/NO
and by ONOO
generated in situ. As shown in
Fig. 7A, ·NO generated by SNN inhibited
MPO/H2O2/NO
- dependent
tyrosine nitration in the aqueous phase by 80% and Y-8 nitration in
the membrane by more than 90%. Under the same conditions, SNN
inhibited SIN-1-mediated aqueous phase nitration by 33% and Y-8
nitration in the membrane by over 80% (Fig. 7B). Decomposed SNN did not inhibit nitration (not shown). These results indicated that
·NO inhibits membrane nitration much more effectively than
tyrosine nitration in the aqueous phase. This is attributed to the
rapid reaction between ·NO and ·NO2 or
·NO and peroxynitrite.

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Fig. 7.
The effect of spermine NONOate on
peroxynitrite- and
MPO/H2O2/NO -induced
nitration of tyrosyl peptides in the membrane. A, Y-8
peptide (0.3 mM) in DLPC liposome (30 mM) or
tyrosine (0.3 mM) was incubated with NaNO2 (0.5 mM), H2O2 (0.2 mM), and
50 nM MPO in phosphate buffer (0.1 M, pH 7.4)
containing DTPA (0.1 mM) at 37 °C for 1 h.
B, Y-8 peptide (0.3 mM) in DLPC liposome (30 mM) or tyrosine (0.3 mM) was incubated with
SIN-1 (1 mM) at room temperature overnight. Spermine
NONOate (1 mM) was added to incubation mixtures in
A and B as indicated. The samples were extracted
and analyzed by HPLC as described under "Experimental
Procedures."
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Spin Trapping of Tyrosyl Radical in Membrane--
To investigate
whether peroxynitrite induces nitration of tyrosyl peptides via a
radical mechanism, we used MNP, a lipophilic spin trap, to detect
tyrosyl peptide-derived radicals. Previously, MNP has been used to
detect and characterize tyrosyl radical formed during
MPO/H2O2/NO
-catalyzed
oxidation of tyrosine in solution (45). As shown in Fig.
8, the addition of peroxynitrite (2 mM) to DLPC liposome (40 mM) incorporated with
Y-4 (0.4 mM) and MNP (20 mM) in a sodium
phosphate buffer (0.1 M, pH 7.4) containing DTPA (0.1 mM) gave rise to an ESR spectrum of a spin adduct that is
motionally restricted. Spectral features show similarity to MTSL-C-4
(see Fig. 2). No ESR spectrum of a spin adduct was obtained with
decomposed peroxynitrite (Fig. 8). The spin adduct was extracted into
methanol, which gave an isotropic ESR (
N = 15.6 G)
spectrum. Because of the poor signal-to-noise ratio and oxygen
broadening, the spectrum could not be resolved further even in the
presence of fully deuterated spin trap (data not shown). It is also
noteworthy that the spectra in Fig. 2 are quite similar to those
obtained by spin trapping during peroxynitrite-induced tyrosyl peptide
nitration (Fig. 8). Thus, the ESR data provide further support for
radical formation from tyrosyl peptides in the presence of active
peroxynitrtie.

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Fig. 8.
Spin trapping of tyrosyl radical formed
during peroxynitrite-induced nitration of tyrosyl peptides in
membrane. Y-4 (0.4 mM) in DLPC (40 mM) was
reacted with peroxynitrite (2 mM) in the presence of MNP
(20 mM) in a phosphate buffer (0.1 M, pH 7.4)
containing DTPA (0.1 mM). The ESR spectrum was recorded
using a modulation amplitude of 5 G, scan time, 20 s; time
constant, 0.64 ms; microwave power, 20 mW. A modulation of 2G was used
to obtained the spectrum of spin adduct in methanol.
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DISCUSSION |
Characterization of Tyrosyl Peptides--
The rationale for this
study was to examine whether protein tyrosyl residues exposed to the
hydrophobic interior of biological membranes are more prone to
nitration than their aqueous counterparts. Biological nitration of
tyrosine residues has been the subject of much interest. At present two
major candidate mechanisms for nitration have been identified that
involve either the formation of peroxynitrite from the
diffusion-limited reaction between nitric oxide and superoxide or the
peroxidatic activity of peroxidase enzymes, using nitrite as the
electron donating substrate. It should be noted that these mechanisms
are not mutually exclusive, nor is nitration the only modification that
results from these agents. This study also attempts to clarify the
mechanisms of hydrophobic tyrosine nitration and to examine how the
source of nitration affects the relative product yields in the
hydrophobic interior of membranes. To examine these issues we have
synthesized model tyrosyl-containing peptides designed to position the
tyrosyl group at varying depths within the membrane. The peptides
consisted of an alanine-leucine repeat that has been shown previously
to form transmembrane helices, anchored at both ends by two lysine residues. Tyrosyl residues were inserted at positions 4, 8, and 12 (termed Y-4, Y-8, and Y-12) to place the nitration target at the
surface of the bilayer, at approximately one-quarter of the way into
the bilayer and at approximately the center of the bilayer. The
position of the residues within the bilayer was confirmed by two
approaches. The first used the intrinsic fluorescence of the tyrosyl
residue and the ability of fatty acid nitroxides to quench this
fluorescence. The second method used a complimentary set of peptides
containing cysteine in place of tyrosine. The cysteine residues were
spin-labeled, and the depth of the label was determined using the
ESR/power saturation technique. In both cases, the data were consistent
with the predicted depth of the tyrosyl residue. The present data
indicate that the peptide was properly inserted into the bilayer, most
likely as a transmembrane
-helix.
The Effect of the Nitration Modality on Tyrosyl Peptide
Nitration--
Nitration of the Y-8 peptide with bolus addition of
chemically synthesized peroxynitrite gave a concentration-dependence
similar to our previous observations using BTBE, a non-anchored
hydrophobic tyrosine analog (15). In both cases, nitration of the
membrane-localized probe was significantly reduced, as compared with
tyrosine in the aqueous phase, at high levels of peroxynitrite.
However, this difference was lost at lower levels of peroxynitrite, and
nitration of membrane-anchored Y-8 exceeded the aqueous phase tyrosine
nitration at the lowest concentration tested (50 µM).
This illustrates the complex relationship between product yield and the
nitration modality that should be taken into consideration when
extrapolating the relevance of in vitro nitration studies to
the biological arena.
We previously observed that BTBE had significantly reduced dimer
formation than tyrosine. In the present study, no dimer product from
peroxynitrite-mediated oxidation of the peptides was observed. The most
likely explanation for this is that the hindered diffusion substantially inhibits radical-radical interactions necessary to form
the tyrosyl dimer. This restriction would be more severe with
membrane-anchored peptides than with BTBE.
The extent of peptide nitration by bolus addition of peroxynitrite did
not show a strong depth dependence, although a trend toward higher
nitration yield at greater depth was apparent. However, in all cases
the yield of nitration product was greater than that observed for
tyrosine. This indicates that the preferential nitration of
membrane-associated tyrosine at low bolus amounts of
peroxynitrite had little depth dependence. In contrast, the
simultaneous formation of nitric oxide and superoxide from SIN-1 gave a
significantly higher yield of peptide nitration compared with tyrosine
and exhibited a marked depth dependence. Nitration from SIN-1 increased
as a function of depth in the membrane. Most significantly, this
pattern was reversed when
myeloperoxidase/H2O2/NO
was
used as the source of the nitrating species. In this case the nitration
of tyrosyl residues was negatively correlated with tyrosyl depth in the
membrane. This not only highlights significant differences in the
mechanism of nitration by these two agents but also gives a potential
way to discriminate between them in a biological milieu.
Mechanism of Peroxynitrite- and
MPO/NO
/H2O2-induced
Transmembrane Nitration: Intermediacy of Nitrogen Dioxide
Radical--
Two major pathways have been suggested to be responsible
for tyrosine nitration in vivo (2, 5, 16). These involve either the nitrative chemistry of peroxynitrite or the catalytic action
of heme peroxidases using H2O2 and nitrite
anion as substrates. Scheme 1 illustrates
the reactions involved in both the oxidation and nitration by either a
peroxynitrite-dependent route (left hand side)
or a myeloperoxidase-dependent pathway (right hand side).

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Scheme 1.
Postulated reaction pathways for
transmembrane nitration of tyrosyl peptides anchored in membranes
catalyzed by peroxynitrite and myeloperoxidase/nitrite
anion/H2O2. The relevant rate constants of
peroxynitrite and ·NO2 with ·NO, tyrosine,
CO2, and thiols are taken from Ref. 46.
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Peroxynitrite exists in equilibrium with peroxynitrous acid as shown in
Reaction 1.
Peroxynitrous acid (ONOOH) has been reported to diffuse into
membranes at rates similar to that of water molecules (36, 38, 40, 41).
Homolytic decomposition of ONOOH in the membrane will form HO·
and ·NO2 (47). Although the involvement of
HO· in tyrosine nitration in the aqueous phase has been shown to be negligible based on hydroxyl radical scavenger experiments, its role
in transmembrane nitration of tyrosyl peptides remains unclear.
Transmembrane nitration mediated by peroxynitrite is more likely to be
a free radical-dependent mechanism.
In the presence of H2O2, MPO forms higher
oxidants (compounds I and II) that will oxidize nitrite into
·NO2 radical and tyrosine to tyrosyl radical.
Nitration of tyrosine occurs following a rapid recombination of
·NO2 and the tyrosyl radical (Scheme 1). The
oxidation of tyrosine by MPO-compound I to tyrosyl radical is deemed to
be essential for tyrosine nitration, as the hydrogen abstraction from
the tyrosyl hydroxyl group by ·NO2 in the aqueous
phase is relatively slow (46). It is conceivable that tyrosyl peptides
incorporated into liposomes are not readily accessible to MPO-compound
I. However, the reaction between ·NO2 and the
tyrosyl hydroxyl group in the hydrophobic interior of the phospholipid
membrane may be more effective.
Nitrogen dioxide radical undergoes hydrolysis very readily in
the aqueous phase forming nitrite and nitrate anions. However, ·NO2 is persistent in the hydrophobic phase and will
participate in one-electron oxidation reactions with tyrosyl peptides
forming the tyrosyl radical that can rapidly combine with
·NO2 to form the nitrated tyrosyl peptide (Scheme 1)
(4, 49-51). Recently, it was shown that ·NO2
generated extracellularly by peroxidatic oxidation of nitrite anion
diffuses into cells and nitrates a tyrosyl residue present within a
green fluorescent protein (52). Nitration of fluorophore quenches the
intrinsic green fluorescent protein fluorescence because of an
electron-withdrawing effect of the nitro group. In the present work, we
show that ·NO2 generated from
MPO/H2O2/NO
diffuses into
the membrane and efficiently nitrates tyrosyl peptide. Nitration yield,
however, decreased with increasing depth of tyrosyl location (i.e. NO2-Y-4 > NO2-Y-8 > NO2-Y-12). These findings suggest that reactive nitrogen
species generated in biological systems can now be quantitated more
accurately by monitoring the nitration of tyrosyl peptides incorporated
into membranes. This trend is opposite the nitration profile observed
with peroxynitrite. Previously, it has been shown that nitration of
tyrosyl peptides by ·NO2 is enhanced in systems
generating tyrosyl radicals (17). Thus, we tested the hypotheses that
(i) Y-4 peptide can be oxidized by compound-I formed by
MPO/H2O2 to generate the tyrosyl radical that
subsequently reacts with ·NO2 to form
NO2-Y-4, and (ii) Y-8 and Y-12 that are buried deeper in
the bilayer are less prone to undergo oxidation by compound-I to form
the tyrosyl radical. However, results indicate that there were no
detectable changes in transmembrane tyrosyl peptides levels (Y-4, Y-8,
and Y-12) in liposome treated with MPO and H2O2
(data not shown). These findings suggest that ·NO2
alone is able to cause nitration in membrane by forming tyrosyl radicals through an electron-transfer or a hydrogen abstraction mechanism (51).
The transmembrane nitration induced by ONOO
is opposite
that observed with
MPO/H2O2/NO
. The
electron-transfer reaction rate between ·NO2 and
tyrosine is dependent on the protonated or unprotonated state of the
phenolic group. ·NO2 is able to oxidize the
phenoxide ion much more readily than the phenolic hydroxyl group. It is
likely that the phenolic hydroxyl group in Y-4 that is anchored closer
to the membrane surface is more ionized than that of Y-12. Thus, the
formation of Y-4 tyrosyl radical in the presence of
·NO2 is probably more efficient, which will, in
turn, accelerate the formation of NO2-Y-4. In the
hydrophobic phase, the protonated form of peroxynitrite (ONOOH) is
likely to be the dominant species. Homolytic decomposition of ONOOH to
·NO2 and ·OH has recently been reported (47).
Tyrosyl peptides in the membrane can be oxidized by ·OH to
tyrosyl radical that will rapidly react with ·NO2 to
form NO2-Y. Homolytic decomposition of peroxynitrite is more likely to occur deeper in the membrane, which may explain the
increased nitration of Y-12 with peroxynitrite. It has been reported
recently (16, 53) that heme peroxidases (e.g. eosinophil peroxidase) could form peroxynitrite at lower pH levels and
·NO2 at physiological pH levels. It is conceivable
that this marked difference in transmembrane tyrosyl nitration profile
may be used to identify the nitrating species in these systems.
Bicarbonate Effect on Peroxynitrite-dependent
Transmembrane Nitration--
Previously, several investigators have
demonstrated that peroxynitrite-dependent nitration and
oxidation product profiles are altered in the presence of
CO2 (22, 23, 33, 34). Peroxynitrite anion reacts with
CO2 to form a transient adduct, nitrosoperoxycarbonate (ONOOCO
), which decomposes in part to form
CO
and ·NO2 radicals (Scheme 1). Both
species can oxidize tyrosine to the tyrosyl radical, which will rapidly
react with ·NO2 or another tyrosyl radical to form
nitrotyrosine and dityrosine. Typically, nitration of tyrosine or
tyrosine analogs is enhanced in the aqueous phase in the presence of
peroxynitrite and CO2 (33-35). However, the situation is
different in membranes. The transmembrane diffusion of
OONOCO
across the bilayer is probably limited
because of its increased rate of decay as compared with
ONOO
(36). In addition, the anionic CO
is not
sufficiently membrane-permeable. This is consistent with the previous
findings that bicarbonate inhibits nitration of hydrophobic phenolic
probes,
-tocopherol and BTBE (13-15).
Implications in Protein Nitration and
Nitrosation--
Emerging literature (6, 7) provides evidence
for selective nitration of tyrosines present in the transmembrane
domains of proteins (e.g. sarcoplasmic reticulum
Ca2+-ATPase, mitochondrial complex-1 protein). These
findings support the notion that hydrophobic tyrosine residues are
potential targets for nitrating species. Tyrosine nitration in
MPO/H2O2/NO
system in
aqueous phase is mediated by MPO/H2O2
oxidant-generated tyrosyl radical. With the hydrophobic transmembrane
tyrosyl nitration, the nitrating species ·NO2 can
clearly facilitate nitration of hydrophobic tyrosines. It has been
suggested that protein tyrosine nitration is enhanced when tyrosine is
in close proximity to negatively charged amino acids (glutamate,
aspartate) and is decreased when tyrosine is located adjacent to easily
oxidizable amino acids such as cysteine or methionine (48, 54). It is
now possible to experimentally verify such effects using specifically
custom-synthesized transmembrane peptides. The biophysical approaches
outlined in the present study should serve as potentially useful tools
to investigate the effect of membrane composition and other factors
controlling transmembrane protein tyrosyl nitration. We have proposed
in Scheme 1 that N2O3 is a likely intermediate
in these reactions. With the availability of hydrophobic transmembrane
cysteinyl peptides, it is now possible to investigate transmembrane
nitrosation reactions initiated by N2O3 (Scheme
1). As shown in the present study, nitric oxide donors inhibit both
myeloperoxidase- and peroxynitrite-induced transmembrane and aqueous
phase nitration reactions. It is conceivable that nitric oxide donors
could be used as anti-nitration and anti-inflammatory therapeutic agents.