J Biol Chem, Vol. 274, Issue 36, 25933-25944, September 3, 1999
Eosinophil Peroxidase Nitrates Protein Tyrosyl Residues
IMPLICATIONS FOR OXIDATIVE DAMAGE BY NITRATING INTERMEDIATES IN
EOSINOPHILIC INFLAMMATORY DISORDERS*
Weijia
Wu
§,
Yonghong
Chen§, and
Stanley L.
Hazen
§¶
From the Departments of
Cell Biology and
¶ Cardiology, Cleveland Clinic Foundation, Cleveland, Ohio 44195 and the § Chemistry Department, Cleveland State University,
Cleveland, Ohio 63119
 |
ABSTRACT |
Eosinophil peroxidase (EPO) has been implicated
in promoting oxidative tissue injury in conditions ranging from asthma
and other allergic inflammatory disorders to cancer and
parasitic/helminthic infections. Studies thus far on this unique
peroxidase have primarily focused on its unusual substrate preference
for bromide (Br
) and the pseudohalide thiocyanate
(SCN
) forming potent hypohalous acids as cytotoxic
oxidants. However, the ability of EPO to generate reactive nitrogen
species has not yet been reported. We now demonstrate that EPO readily
uses nitrite (NO2
), a major
end-product of nitric oxide (·NO) metabolism, as substrate to
generate a reactive intermediate that nitrates protein tyrosyl residues
in high yield. EPO-catalyzed nitration of tyrosine occurred more
readily than bromination at neutral pH, plasma levels of halides, and
pathophysiologically relevant concentrations of
NO2
. Furthermore, EPO was
significantly more effective than MPO at promoting tyrosine nitration
in the presence of plasma levels of halides. Whereas recent studies
suggest that MPO can also promote protein nitration through indirect
oxidation of NO2
with HOCl, we
found no evidence that EPO can indirectly mediate protein
nitration by a similar reaction between HOBr and
NO2
. EPO-dependent
nitration of tyrosine was modulated over a physiologically relevant
range of SCN
concentrations and was accompanied by
formation of tyrosyl radical addition products (e.g.
o,o'-dityrosine, pulcherosine, trityrosine). The potential role
of specific antioxidants and nucleophilic scavengers on yields of
tyrosine nitration and bromination by EPO are examined. Thus, EPO may
contribute to nitrotyrosine formation in inflammatory conditions
characterized by recruitment and activation of eosinophils.
 |
INTRODUCTION |
Eosinophils play a central role in host defenses against a variety
of cancers and both parasitic and helminthic infections (1-3).
Increased levels of circulating and tissue eosinophils are also
implicated in promoting cellular injury during asthma and other
allergic inflammatory disorders (1-6). Eosinophils are thought to
mediate many of their cytotoxic and tissue-destroying effects through
their exceptional ability to generate oxidizing species (1-4, 7-9).
Indeed, the respiratory burst of eosinophils generates several times as
much superoxide (O
2) and hydrogen peroxide
(H2O2) as a corresponding number of neutrophils
(9, 10). Despite their known proclivity for producing reactive oxygen species and a wealth of clinical evidence linking eosinophils to host
defenses and inflammatory tissue injury, structural identification of
specific oxidation products formed by these leukocytes is lacking.
Eosinophil peroxidase (EPO),1
an abundant heme protein secreted from activated eosinophils, plays a
central role in oxidant production by eosinophils (1-4, 8, 11, 12).
EPO amplifies the oxidizing potential of H2O2
produced during the respiratory burst by using it as a co-substrate to
generate cytotoxic oxidants. Studies thus far have focused on the
unusual substrate preference of EPO for physiological levels of bromide
(Br
) (8, 13-17) and the pseudohalide thiocyanate
(SCN
) (18-20), even in the presence of a vast molar
excess of chloride (Cl
), as is seen in vivo.
Studies with proteins incubated in the presence of radioactive
Br
or SCN
salts and either activated
eosinophils or the EPO-H2O2 system have
demonstrated covalent incorporation of radiolabel into target proteins
(13, 14). However, structural identification of the oxidation products
formed on proteins following exposure to EPO-generated oxidants is
essentially unexplored. We recently identified 3-bromotyrosine and
3,5-dibromotyrosine as major products of protein oxidation by the
EPO-H2O2-Br
system (21). The role
of brominating oxidants in promoting tissue injury in eosinophilic
inflammatory disorders has not yet been established.
Another potential pathway for oxidative tissue damage by eosinophils
may involve formation of nitrating intermediates by EPO. Immunohistochemical studies using antibodies raised against
nitrotyrosine, a global marker for protein damage by reactive nitrogen
species, intensely stain eosinophils present in the inflamed lung
tissues of asthmatics (22). Klebanoff (23) demonstrated that the
antimicrobicidal properties of eosinophil
peroxidase-H2O2 systems are enhanced in the
presence of nitrite (NO2
), the
autoxidation product of ·NO. Moreover, recent studies have shown
that myeloperoxidase (MPO), a neutrophil- and monocyte-specific
peroxidase, can use NO2
and
H2O2 as substrates to generate a reactive
intermediate capable of nitrating phenolic compounds and protein
tyrosyl residues (25-28). However, MPO and EPO are distinct gene
products with divergent physical properties. Although both peroxidases
contain heme prosthetic groups, structural studies have established
that MPO contains a six-coordinate, high spin chlorin, while EPO
possesses a high spin, six-coordinate protoporphyrin (24). Moreover,
although human MPO is tetrameric and comprised of two heavy and two
light chains, EPO is dimeric, having only one heavy and one light chain (7, 24). Whether these structural differences underlie the distinct
halide and substrate specificities observed between MPO and EPO remains
unknown. Furthermore, although increased levels of
NO2
and eosinophils have been reported
in a variety of conditions, the ability of EPO to generate nitrating
intermediates has not yet been explored.
The biological roles and potential targets of mammalian peroxidases are
defined by their unique and often nonoverlapping substrate preferences
(29, 30). We therefore sought to determine whether the
EPO-H2O2 system of eosinophils could promote
protein oxidative damage through formation of reactive nitrogen
species. We now show that isolated EPO readily uses
NO2
as substrate to generate a
reactive intermediate that nitrates protein tyrosyl residues in high
yield. We demonstrate that EPO is more efficient than MPO at nitrating
tyrosine and that EPO-dependent protein nitration is a
preferred activity of the enzyme. Finally, we provide evidence that EPO
also generates tyrosyl radical and that tyrosine nitration by EPO may
involve a tyrosyl radical intermediate.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Organic solvents, H3PO4,
NaH2PO4, Na2HPO4, and
H2O2 were obtained from Fisher. Methane
sulfonic acid and bromine were purchased from Fluka Chemical Co.
(Ronkonkoma, NY). Constant boiling HCl was obtained from Pierce.
L-[13C6]Tyrosine and
L-[2H4]tyrosine were
purchased from Cambridge Isotopes, Inc. (Andover, MA). All other
reagents were purchased from Sigma unless otherwise indicated.
Isolation and Characterization of EPO and MPO--
Porcine EPO
was isolated according to the method of Jorg (31) employing guaiacol
oxidation as the assay (32). Purity of EPO preparations was assured
before use by demonstrating a RZ of >0.9
(A415/A280),
SDS-polyacrylamide gel electrophoresis analysis with Coomassie Blue
staining, and in-gel tetramethylbenzidine peroxidase staining to
confirm no contaminating MPO activity (33). MPO was initially purified
from detergent extracts of human leukocytes by sequential lectin
affinity and gel filtration chromatography as described (34). Trace
levels of contaminating EPO were then removed by passage over a CM-52
ion exchange column (20). Purity of isolated MPO was established by
demonstrating a RZ of 0.87 (A430/A280),
SDS-polyacrylamide gel electrophoresis analysis with Coomassie Blue
staining, and in-gel tetramethylbenzidine peroxidase staining to
confirm no contaminating EPO activity (33). Enzyme concentrations were
determined spectrophotometrically utilizing extinction coefficients of
89,000 and 112,000 M
1 cm
1/heme
of MPO (35) and EPO (36, 37), respectively. The concentration of the
MPO dimer was calculated as half of the indicated concentration of
hemelike chromophore.
General Procedures--
SDS-polyacrylamide gel electrophoresis
analysis was performed as described by Laemmli (38). Protein content
was measured by the Markwell-modified Lowry assay with bovine serum
albumin (BSA) as the standard (39). HOBr free of Br
and
bromate was prepared from liquid bromine the day of use as described
(40). HOBr was quantified spectrophotometrically (
331 = 315 M
1 cm
1) (41) as its
conjugate base, hypobromite (OBr
), immediately prior to
use. The concentration of reagent H2O2 (
240 = 39.4 M
1
cm
1) (42) was determined spectrophotometrically.
Production of H2O2 by the glucose/glucose
oxidase system was determined by oxidation of Fe(II) and formation of a
Fe(III)-thiocyanate complex (25). Western blot analysis of nitrated
proteins was determined as described using immunopurified rabbit
polyclonal antibody against nitrotyrosine (Upstate Biotechnology, Inc.,
Lake Placid, NY) (43). The specificity of the primary antibody was
confirmed by blocking immunoreactivity in coincubations with either 10 mM nitrotyrosine or 1 mM of the tripeptide
Gly-nitro-Tyr-Ala as described (43). o,o'-Dityrosine, pulcherosine, trityrosine, and isodityrosine were prepared and quantified as described previously (44). 3-Bromotyrosine and 3,5-dibromotyrosine standards were synthesized from
L-tyrosine and isolated by preparative HPLC as
recently described (21). [13C6]Ring-labeled
analogs of 3-bromotyrosine and 3,5-dibromotyrosine were similarly
prepared using
L-[13C6]tyrosine as
starting material. UV-visible studies on authentic and EPO-generated
3-nitrotyrosine were performed on a
Bio Spectrophotometer (Perkin-Elmer). 3-Nitro-[13C6]tyrosine,
3-chloro-[13C6]tyrosine, and o,
o'-[13C12]dityrosine standards
for gas chromatography-mass spectrometry (GC/MS) identification of
products were synthesized and quantified as described previously
(45).
5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB) Formation by
EPO--
5-Thio-2-nitrobenzoic acid (TNB) was prepared fresh by
reduction of 1 mM DTNB in 100 ml of sodium phosphate buffer
(50 mM, pH 7.0) with 4 µl of 2-mercaptoethanol (46). EPO
(14.2 nM) was then incubated at 37 °C in sodium
phosphate buffer (50 mM, pH 7.0) with TNB (53 µM) in the absence or presence of the indicated concentrations of Br
, SCN
,
NO2
, or Cl
. Oxidation of
TNB to DTNB was initiated by the addition of
H2O2 (30 µM) and followed
spectrophotometrically at 412 nm (
412 = 27,200 M
1 cm
1) (46).
Oxidation of Free L-Tyrosine and Protein-bound
Tyrosyl Residues--
Unless otherwise specified, reactions were
initiated by the addition of oxidant (H2O2,
HOBr or glucose to the glucose/glucose oxidase system) and performed in
sodium phosphate buffer (20 mM, pH 7.0) at 37 °C for 60 min under the conditions described in the figure and table legends.
Reactions were stopped by the addition of phenol (1% final) and
catalase (30 nM; for glucose/glucose oxidase studies) to
the reaction mixture. The pH dependence of 3-nitrotyrosine formation
was assayed in phosphate buffer (50 mM final) composed of
mixtures of phosphoric acid and monobasic and dibasic sodium phosphate.
The pH of each reaction mixture was determined at the end of the
incubation and did not change by more than 0.2 pH units over the course
of the reaction. Experiments utilizing the glucose/glucose oxidase
system for H2O2 generation were performed in
the presence of 100 µg/ml glucose and 20 ng/ml glucose oxidase (grade
II; Roche Molecular Biochemicals) at 37 °C for the indicated times.
Preliminary studies demonstrated that under these conditions, a
constant flux of H2O2 (0.18 µM/min) was generated.
Protein Hydrolysis--
Preliminary studies confirmed that
removal of NO2
prior to acidification
of proteins was essential to avoid artifactual nitration of proteins.
Proteins oxidized in vitro were therefore prepared for
analysis by first precipitating and desalting them in a single-phase extraction mixture composed of
H2O/methanol/H2O-saturated diethyl ether
(1:3:7, v/v/v) as described (47). Proteins were typically hydrolyzed by
incubating the desalted protein pellet with 6 N HCl (0.5 ml) supplemented with 1% phenol for 24 h (47). Prior to
initiating hydrolysis, acid mixtures were degassed under vacuum and
then sealed under a blanket of argon. When chlorotyrosine and/or
bromotyrosine determinations were performed, formation of trace levels
of halogenated tyrosine analogs during acid hydrolysis was avoided by
hydrolysis in 4 N methane sulfonic acid (0.5 ml) supplemented with 1% phenol for 24 h at 100 °C under a blanket of argon (21). Control experiments utilizing proteins supplemented with
L-[2H4]tyrosine as
internal standard added prior to acid hydrolysis and then analyzed by
GC/MS (48) demonstrated no detectable intrapreparative formation of
3-nitro-[2H3]tyrosine,
3-chloro-[2H3]tyrosine, or
3-bromo-[2H3]tyrosine under these conditions.
Reverse Phase HPLC Quantification of Tyrosine Oxidation
Products--
Quantification of oxidation products from free
L-tyrosine (3-nitrotyrosine, 3-bromotyrosine,
3,5-dibromotyrosine, 3-chlorotyrosine, and o,o'-dityrosine)
was performed on a Beckman Gold HPLC system equipped with diode array
and fluorescence detectors. Separations were performed on a C18 column
(Beckman Ultrasphere, 5 µm, 4.6 × 250 mm) equilibrated with
solvent A (0.1% trifluoroacetic acid, pH 2.5). L-Tyrosine
and its oxidation products were eluted at a flow rate of 1 ml/min with
a linear gradient generated with solvent B (0.1% trifluoroacetic acid
in methanol, pH 2.5) as follows: 0% solvent B for 5 min, 0-100%
solvent B over 30 min, 100% solvent B for 10 min. Products were
quantified employing standard curves constructed with authentic
synthetic standards.
N
-acetyl-L-tyrosine-derived
oxidation products were isolated, and their structures were confirmed
by on-line electrospray ionization mass spectrometric analysis as
described below. Routine quantification of
N
-acetyl-L-tyrosine oxidation
products was determined by reverse phase HPLC with UV detection
(A280) as above, utilizing external calibration
curves constructed with known amounts of their nonacetylated counterparts (and assuming identical extinction coefficients). The
retention times of
N
-acetyl-L-tyrosine analogs of
tyrosine oxidation products were determined using authentic standards
isolated at the time of on-line liquid chromatography/mass
spectrometric analysis (LC/MS).
3-Nitrotyrosine, 3-bromotyrosine, 3,5-dibromotyrosine,
o,o'-dityrosine, and 3-chlorotyrosine in protein
hydrolysates were routinely quantified by reverse phase HPLC with
electrochemical (coulometric) detection on an ESA (Cambridge, MA)
CoulArray HPLC instrument equipped with UV detector and electrochemical
cells (six channels) arranged in series and set to increasing specified potentials: channel 1 (300 mV); channel 2 (350 mV); channel 3 (480 mV);
channel 4 (650 mV); channel 5 (800 mV); channel 6 (880 mV).
Chromatographic separation of amino acids was performed on a a Projel
TSK ODS-80 TM column (5 µm, 4.6 × 250 mm) equilibrated with
mobile phase A (15 mM lithium phosphate, 3 mg/liter lithium dodecyl sulfate, pH 3.2). Products were eluted at a flow rate of 1 ml/min with a nonlinear gradient generated with mobile phase B (50%
methanol, 15 mM lithium phosphate, 3 mg/liter lithium
dodecyl sulfate, pH 3.2) as follows: isocratic elution at 0% mobile
phase B for 10 min, 0-15% mobile phase B over 10 min, isocratic
elution at 15% mobile phase B for 10 min, 15-20% mobile phase B over
10 min, isocratic elution at 20% mobile phase B for 10 min, 20-100% mobile phase B over 20 min, isocratic elution at 100% mobile phase B
for 10 min. Peak identity was established by demonstrating the appropriate retention time, redox potential, and ratio of integrated currents in adjacent channels and by the method of standard additions for each analyte. L-Tyrosine, 3-nitrotyrosine,
3-bromotyrosine, 3,5-dibromotyrosine, o,o'-dityrosine, and
3-chlorotyrosine standards (0.5-50 pmol each on column) were also
dissolved together and used to generate external calibration curves.
The experimental error in data obtained by this assay is less than
±5%. Control experiments comparing quantitation of the tyrosine
analogues by stable isotope dilution GC/MS (45) versus
reverse phase with electrochemical detection demonstrated comparable results.
Mass Spectrometry--
LC/MS was performed using electrospray
ionization (ESI) and detection with a Quatro II triple quadrupole mass
spectrometer (Micromass, Inc.) interfaced with an HP 1100 high
performance liquid chromatograph (Hewlett Packard, Wilmington, DE)
equipped with diode array detector. L-Tyrosine oxidation
products were resolved on an Ultrasphere C18 column (Beckman; 5 µm,
4.6 × 250 mm) at a flow rate of 1 ml/min and a linear gradient
between H2O (with 0.3% formic acid) and methanol (with
0.3% formic acid) over 30 min. Column eluent was split (970 µl/min
to diode array, 30 µl/min to mass detector) and analyzed in the
positive ion mode with a cone potential of 30 eV. GC/MS analyses of
HPLC-isolated L-tyrosine oxidation products were performed
following derivatization to their n-propyl
perheptafluorylbutyryl or n-propyl perpentafluorylproprionyl derivatives (49). 3-Nitrotyrosine was also analyzed by GC/MS analysis
as its n-propyl perheptafluorylbutyryl derivative following reduction to 3-aminotyrosine (50). Negative ion chemical ionization GC/MS studies were performed utilizing a Perkin Elmer (Norwalk, CT)
TurboMass spectrometer equipped with chemical ionization probe.
 |
RESULTS |
3-Nitrotyrosine and Tyrosyl Radical Addition Products Are Formed
during L-Tyrosine Oxidation by the Eosinophil
Peroxidase-H2O2-NO2
System--
The addition of purified EPO and
H2O2 to reaction buffer (20 mM
sodium phosphate, 100 µM diethylenetriaminepentaacetic
acid, pH 7.0) supplemented with L-tyrosine
generated several new species with distinct retention times on reverse
phase HPLC analysis with diode array detection (Fig.
1, upper panel,
peaks a-c). The ultraviolet absorption maximum
of each peak was nearly indistinguishable from L-tyrosine (
max 275-285 nm).
LC/MS analyses were consistent with formation of the tyrosyl radical
addition products o,o'-dityrosine and isodityrosine (peak
a; m/z 361 (M + H)+),
pulcherosine (peak b; m/z
540 (M + H)+), and trityrosine (peak
c; m/z 540 (M + H)+) (44).
Likewise, HPLC analysis with UV and fluorescence detection demonstrated
co-migration with authentic synthetic standards prepared as described
under "Experimental Procedures." Following the addition of
NO2
, the reaction mixture developed an
intense yellow color. Reverse phase HPLC analysis demonstrated the
formation of an additional major (peak I) and
minor (peak d) product (Fig. 1, bottom
panel) as well as enhanced production of the tyrosyl radical
addition products (peaks a-c). The UV-visible
spectra of the new products (peaks I and
d) both illustrated similar absorbance maxima in the visible
wavelength range (
max 350-355 nm), consistent with nitration of the aromatic amino acid (51). Use of heat-inactivated EPO
or omission of either L-tyrosine or
H2O2 from the reaction mixture both resulted in
no detectable production of the products. The reaction products formed
(peak I and peaks a-d)
were stable to treatment with acid (4 N HCl at 100 °C
for 24 h), incubation at 37 °C with
H2O2, and the addition of a molar excess of
either reducing agents or nucleophilic scavengers (e.g.
NaCNBH3, methionine, 2-mercaptoethanol, taurine, ammonium
acetate).

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Fig. 1.
Reverse-phase HPLC separation of
L-tyrosine oxidation products generated by eosinophil
peroxidase. Top, L-Tyrosine (500 µM) was incubated with eosinophil peroxidase (57 nM) and H2O2 (500 µM)
in sodium phosphate buffer (20 mM, pH 7.0) at 37 °C for
60 min. Products were subsequently analyzed by reverse phase HPLC as
described under "Experimental Procedures." Bottom,
L-tyrosine (500 µM) was incubated with
eosinophil peroxidase (57 nM), H2O2
(500 µM), and NO2 (1 mM) in sodium phosphate buffer (20 mM, pH 7.0)
at 37 °C for 60 min. Products were then analyzed by reverse phase
HPLC as described under "Experimental Procedures."
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To characterize the structures of the L-tyrosine
oxidation products generated by the
EPO-H2O2-NO2
system, a variety of analytical techniques were employed. The pH
dependence of the UV-visible absorbance spectrum of HPLC-isolated peak
I is illustrated in Fig. 2
(upper panel) and is identical to that observed
with authentic 3-nitrotyrosine. To confirm the structure of the major
L-tyrosine oxidation product (peak I)
as 3-nitrotyrosine, LC/MS analysis was performed. The retention time and positive ion mass spectrum of peak I was
identical to that of authentic 3-nitrotyrosine and demonstrated a
m/z of 227 (M + H)+ (Fig. 2,
lower panel). LC/MS analysis of the minor
products generated during oxidation of
L-tyrosine by the complete
EPO-H2O2-NO2
system was consistent with the prior structural assignments of peaks a-c and suggested that peak d
was composed of a nitrated analog of o,o'-dityrosine
(e.g. 3-nitro-o,o'-dityrosine,
m/z 406 (M + H)+). These results,
combined with the chemical stability and fluorescence spectra (data not
shown) of the compounds formed, suggest that the major compounds
generated during oxidation of free L-tyrosine by
EPO and H2O2 are tyrosyl radical addition
products. In contrast, in the presence of EPO,
H2O2, and NO2
,
3-nitrotyrosine is the major product formed.

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Fig. 2.
UV-visible spectra and positive ion
electrospray ionization mass spectrum of 3-nitrotyrosine generated by
the
EPO-H2O2-NO2
system. Top, the major L-tyrosine oxidation
product generated by the
EPO-H2O2-NO2
system (peak I, Fig. 1, bottom) was
isolated by reverse phase HPLC as described under "Experimental
Procedures." The isolated product was dried, resuspended at the
indicated pH, and then analyzed by UV-visible spectroscopy (200-600
nm). Bottom, the major L-tyrosine oxidation
product generated by the
EPO-H2O2-NO2
system (peak I in Fig. 1, bottom) was
analyzed by LC/MS as described under "Experimental Procedures." The
positive ion ESI mass spectrum is shown. The structure and predicted
m/z of protonated 3-nitrotyrosine is also shown
(inset).
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Characterization of Reaction Requirements for
EPO-dependent Formation of 3-Nitrotyrosine--
The
reaction requirements for EPO-catalyzed nitration of
L-tyrosine yielding 3-nitrotyrosine are
illustrated in Fig. 3. At pH 7.0 and 100 µM L-tyrosine and
NO2
, 3-nitrotyrosine synthesis by EPO
(following the bolus addition of 100 µM
H2O2) was linear over the first 15 min and
reached a plateau within 30 min (Fig. 3A). At higher
L-tyrosine concentrations, the yield of
3-nitrotyrosine decreased, consistent with the competing bimolecular
mechanism of o,o'-dityrosine formation. EPO-catalyzed nitration was optimal between pH 6 and 6.5, where approximately 0.35 mol of product was formed for each mole of oxidant consumed (Fig.
3B). A dose-dependent increase in the synthesis
of 3-nitrotyrosine was observed over a (patho)physiologically relevant
range of NO2
concentrations (Fig.
3C). The H2O2 concentration
dependence for 3-nitrotyrosine production by EPO yielded a similarly
shaped plot and overall yield (Fig. 3D). At high
concentrations of H2O2, however, the total
amount of 3-nitrotyrosine formed decreased, consistent with either
substrate inhibition or an interaction between
H2O2 and an intermediate in 3-nitrotyrosine
formation (data not shown).

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Fig. 3.
Reaction characteristics of 3-nitrotyrosine
formation by the eosinophil peroxidase. Reactions (0.5 ml) were
performed at 37 °C for 60 min in sodium phosphate buffer (50 mM, pH 7.0) supplemented with L-tyrosine (100 µM), H2O2 (100 µM),
NO2 (100 µM) and
eosinophil peroxidase (57 nM) (unless otherwise indicated).
Reactions were terminated by the addition of phenol (1%), and the
extent of 3-nitrotyrosine formation was determined as described under
"Experimental Procedures." Results represent the mean values from
two or three independent experiments.
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3-Nitrotyrosine Is a Preferred Product of Eosinophil Peroxidase at
Physiologically Relevant Concentrations of L-Tyrosine,
Halides, and NO2
--
Thiocyanate
(SCN
) and bromide (Br
) are reported to be
preferred substrates for EPO (13-16, 18). To estimate the relative substrate preferences of isolated EPO for
NO2
versus other reported
substrates for the enzyme, we examined the initial rate of DTNB
formation in reaction mixtures containing EPO, TNB,
H2O2, and various concentrations of anionic
substrates (SCN
, Br
,
NO2
, Cl
) at neutral pH.
The relative rates of TNB oxidation observed by EPO were as follows:
SCN
> Br
> NO2
Cl
(Fig.
4). Although EPO does not conform to
Michaelis-Menten kinetics (e.g. high concentrations of
H2O2 inhibit tyrosine nitration), a measure of
the relative ability of EPO to discriminate in favor of a particular
substrate in the presence of a mixture of competing substrates can be
calculated as an apparent specificity constant (kx
) for each substrate (33). Because
high levels of H2O2 can inhibit oxidant
production, we estimated the kinetic parameters under conditions that
minimize inhibition and mimic physiological conditions (e.g.
neutral pH and low concentrations of H2O2)
(Table I). SCN
was the
preferred substrate for EPO, demonstrating an approximately 2.7-fold
higher apparent specificity constant than Br
, the next
best substrate examined. EPO preferred Br
approximately
4-fold more than NO2
(Table I).
Implicit in this type of kinetic analysis is the assumption that the
reactive nitrogen species formed by the
EPO-H2O2-NO2
system oxidize TNB at a rate and stoichiometry comparable with that of
other hypohalous acids formed by the enzyme.

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Fig. 4.
Eosinophil peroxidase-dependent
oxidation of TNB in the presence of Br ,
NO2 , Cl , or
SCN . Eosinophil peroxidase (14.2 nM) was
mixed with 53 µM TNB and the indicated concentrations of
Br ( ), NO2 ( ),
Cl ( ), or SCN ( ). Reactions were
initiated by the addition of 30 µM
H2O2, and DTNB formation was monitored
spectrophotometrically at 412 nm as described under "Experimental
Procedures." Rates of DTNB formation were corrected for TNB oxidation
in the absence of anionic substrate (0.26 µM/min). Data
represent the mean values for three independent experiments.
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Table I
Kinetic parameters for substrates for eosinophil peroxidase
The apparent Vmax and Km values
for chloride, nitrite, bromide, and thiocyanate were determined by
linear regression analysis of double reciprocal plots for the rate of
TNB formation versus anion concentration for experiments
performed at neutral pH and 30 µM H2O2 as
in Fig. 4. The catalytic rate constants (kobs) were
calculated by dividing Vmax values by the
concentration of EPO. The specificity constants
(kx ) were determined by dividing
kobs by Km.
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As an alternative means of assessing the potential significance of
EPO-dependent tyrosine nitration, we performed product analyses of the major L-tyrosine oxidation products formed
by the enzyme system (Table I). When plasma levels of
L-tyrosine and halides were incubated with isolated EPO,
H2O2, and concentrations of
NO2
as might be observed in
inflammatory tissues or fluids (tyrosine = Br
= NO2
= 100 µM,
Cl
= 100 mM; Refs. 25 and 52-56),
3-nitrotyrosine was formed in high yield (Table
II). In fact, 3-nitrotyrosine was the
major product formed and was present at 2-3-fold greater levels than either 3-bromotyrosine or o,o'-dityrosine (Table II).
Despite that vast molar excess of Cl
, no detectable
formation of 3-chlorotyrosine was observed under all of the conditions
examined, consistent with the low apparent specificity constant
observed (Tables I and II). The highest yields of 3-nitrotyrosine
production were observed during incubations in the absence of
Br
, consistent with the unusual substrate preference of
EPO for this halide.
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Table II
L-Tyrosine oxidation by eosinophil peroxidase in the
presence of physiological concentrations of Cl , Br ,
and NO2
L-tyrosine (100 µM) was incubated with
eosinophil peroxidase (57 nM), H2O2 (100 µM), and the indicated anionic substrates (Br = NO2 = 100 µM, Cl = 100 mM) in 1 ml of sodium phosphate buffer (20 mM,
pH 7.0) at 37 °C for 60 min. The quantities of
o,o'-dityrosine, 3-bromotyrosine, 3-nitrotyrosine, and
3-chlorotyrosine formed were then determined by reverse phase HPLC with
UV detection as described under "Experimental Procedures." Results
are the mean ± S.D. of three independent experiments. The limit
of detection for these analyses is <0.1 nmol for each analyte
examined.
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3-Nitrotyrosine Formation by Eosinophil Peroxidase Occurs in the
Presence of Nucleophilic Scavengers and Is Inhibited by Ascorbate and
Urate--
Reactive oxidants and diffusible radical species generated
by peroxidases in vivo will encounter a variety of
nucleophilic scavengers and antioxidant compounds. To characterize the
nature of the reactive halogenating and nitrating intermediates
generated by EPO, we incubated the enzyme with
H2O2, Br
, and
NO2
under a variety of conditions and
then quantified 3-bromotyrosine and 3-nitrotyrosine production. Again,
when L-tyrosine was incubated with EPO,
H2O2 (100 µM), and equivalent
amounts of Br
and NO2
(100 µM each), 3-nitrotyrosine was the major oxidation
product formed and was produced in 2-3-fold higher yield than
3-bromotyrosine (Fig. 5). The addition of
methionine, a potent scavenger of reactive halogenating species,
completely ablated 3-bromotyrosine production. In contrast, the
addition of the thiol ether-containing amino acid to the reaction
mixture had no significant effect on 3-nitrotyrosine production (Fig.
5). Likewise, the addition of reduced thiol compounds such as
glutathione totally inhibited tyrosine bromination but only partially
blocked aromatic nitration by the peroxidase. The addition of either
ascorbic acid or uric acid potently inhibited both 3-bromotyrosine and
3-nitrotyrosine production. The addition of primary amine-containing
species (e.g. N
-acetyl lysine) to the
reaction mixture did not attenuate either 3-bromotyrosine or
3-nitrotyrosine production. These results are consistent with our
recent report that N-monobromamines serve as brominating
intermediates for tyrosine (21). They also suggest that the nitrating
intermediate formed by the
EPO-H2O2-NO2
system is not scavenged by amine groups. Finally, these results suggest
that aromatic nitration reactions may be mediated by EPO in
inflammatory fluids and tissues where
NO2
levels are increased and
antioxidants such as ascorbate and urate may be depleted.

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Fig. 5.
Effect of nucleophilic scavengers and
antioxidants on the yield of eosinophil peroxidase-mediated nitration
and bromination of L-tyrosine. L-Tyrosine
(100 µM) was incubated with eosinophil peroxidase (57 nM), H2O2 (100 µM),
NO2 (100 µM),
Br (100 µM), and the indicated reagents
(100 µM) in sodium phosphate buffer (20 mM,
pH 7.0) at 37 °C for 60 min. The content of 3-nitrotyrosine and
3-bromotyrosine formed was then determined by reverse phase HPLC as
described under "Experimental Procedures."
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Eosinophil Peroxidase Nitrates Protein Tyrosyl Residues in High
Yield--
The ability of EPO to nitrate protein tyrosyl residues was
first examined by incubating a target protein (e.g. BSA)
with the complete
EPO-H2O2-NO2
system and then performing SDS-polyacrylamide gel electrophoresis analysis with Western blot analysis using affinity-purified polyclonal antibodies specific for 3-nitrotyrosine (43). A
NO2
-dependent increase in
the intensity of staining was observed following exposure of the
protein to the enzymatic system (Fig. 6).
To confirm that 3-nitrotyrosine was produced in proteins exposed to the
EPO-H2O2-NO2
system, protein hydrolysates were directly analyzed for 3-nitrotyrosine production by GC/MS in the negative-ion chemical ionization mode employing synthetic 3-nitro-[13C6]tyrosine as
an internal standard, as described under "Experimental Procedures."
Ions with the appropriate mass-to-charge ratio
(m/z 464 (M)
and 444 (M
HF)
) and identical retention time to that observed with their
corresponding stable isotope-labeled fragment ions were readily
observed for the n-propyl-perheptafluorylbutyryl derivative
of 3-nitrotyrosine. Moreover, reduction of the products prior to
analysis by GC/MS demonstrated ions with identical retention time and
appropriate m/z for that of the
n-propyl perheptafluorylbutyryl derivative of
3-aminotyrosine (m/z 806 (M
HF)
,
m/z 762 (M
HF
CO2)
and m/z 628 (M
heptafluorylbutyryl)
). Collectively, these results
unambiguously confirm that EPO catalyzes nitration of protein tyrosyl
residues in the presence of the co-substrates, H2O2 and
NO2
.

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Fig. 6.
Nitration of protein tyrosine residues by
eosinophil peroxidase. BSA (0.4 mg/ml) was incubated with
eosinophil peroxidase (57 nM), H2O2
(100 µM), and the indicated concentrations of
NO2 in sodium phosphate buffer (20 mM, pH 7.0) at 37 °C for 60 min. After incubation, 1.2 µg of protein was loaded on 10% SDS-polyacrylamide gels for
electrophoresis, and proteins were transferred and immunostained using
a rabbit polyclonal antibody against 3-nitrotyrosine (top)
or stained with Coomassie Blue (bottom).
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To characterize the significance of EPO-dependent protein
nitration, the content of protein-bound 3-nitrotyrosine produced by the
enzyme was determined by reverse phase HPLC with electrochemical detection as described under "Experimental Procedures." BSA was incubated with the complete
EPO-H2O2-NO2
system, while the concentrations of either
NO2
(Fig.
7, upper panel) or
H2O2 (Fig. 7, lower
panel) were varied. Nitration of protein tyrosyl residues
occurred readily in a concentration-dependent manner for both
co-substrates. The overall yield of EPO-catalyzed nitration of protein
tyrosyl residues as a function of each substrate is shown in Fig.
8. The highest yields (10-20%) of
protein nitration were noted when NO2
concentrations were high and H2O2
concentrations were low. These results suggest that protein nitration
would be highest under conditions where H2O2
production occurs at a low rate, similar to the conditions that are
likely to be present in vivo.

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Fig. 7.
Characterization of
NO2 and
H2O2 concentration dependences of eosinophil
peroxidase-mediated nitration of protein tyrosyl residues.
Top, BSA (0.4 mg/ml) was incubated with eosinophil
peroxidase (57 nM), H2O2 (50 µM total, added in 10 µM increments at
10-min intervals), and the indicated concentrations of
NO2 in sodium phosphate buffer (20 mM, pH 7.0) for 60 min at 37 °C. Oxidation products were
then desalted and subjected to acid hydrolysis, and the content of
3-nitrotyrosine was determined by reverse phase HPLC with
electrochemical detection as described under "Experimental
Procedures." Bottom, BSA (0.4 mg/ml) was incubated with
eosinophil peroxidase (57 nM), NaNO2 (100 µM), and the indicated concentrations of
H2O2 (added in 10 µM increments
at 10 min intervals) in sodium phosphate buffer (20 mM, pH 7.0) for 60 min at 37 °C. 3-Nitrotyrosine
production was then determined as described under "Experimental
Procedures." Data represent the mean ± S.D. of results from
three independent experiments.
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Fig. 8.
Overall yield of protein tyrosyl residue
nitration by eosinophil peroxidase at physiological concentrations of
NO2 and H2O2.
The overall yield of EPO-dependent nitration of
protein tyrosyl residues for the experiments described in Fig. 8 was
calculated as the moles of 3-nitrotyrosine formed divided by the moles
of oxidant (H2O2) added to the reaction
mixture. Data represent the mean results from three independent
experiments.
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To more fully examine the potential physiological significance of
protein nitration by EPO, we quantified the levels of formation of
multiple tyrosyl residue oxidation products (3-nitrotyrosine, 3-bromotyrosine, 3-chlorotyrosine, and o,o'-dityrosine) in
proteins incubated with isolated EPO, a hydrogen peroxide-generating
system (glucose/glucose oxidase), and concentrations of halides and
NO2
as might be observed in
inflammatory fluids (i.e. 100 mM
Cl
, 100 µM Br
, and 100 µM NO2
). Again, EPO
nitrated protein tyrosyl residues to a greater extent than that of all
other tyrosine oxidation products examined (Table III). Under the conditions employed, only
minimal amounts of o,o'-dityrosine and no detectable levels
of 3-chlorotyrosine were formed (Table III). EPO-catalyzed bromination
of protein tyrosyl residues occurred, albeit at a level approximately
one-third that of tyrosine nitration. Generation of the tyrosine
oxidation products demonstrated an absolute requirement for EPO and the
H2O2-generating system. Collectively, these
results demonstrate that EPO uses NO2
as a preferred substrate to form a reactive nitrogen intermediate(s) even in the presence of physiological levels of halides.
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Table III
Oxidation of protein tyrosyl residues by eosinophil peroxidase in the
presence of physiological concentrations of Cl , Br ,
and NO2
BSA (0.4 mg/ml) was incubated with eosinophil peroxidase (57 nM),
glucose (100 µg/ml), glucose oxidase (20 ng/ml), and the indicated
anionic substrates (Br = NO2 = 100 µM, Cl = 100 mM) in sodium
phosphate buffer (20 mM, pH 7.0) at 37 °C for 4 h.
Following reaction, protein was desalted and subjected to acid
hydrolysis, and the content of 3-nitrotyrosine, 3-bromotyrosine,
o,o'-dityrosine, and 3-chlorotyrosine was determined by
reverse phase HPLC with electrochemical detection as described under
"Experimental Procedures." Results are the mean ± S.D. of
three independent experiments. The limit of detection for these
analyses is <0.05 mmol/mol tyrosine for each analyte examined.
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The pseudohalide SCN
is a preferred substrate of EPO
(18-20). We were therefore interested in examining the potential
influence of SCN
levels on EPO-catalyzed nitration.
Extracellular fluid and tissue levels of this unusual anion have not
been reported; however, plasma levels of SCN
vary
considerably (normal values are <69 µM) depending upon
diet and smoking habits in normal individuals (57). In a separate set
of experiments, either free L-tyrosine (Fig.
9, left) or BSA (Fig. 9,
right) was incubated with
NO2
(100 µM) and plasma
levels of halides (100 mM Cl
and 100 µM Br
) in the absence and presence of
varying amounts of SCN
, and the content of
3-nitrotyrosine was determined. EPO-mediated nitration was attenuated
by the addition of SCN
over the reported range of plasma
levels of SCN
(Fig. 9). In the presence of equivalent
levels of NO2
and SCN
(100 µM each), 3-nitrotyrosine formation was
significantly inhibited; however, nitration of both free and
protein-bound tyrosine residues was still detectable.

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Fig. 9.
Inhibitory effect of SCN on
3-nitrotyrosine production by eosinophil peroxidase.
Left, L-tyrosine (100 µM) was
incubated with eosinophil peroxidase (57 nM),
H2O2 (100 µM), and
NO2 (100 µM) in the
presence of the indicated concentrations of NaSCN in sodium phosphate
buffer (20 mM, pH 7.0) at 37 °C for 1 h. The
content of 3-nitrotyrosine formed was then determined as described
under "Experimental Procedures." Data represent the mean ± S.D. of results from three independent experiments. Right,
BSA (0.4 mg/ml) was incubated with eosinophil peroxidase (57 nM), glucose (100 µg/ml), glucose oxidase (20 ng/ml), and
NO2 (100 µM) in the
presence of the indicated concentrations of NaSCN in sodium phosphate
buffer (20 mM, pH 7.0) at 37 °C overnight. The content
of protein-bound nitrotyrosine formed was then determined as described
under "Experimental Procedures." Data represent the mean ± S.D. of results from three independent experiments.
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Eosinophil Peroxidase Is More Effective Than Myeloperoxidase at
Generating Nitrotyrosine in the Presence of
NO2
, H2O2,
and Plasma Levels of Halides--
Since MPO-dependent
formation of nitrotyrosine may also contribute to protein nitration at
sites of inflammation (25-28, 58, 59), we sought to compare the
ability of isolated MPO and EPO to nitrate target proteins. BSA was
incubated with equal concentrations of each peroxidase in the presence
of plasma levels of halides (100 mM Cl
and
100 µM Br
) and an
H2O2-generating system (glucose/glucose
oxidase) to more closely mimic the low steady flux of peroxide that
might be formed in vivo. Kinetic analyses comparing the
ability of each peroxidase to use NO2
as substrate for protein nitration demonstrated that EPO formed at
least 4-fold more 3-nitrotyrosine on target proteins than MPO at all
times examined (Fig. 10,
left). Moreover, regardless of the concentration of
NO2
examined (0-100
µM), EPO was significantly more effective than MPO at
nitrating protein tyrosyl residues (Fig. 10, right). Thus, at sites of eosinophilic inflammatory disorders, EPO may contribute to
3-nitrotyrosine formation.

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Fig. 10.
Comparison of protein nitration by
eosinophil peroxidase and myeloperoxidase. Left, BSA
(0.4 mg/ml) was incubated with equivalent amounts (57 nM
each) of either EPO or MPO, NaNO2 (100 µM),
glucose (100 µg/ml), and glucose oxidase (20 ng/ml) in sodium
phosphate buffer (50 mM, pH 7.0) supplemented with NaCl
(100 mM) and NaBr (100 µM) at 37 °C for
the indicated periods of time. Under these conditions, a constant flux
of H2O2 (0.18 µM/min) is
generated by the glucose/glucose oxidase system. Reactions were stopped
by the addition of phenol (1% final concentration) and then immediate
delipidation/desalting of proteins by a single phase extraction mixture
as described under "Experimental Procedures." The content of
3-nitrotyrosine was then determined by reverse phase HPLC with
electrochemical detection as described under "Experimental
Procedures." Right, BSA (0.4 mg/ml) was incubated with 57 nM of either EPO or MPO, glucose (100 µg/ml), and glucose
oxidase (20 ng/ml) in the presence of the indicated concentrations of
NO2 in sodium phosphate buffer (50 mM, pH 7.0) supplemented with NaCl (100 mM) and
NaBr (100 µM) for 12 h at 37 °C. The content of
3-nitrotyrosine was then determined by reverse phase HPLC with
electrochemical detection. Data represent the mean ± S.D. of
results from three independent experiments.
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The Mechanism of Eosinophil Peroxidase-dependent
Production of 3-Nitrotyrosine Involves Formation of a Tyrosyl Radical
Intermediate--
To examine the mechanism of protein tyrosyl residue
nitration by the
EPO-H2O2-NO2
system, we initially performed a variety of experiments utilizing N
-acetyl-L-tyrosine, a low
molecular weight surrogate for a protein-bound tyrosyl residue. In the
absence of NO2
, incubation of
N
-acetyl-L-tyrosine with EPO and
H2O2 resulted in formation of the
N
-acetyl dityrosine analog (Table
IV). The addition of Br
to
the reaction mixtures resulted in production of the
N
-acetyl-L-tyrosine analogs of
3-bromotyrosine and 3,5-dibromotyrosine but no detectable production of
the N
-acetyl-L-tyrosine analog of
the tyrosyl radical-addition product o,o'-dityrosine. In
contrast, the addition of NO2
to
N
-acetyl-L-tyrosine and the
EPO-H2O2 system formed predominantly the
N
-acetyl-L-tyrosine analog of
3-nitrotyrosine and modest levels of the
N
-acetyl-L-tyrosine analog of
o,o'-dityrosine (Table IV). The addition of both
NO2
and Br
to
N
-acetyl-L-tyrosine and the
EPO-H2O2 system formed all of the expected tyrosine oxidation products
(N
-acetyl-L-tyrosine analogs of
3-nitrotyrosine, o,o'-dityrosine, 3-bromotyrosine, and
3,5-dibromotyrosine). Thus, in every instance where tyrosine nitration
occurred, concurrent formation of tyrosyl radical addition products was
observed. In contrast, during tyrosine bromination (in the absence of
NO2
), no formation of tyrosyl radical
addition products (i.e.
N
-acetyl-L-tyrosine analog of
o,o'-dityrosine) could be detected. These results are
consistent with 3-nitrotyrosine production through an addition reaction
between an intermediate tyrosyl radical and reactive nitrogen species
such as ·NO2, the one electron oxidation product of
NO2
.
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Table IV
Oxidation of N -acetyl tyrosine by eosinophil
peroxidase-generated oxidants and by HOBr with or without
NO2
N -Acetyl tyrosine (N -Ac-Y,
250 nmol) was mixed with the indicated additions in 0.5 ml of sodium
phosphate buffer (20 mM, pH 7.0). Reactions were initiated
by the addition of 250 nmol of oxidant (H2O2 or HOBr)
and incubated at 37 °C for 1 h. The quantities of the
N -acetyl derivatives of
o,o'-dityrosine (N -Ac-di-Y),
3-nitrotyrosine (N -Ac-3-NO2-Y),
3-bromotyrosine (N -Ac-3-Br-Y), and
3,5-dibromotyrosine (N -Ac-3,5-diBr-Y) formed were
then determined by reverse phase HPLC with UV absorbance
(A280) detection as described under "Experimental
Procedures." Product identity was confirmed by concomitant on-line
ESI-MS analyses as described under "Experimental Procedures." The
concentrations of the following reagents (when present) were as
follows: eosinophil peroxidase (57 nM), NaBr (1 mM), NaNO2 (1 mM). Results shown are
the mean ± S.D. of three independent experiments. The limit of
detection for these analyses is <0.1 nmol for each analyte examined.
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Co-incubation of HOCl and NO2
has been
reported to form a nitrating and chlorinating intermediate, presumably
NO2Cl (58). Moreover, we observed modest increases in the
levels of free and protein-bound nitrotyrosine formed during incubation
with the EPO-H2O2 system in the presence of
NO2
and Cl
compared with
NO2
alone (Tables II and III).
However, the mechanism for the increase does not appear to involve
reaction of HOCl with NO2
, since
comparable increases in tyrosine nitration were observed in the
presence of Cl
during incubations that contained excess
methionine, a potent scavenger of HOCl and other halogenating
intermediates (data not shown). To determine if EPO might indirectly
promote protein nitration by forming a nitrating intermediate by
oxidation of NO2
with HOBr, we
performed the following experiment.
N
-Acetyl-L-tyrosine was incubated
with HOBr in the presence and absence of
NO2
, and the extent of tyrosine
nitration was determined. No detectable formation of the
3-nitrotyrosine analog was observed, even in the presence of 1 mM concentrations of both HOBr and
NO2
(Table IV). Similar results were
observed in reactions performed under acidic (pH 4) conditions (data
not shown). Collectively, these results suggest that the nitrating
intermediate formed by EPO arises from direct oxidation of
NO2
and not by secondary oxidation of
NO2
by HOCl, HOBr, or some other
halogenating agent.
In a final series of experiments, we sought to further explore the
potential role of a tyrosyl radical intermediate in 3-nitrotyrosine formation by monitoring the extent of aromatic nitration using tyrosine
analogs that do not form tyrosyl radicals. Because of its acidic
character, the phenoxyl hydrogen of L-tyrosine is the preferred site of hydrogen atom abstraction from tyrosine;
consequently, O-methyl-L-tyrosine is resistant
to formation of tyrosyl radical (60). If 3-nitrotyrosine production by
the
EPO-H2O2-NO2
system instead occurred by an electrophilic addition reaction (e.g. through a NO2+
intermediate), use of the O-methyl-L-tyrosine
analog should not significantly effect the yield of 3-nitrotyrosine
formation. Incubation of the complete
EPO-H2O2-NO2
system with L-tyrosine readily formed 3-nitrotyrosine; in
contrast, no nitrated products were formed from
O-methyl-L-tyrosine at all concentrations of
NO2
examined, as determined by reverse
phase HPLC with on-line UV and ESI/MS (Fig.
11). Collectively, these results
strongly support the hypothesis that the
EPO-H2O2-NO2
system nitrates tyrosyl residues through a tyrosyl radical
intermediate.

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Fig. 11.
Examination of eosinophil
peroxidase-mediated nitration of L-tyrosine
versus O-methyl-L-tyrosine. Either
L-tyrosine (100 µM) or
O-methyl-L-tyrosine (100 µM) were
incubated with eosinophil peroxidase (57 nM),
H2O2 (100 µM), and the indicated
concentrations of NO2 in sodium
phosphate buffer (20 mM, pH 7.0) at 37 °C for 60 min.
The content of 3-nitrotyrosine and O-methyl-3-nitrotyrosine
formed was then quantified by reverse phase HPLC as described under
"Experimental Procedures." Results represent the mean of triplicate
determinations from a typical experiment performed in duplicate.
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DISCUSSION |
Eosinophils play an essential role in tissue surveillance and host
defense mechanisms (1-3). These cells have evolved enzymatic mechanisms to inflict oxidative damage upon invading parasites, pathogens, and cancer cells. The reactive species they form, however, also have the potential to harm host tissues and contribute to inflammatory tissue injury. Microbicidal and cytotoxic oxidants generated by the EPO-H2O2 system are thought to
participate in