Kinetics of 3-nitrotyrosine modification on exposure to hypochlorous acid

Article abstract of DOI:10.3109/10715762.2014.954110

The markers 3-nitrotyrosine and 3-chlorotyrosine are measured as surrogates for reactive nitrogen species and hypochlorous acid respectively, which are both elevated in inflamed human tissues. Previous studies reported a loss of 3-nitrotyrosine when exposed to hypochlorous acid, suggesting that observations of 3-nitrotyrosine underestimate the presence of reactive nitrogen species in diseased tissue (Whiteman and Halliwell, Biochemical and Biophysical Research Communications, 258, 168-172 (1999)). This report evaluates the significance of 3-nitrotyrosine loss by measuring the kinetics of the reaction between 3-nitrotyrosine and hypochlorous acid. The results demonstrate that 3-nitrotyrosine is chlorinated by hypochlorous acid or chloramines to form 3-chloro-5-nitrotyrosine. As 3-nitrotyrosine from in vivo samples is usually found within proteins rather than as free amino acid, we also examined the reaction of 3-nitrotyrosine modification in the context of peptides. The chlorination of 3-nitrotyrosine in peptides was observed to occur up to 700-fold faster than control reactions using equivalent amino acid mixtures. These results further advance our understanding of tyrosine chlorination and the use of 3-nitrotyrosine formed in vivo as a biomarker of reactive nitrogen species.

Full text of DOI:10.3109/10715762.2014.954110

Free Radical Research, November 2014; 48(11): 1355–1362
© 2014 Informa UK, Ltd.
ISSN 1071-5762 print/ISSN 1029-2470 online
DOI: 10.3109/10715762.2014.954110
ORIGINAL ARTICLE
Kinetics of 3-nitrotyrosine modification on exposure to hypochlorous acid
M. P. Curtis & J. W. Neidigh
Department of Basic Sciences, School of Medicine, Loma Linda University, Loma Linda, CA, USA
Abstract
The markers 3-nitrotyrosine and 3-chlorotyrosine are measured as surrogates for reactive nitrogen species and hypochlorous acid respectively,
which are both elevated in inflamed human tissues. Previous studies reported a loss of 3-nitrotyrosine when exposed to hypochlorous
acid, suggesting that observations of 3-nitrotyrosine underestimate the presence of reactive nitrogen species in diseased tissue (Whiteman
and Halliwell, Biochemical and Biophysical Research Communications, 258, 168–172 (1999)). This report evaluates the significance of
3-nitrotyrosine loss by measuring the kinetics of the reaction between 3-nitrotyrosine and hypochlorous acid. The results demonstrate that
3-nitrotyrosine is chlorinated by hypochlorous acid or chloramines to form 3-chloro-5-nitrotyrosine. As 3-nitrotyrosine from in vivo samples
is usually found within proteins rather than as free amino acid, we also examined the reaction of 3-nitrotyrosine modification in the context of
peptides. The chlorination of 3-nitrotyrosine in peptides was observed to occur up to 700-fold faster than control reactions using equivalent
amino acid mixtures. These results further advance our understanding of tyrosine chlorination and the use of 3-nitrotyrosine formed
in vivo as a biomarker of reactive nitrogen species.
Keywords: 3-chlorotyrosine, chloramine, biomarker, reactive species, protein damage
Abbreviations: 3ClY, 3-chlorotyrosine; AcHis, N-α-acetylhistidine; AcHisCl, N-α-acetyl-N-δ-chlorohistidine; AcLys, N-α-acetyllysine;
AcLysCl, N-α-acetyl-N-ε-chlorolysine; AcTyr, N-acetyltyrosine; Ac3ClTyr, N-acetyl-3-chlorotyrosine; AcNO2Y, N-acetyl-3-nitrotyrosine;
AcClNO2Y, N-acetyl-3-chloro-5-nitrotyrosine; ClNO2Y, 3-chloro-5-nitrotyrosine; Cl2Y, 3,5-dichlorotyrosine; HOCl, hypochlorous acid;
NO2Y, 3-nitrotyrosine; RNS, reactive nitrogen species; V0, initial reaction rate
Introduction
hydrogen peroxide and generate reactive nitrogen species
capable of nitrating tyrosine residues to form NO2Y [9,10].
Resting human neutrophils isolated from peripheral blood
do not normally express inducible nitric oxide synthase
[11]. However, activated neutrophils exposed to cytokines
can express inducible nitric oxide synthase, produce nitric
oxide, and increase the level of reactive nitrogen species,
suggesting that recruitment of neutrophils during inflam-
mation can increase both tyrosine nitration and chlorina-
tion [12]. The observation of both 3ClY and NO2Y in
diseased tissues of patients with atherosclerosis, cardio-
vascular disease or neurodegeneration, confirms the pres-
ence of active MPO in tissues with reactive nitrogen
species [13–15]. The kinetics of NO2Y reacting with
HOCl or HOCl-derived chloramines is therefore impor-
tant for estimating the loss of the marker NO2Y in these
diseased tissues when both reactive nitrogen species and
active myeloperoxidase are present.
Reactive chemicals are implicated in the pathology of
human diseases associated with aberrant or chronic inflam-
mation including cancer, neurodegenerative diseases, and
cardiovascular disease [1–4]. Myeloperoxidase catalyzes
the oxidation of chloride ions by hydrogen peroxide to
form hypochlorous acid (HOCl) that damages biological
molecules including tyrosine, which is free in solution and
tyrosine residues in proteins, to form 3-chlorotyrosine
(3ClY) [5]. Reactive nitrogen species formed when nitric
oxide is oxidized, including peroxynitrite and nitrogen
dioxide, damage biological molecules including free or
protein-bound tyrosine, to produce 3-nitrotyrosine (NO2Y)
[4,6]. The markers NO2Y and 3ClY are measured as sur-
rogates, respectively, of reactive nitrogen species and
HOCl formed in vivo because these products of tyrosine
damage are chemically stable and readily measured
with existing analytical methods [6,7]. Whiteman and
Halliwell, however, demonstrated in vitro that NO2Y is
lost in the presence of HOCl, suggesting that observed
NO2Y underestimates the levels of reactive nitrogen spe-
cies in tissues that also produce HOCl [8].
Experimental procedures
Materials
Coincident formation of reactive halogen and reactive
nitrogen species occurs when the enzymes myeloperoxi-
dase and nitric oxide synthase are both present. Myeloper-
oxidase can consume nitric oxide in the presence of
N-Acetyl-L-tyrosine (AcTyr), N-α-acetyl-L-lysine (AcLys),
and N-acetyl-L-histidine (AcHis) were purchased from
Novabiochem (San Diego, CA). FMOC-amide resin was
Correspondence: Jonathan W. Neidigh, Department of Basic Sciences, School of Medicine, Loma Linda University, Loma Linda, CA 92350,
USA. Tel: ϩ909-558-1000 Ext. 48559. Fax: ϩ909-558-4887. E-mail: jneidigh@llu.edu
(Received date: 16 July 2014; Accepted date: 9 August 2014; Published online: 8 September 2014)

1356 M. P. Curtis & J. W. Neidigh
1
purchased from Applied Biosystems, Inc. (Foster City,
CA). FMOC-OSu, FMOC-Lys(Boc)-OH, FMOC-His
(Trt)-OH, and all other FMOC amino acids were pur-
chased from Advanced ChemTech (Louisville, KY),
with the exception of FMOC-Tyr(NO2)-OH which was
synthesized as described below. All other laboratory chem-
icals including 3-chloro-L-tyrosine, 3-nitro-L-tyrosine,
L-methionine, sodium hypochlorite, N-acetylglycine,
N-acetyl-L-alanine, and N-acetyl-L-glutamic acid were
purchased from Sigma-Aldrich (St. Louis, MO).
954110). The H NMR was consistent with AcClNO2Y.
1H NMR (500 MHz, DMSO): 12.77 (s, 1H, -CO2H), 10.91
(s, 1H, -OH), 8.21 (d, Jϭ8.3 Hz, 1H, NH), 7.79 (d,
Jϭ2.0 Hz, 1H, H6), 7.70 (d, Jϭ2.0 Hz, 1H, H2), 4.43
(m, 1H, Hα), 3.05 (dd, Jϭ4.8, 13.8 Hz, 1H, Hβ), 2.83
(dd, Jϭ9.7, 13.8 Hz, 1H, Hβ), 1.80 (s, 3H, CH3).
Synthesis of FMOC-3-nitrotyrosine
An FMOC protecting group was added to the N-terminus
of NO2Y by reacting with equimolar FMOC-OSu and a
3-fold excess sodium bicarbonate in a 50:50 mixture of
water and acetone. After stirring overnight at room tem-
perature, the aqueous layer was extracted four times with
diethyl ether. The combined diethyl ether solution was
back-extracted twice with 5% sodium bicarbonate. The
combined aqueous layers were acidified by adding con-
centrated HCl until a precipitate formed. The product was
extracted with ethyl acetate twice, combined, and dried
using anhydrous magnesium sulfate. Finally, the solvent
was removed by rotary evaporation, leaving a golden pow-
der for use in peptide synthesis.
Synthesis of N-acetyl-3-nitrotyrosine
3-Nitro-L-tyrosine was acetylated with excess acetic anhy-
dride in acetone at room temperature and left to react over-
night. The product formed was N-acetyl-3-nitrotyrosine
(AcNO2Y) and it was found soluble in acetone. The ace-
tone was evaporated using a rotary evaporator and any
remaining acetic anhydride was hydrolyzed by adding
water. The solution was added to a SupelClean, LC-18
packing, solid phase extraction (SPE) column (Supelco,
Bellefonte, PA). The column was washed with 0.1% trif-
luoroacetic acid (TFA) and subsequent washes were com-
posed of increasing concentrations of methanol (5%
increments) in 0.1% TFA. The aliquots were analyzed by
analytic HPLC to determine the purity. Fractions, which
were more than 98% pure, were combined and lyophilized.
The compound gave the expected UV/Vis maximum
absorption at 279 and 360 nm [16]. The pKa value deter-
mined by UV/Vis spectroscopy was 7.13 (see Supplemen-
tary Material available online at http://informahealthcare.
com/doi/abs/10.3109/10715762.2014.954110). The 1H
Peptide synthesis and purification
Peptides containing histidine or lysine residues in close
proximity to the NO2Y residue were synthesized on an
ABI model 433A peptide synthesizer using fast FMOC
chemistry. Following synthesis, the N-terminus was
acetylated using acetic anhydride. The protecting groups
and resin support were cleaved from the peptides using
a
mixture of TFA/phenol/water/triisopropylsilane
1
NMR spectra were consistent with AcNO2Y. H NMR
(88:5:5:2). The resin was removed by filtration, the cleav-
age reaction was concentrated by rotary evaporation, and
then the peptide was precipitated by adding cold diethyl
ether to the cleavage mixture. The ether was removed
following centrifugation to pellet the precipitated pep-
tide. After washing the crude peptide with cold diethyl
ether three times, the peptides were purified by reverse
phase HPLC using a C18 column (Varian Dynamax,
250ϫ21.4 mm, 300 Å, 5 μM) with a gradient of 20–60%
mobile phase B (acetonitrile with 0.085% TFA), where
mobile phase A contained aqueous 0.1% TFA. The
sequence of the purified peptides was confirmed by
MS/MS using a Thermo Finnigan LCQ Deca XP mass
spectrometer, and a purity of more than 95% was verified
by analytical HPLC (data not shown).
(500 MHz, DMSO): 12.72 (s, 1H, -CO2H), 10.79 (s, 1H,
-OH), 8.18 (d, Jϭ8.1 Hz, 1H, NH), 7.74 (d, Jϭ2.0 Hz,
1H, H2), 7.41 (dd, Jϭ2.0, 8.5 Hz, 1H, H6), 7.05 (d,
Jϭ8.5 Hz, 1H, H5), 4.38 (m, 1H, Hα), 3.01 (dd, Jϭ4.7,
13.9 Hz, 1H, Hβ), 2.80 (dd, Jϭ9.4, 13.9 Hz, Hβ), 1.78
(s, 3H, CH3).
Synthesis of N-acetyl-3-chloro-5-nitrotyrosine
3-Chloro-L-tyrosine was acetylated as described above.
The crude product, N,O-diacetyl-3-chlorotyrosine, was
produced after an overnight reaction and found soluble in
acetone. The acetone was evaporated and the crude com-
pound was then dissolved in water. Excess sodium nitrate
(approximately 3-fold) was added, the solution was placed
on ice, and then concentrated sulfuric acid was added
dropwise to hydrolyze the phenol acetate ester and nitrate
the phenol, giving N-acetyl-3-chloro-5-nitrotyrosine
(AcClNO2Y). The solution was allowed to react for 2 h
before being purified by SPE as described above. Ana-
lytical HPLC showed that the final purity was more than
98%. The UV/Vis spectrum showed absorbance maxi-
mums at 285 nm and 358 nm under acidic conditions. The
pKa value determined by UV/Vis spectroscopy was
5.39 (see Supplementary Material available online at http://
informahealthcare.com/doi/abs/10.3109/10715762.2014.
Methods
The purchased sodium hypochlorite stock was stored at
4°C. The pKa of HOCl is 7.5 resulting in almost equal
concentrations of hypochlorous acid and its conjugate
base, hypochlorite, at physiological pH. The term
“hypochlorous acid” is thus used to refer to both the acid
and its conjugate base. The concentration of the sodium
hypochlorite stock was determined daily using the absor-
bance at 290 nm (ε290 nm ϭ350 MϪ1 cmϪ1 ), for a fresh
dilution of the stock into 0.1 M NaOH [17]. The stock

Nitrotyrosine chlorination 1357
concentrations of chemicals containing NO2Y or 3-chloro-
5-nitrotyrosine (ClNO2Y) were determined following
dilution into 0.1 M HCl and using UV/Vis spectroscopy
using a Restek Ultra IBD column (C18, 3 μm, 150
ϫ2.1 mm). The initial solvent mix was 95% mobile phase
A (0.1% trifluoroacetic acid in water) and 5% mobile
phase B (0.085% trifluoroacetic acid in acetonitrile) for
5 min, followed by a linear gradient to 50% mobile phase
B over 20 min at a flow rate of 100 μL/min. The gradient
stayed constant for 2.5 min, followed by a linear decrease
to 5% B in 2.5 min. AcNO2Y and AcClNO2Y were quan-
titated at 360 nm.
Ϫ1
with molar absorptivity values of ε360 nm ϭ2790 MϪ1 cm
and ε358 nm ϭ2496 MϪ1 cmϪ1 , respectively [16]. The molar
absorptivity value of ClNO2Y was determined as a ratio
of the published value for NO2Y by mixing AcClNO2Y
and AcNO2Y stock solutions with known UV spectra,
vacuum centrifuging to remove water, dissolving in deu-
terated DMSO, collecting a 1D NMR spectrum, and cal-
culating the ratio of both compounds based on pairs of
integrated peaks corresponding to the same resonance.
Determination of initial reaction rate and reaction order
The peptides were reacted with HOCl and quenched with
excess cysteine at 37°C in 10 mM phosphate buffer at pH
7.4. The reported initial reaction rate of NO2Y chlorina-
tion, V0, was determined using only data points where
product formation was a linear function of time. The V0
values of 12–15 reactions with varying initial concentra-
Reactions of AcNO2Y with HOCl or chloramines
All reactions contained 200 μM HOCl, 350–1000 μM
AcNO2Y, and 100 mM phosphate buffer at the indicated
pH. In some previous studies, the chloramine was formed
first, followed by the addition of the tyrosine analog [18].
Preliminary reactions with AcNO2Y showed no statisti-
cally significant differences between adding the tyrosine
analog or HOCl last. For reactions with lysine or histidine
chloramines, a 5-fold excess AcLys or AcHis was added
to the AcNO2Y solution before the addition of HOCl, as
described in our previous publication [19]. All reactions
with HOCl or chloramines were quenched with 10-fold
excess methionine or cysteine, respectively, at time points
ranging between 5 s and 2 h.
tions of peptide and HOCl were used to solve for the reac-
A
*
*
tion order using the equation V0 ϭk
[Peptide]0
B
[Chloramine]0 , where A and B are the rate order of pep-
tide and chloramine, respectively. Because HOCl reacts
with lysine forming a chloramine significantly faster than
any measurable formation of ClNO2Y, the initial concen-
tration of chloramine peptide is equivalent to the initial
concentration of HOCl [20].
Analysis of kinetic data and determination of rate
constants
UV/Vis spectroscopy of AcClNO2Y chlorination by HOCl
Microsoft Excel was used to analyze all kinetic data using
a model comparison method as previously described [19].
A time interval of 0.1 s was used to numerically model
the differential equations. The chemical models for chlo-
rination of NO2Y by HOCl or chloramines are presented
in the Results section. The sum of the squared differences
(SSD) was calculated between the experimental and mod-
eled concentrations for time points in a kinetics experi-
ment. The Solver tool in Excel was used to determine the
optimized second order rate constants that minimized the
SSD, giving SSDopt. The error in each rate constant was
estimated by determining the rate constant that gave
The UV-spectra of AcClNO2Y reacting with HOCl was
measured on a Varian (Palo Alto, CA) Cary 300 Bio UV/
Vis spectrophotometer in a 1-cm micro quartz cell. AcCl-
NO2Y (185 μM) was reacted with 720 μM HOCl at 37°C
and the spectra between 350 and 550 nm were measured
every 24 s.
Reaction of peptides with HOCl
HOCl was reacted with excess peptide at 20–240 μM pep-
tide and 10–120 μM HOCl in 10 mM phosphate buffer at
the indicated pH at 37°C and quenched with 10-fold excess
cysteine at time points between 5 s and 2 h.
*
SSDϭSSDopt (F (P/(N-P)) ϩ1), where F is the critical
value of the F distribution, P is the number of model
parameters, and N is the number of data points. We chose
a value of F corresponding to the 95% confidence level.
The rate constant values are reported as middle value Ϯ
HPLC quantitation of reaction products
*
The concentration of products and reactants in kinetic
samples were measured using a Thermo Finnigan
(Waltham, MA) Surveyor HPLC system with a MS Pump,
Autosampler, and PDA detector (200–600 nm). Standard
curves were generated using stocks (Ͼ98% pure) of
AcNO2Y and AcClNO2Y (r2 Ͼ0.999). Peptides with
NO2Y and ClNO2Y residues were quantitated using the
AcNO2Y and AcClNO2Y, respectively, along with stan-
dard curves and the same HPLC method, assuming that
the different retention time and small change in solvent
composition would not affect analyte absorbance at
360 nm. The separation of each analyte was accomplished
1/2 range; the middle value was calculated from the high
and low SSD values that defined the range of certainty
(95% confidence level) and were within 1% of the opti-
mized value in all cases.
Results
HOCl reacts with NO2Y to form ClNO2Y
The published chlorination of 3-substituted tyrosine ana-
logs by HOCl suggested that the loss of 3-nitrotyrosine

1358 M. P. Curtis & J. W. Neidigh
observed by Whiteman and Halliwell [8] resulted in the
formation of 3-chloro-5-nitrotyrosine [19]. To avoid the
competing reaction of HOCl with the amino group of
3-nitrotyrosine, the acetylated analogs of NO2Y and
ClNO2Y were synthesized [21]. The reaction of AcNO2Y
with HOCl to form AcClNO2Y was monitored by HPLC
(see Supplementary Material available online at http://
informahealthcare.com/doi/abs/10.3109/10715762.2014.
954110). The observed product peak had the same reten-
tion time and UV spectrum as observed for the authentic
standard of AcClNO2Y.
with AcLysCl was too slow to produce any measureable
AcClNO2Y after 2 h but AcClNO2Y was observed a day
later (data not shown).
HOCl reacts with AcClNO2Y to form unknown products
We investigated the reaction of AcClNO2Y with HOCl by
HPLC and UV/Vis spectroscopy. No product peaks were
identified by UV/Vis-detected HPLC and the measured
loss of AcClNO2Y was less than the initial HOCl intro-
duced. This data indicates that an unknown product forms
with no significant UV/Vis absorbance between 200 and
600 nm and that it reacts more readily with HOCl than
AcClNO2Y. We followed the loss of AcClNO2Y in the
presence of HOCl by UV/Vis spectroscopy at 37°C, pH
7.4 (Figure 2). The rate of AcClNO2Y loss decreases as
the reaction progresses, suggesting that the unknown
products successfully compete with AcClNO2Y for reac-
tion with HOCl. We also determined that an approximately
6-fold excess of HOCl is required to completely degrade
AcClNO2Y as determined by UV/Vis spectroscopy (data
not shown).
We monitored the reactions between AcNO2Y and
HOCl, N-acetylhistidine chloramine (AcHisCl), or N-α-
acetyllysine chloramine (AcLysCl) at pH 7.4 and 37°C
between 5 s and 120 min. AcNO2Y, at concentrations of
approximately 500, 750, and 1000 μM, was reacted with
200 μM HOCl, both in the presence and absence of excess
AcHis or AcLys. The reaction of 750 μM AcNO2Y with
200 μM HOCl (Figure 1a) or AcHisCl (Figure 1b) results
in AcClNO2Y production. The reaction rate is pH
sensitive with a maximum rate at 7.4 (see Supplementary
Materials available online at http://informahealthcare.
com/doi/abs/10.3109/10715762.2014.954110). The reaction
Figure 2. UV/Vis analysis of AcClNO2Y reacting with HOCl.
(A) UV spectrum of 185 μM AcClNO2Y reacting with 720 μM
HOCl at 37°C, pH 7.4. The initial spectrum is shown as a bold line
while the arrow shows the changes in the spectra collected every
subsequent 24 s. (B) The consumption of 185 μM AcClNO2Y by
720 μM HOCl at 37°C, pH 7.4. The concentration of AcClNO Y
was based on the absorbance at 430 nm and a molar absorptiv2ity
value of ε430 ϭ4205 MϪ1 cmϪ1 . The change in the rate of AcClNO2Y
degradation indicates multiple reactions going on, likely between
HOCl and the product of AcClNO2Y chlorination. It takes
approximately a 6-fold excess HOCl to completely degrade
AcClNO2Y (data not shown).
Figure 1. Kinetics of AcNO Y chlorination by HOCl or AcHisCl.
(A) 780 μM AcNO2Y or (B)2730 μM AcNO2Y and 1000 μM AcHis
were reacted with 200 μM HOCl at 37°C for the indicated time; the
reaction was stopped with excess methionine, and the products were
quantitated by UV-detected HPLC. The solid lines in each panel
represent the modeled reaction progress with optimized rate
constants. The rate constant k1 for AcNO2Y reacting with HOCl in
panel A is 24.5 MϪ1 sϪ1 . The rate constant k1 for AcNO2Y reacting
with AcHisCl in panel B is 1.0 MϪ1 sϪ1 . Similar experiments were
done for AcLysCl but no product was measured after 2 h of
reaction.

Nitrotyrosine chlorination 1359
Table I. Rate constants for chlorination of tyrosine, 3ClY, and
NO2Y.a
Chlorinating
species
AcTyrb
Ac3ClTyrb
AcNO2Y
Ϫ1
Ϫ1
Ϫ1
(MϪ1 s
)
(MϪ1 s
)
(MϪ1 s
)
HOCl
HisCl
LysCl
71Ϯ8
238Ϯ27
24.5Ϯ1 .2
1.1Ϯ0 .2
NDc
3.0Ϯ0.3
10.4Ϯ1.0
0.004Ϯ0.003
0.008Ϯ0.002
aReactions were performed at 37°C, pH 7.4, in 100 mM phosphate buffer.
bRate constants were reported previously in reference [19].
cProduct was not detected after 2 h of reaction.
Figure 3. Acceleration of NO Y chlorination by His chloramine in
a peptide. 75 μM Ac-HGNY(2NO )AE-NH or an analogous amino
acid mixture were reacted with 237.5 μM2 HOCl at 37°C for the
indicated times; the reaction was stopped with excess cysteine and
the products were quantitated by UV-detected HPLC. At the
concentrations indicated here, the rate of NO2Y chlorination in the
peptide is approximately 700-fold faster than in the analogous
amino acid mixture.
Determination of kinetic rate constants
Only the initial 1 and 5 min, respectively, were used to
determine the kinetics of AcNO2Y chlorination by HOCl
and AcHisCl (Table I). At these initial time points, the
total concentrations of AcNO2Y and AcClNO2Y are con-
stant, indicating that the possible reaction of HOCl with
AcClNO2Y is not sufficient to alter the observed concen-
trations or calculated rate constants. Including the decom-
position of HOCl and/or the degradation of AcHisCl in
the kinetic model did not result in significant changes to
the calculated rate constants [18,22,23].
kinetics were measured to minimize the effect of
ClNO2Y reacting with HOCl or chloramines. Initial
experiments found that the reaction kinetics were inde-
pendent of the initial peptide concentration and changed
only with the HOCl concentration when excess peptide
was present. Since HOCl rapidly reacts with the peptide
amines to form a chloramine [20], the reaction is first
order with respect to the peptide-chloramine concentra-
tion (Figure 4).
Rate of NO2Y chlorination by HOCl increase in peptides
Previous studies indicate that the chlorination of tyrosine
residues in polypeptides is facilitated by nearby histidine
or lysine residues [18,19,24]. We reacted the synthetic
peptide, Ac-HGNY(NO2) AE-NH2, with a 0.5-fold con-
centration of HOCl and followed product formation by
HPLC (see Supplementary Materials available online at
http://informahealthcare.com/doi/abs/10.3109/10715762.
2014.954110), as shown in Figure 3 and summarized
in Table II. An amino acid mixture of N-acetyl amino
acids that match the residue composition of the peptide
Ac-HGNY(NO2)AE-NH2 was also reacted with 0.5-fold
concentration of HOCl as a control reaction. The produc-
tion of ClNO2Y in the peptide was approximately 700-fold
faster than in the amino acid mixture at the concentrations
and reaction conditions examined.
Discussion
Reactive nitrogen species (RNS) chemically damage
tyrosine residues to form 3-nitrotyrosine (NO2Y), a sur-
rogate marker used to investigate the role of RNS in human
diseases. However, Whiteman and Halliwell [8] reported
that NO2Y disappears when exposed to HOCl, suggesting
that the observed NO2Y underestimates the role of RNS
in tissues when chlorinating species are also present.
Whiteman and Halliwell [8] did not report the resulting
product when NO2Y reacts with HOCl, but subsequent
studies [19,24–26] on the reaction of HOCl with 3ClY to
form Cl2Y suggested that the expected product should be
ClNO2Y. This expectation is now confirmed since the
reaction of AcNO2Y with HOCl forms a new product that
has the same HPLC retention time and UV-spectra as
authentic AcClNO2Y.
The kinetics of NO2Y chlorination by HOCl in peptides
is a first order reaction
To probe the mechanism whereby neighboring lysine or
histidine residues facilitate NO2Y chlorination, we var-
ied the peptide and HOCl concentrations to determine
the order of the reaction kinetics. The initial reaction
As observed with Cl2Y [19], ClNO2Y also reacts with
HOCl. The complex kinetics of ClNO2Y loss indicate
that the unknown compound(s) formed upon exposure to
HOCl also react with HOCl (Figure 2). Previous reports
demonstrate that HOCl and chloramines chlorinate phe-
nolic compounds, degrading the aromatic phenol and
eventually producing chloroform [27,28]. As the focus
of this report is the physiological significance of NO2Y
loss, we did not identify the unknown products or
reaction kinetics when AcClNO2Y reacts with HOCl.
The reported kinetic rates used excess NO2Y and were
Table II. Rate constants for chlorination of NO2Y within a peptide.a
Ϫ2
Peptide
Rate constant (s
)
Ϫ2
Ac-HGN-Y(NO2)-AE-NH2
Ac-KGN-Y(NO2)-AE-NH2
2.60Ϯ0.53ϫ1 0
2.54Ϯ0.03ϫ1 0
2.38Ϯ0.07ϫ1 0
Ϫ5
Ϫ4
b
Ac-KGN-Y-AE-NH2
aReactions were performed at 37°C, pH 7.4, in 10 mM phosphate buffer.
bData from reference [19].

1360 M. P. Curtis & J. W. Neidigh
7.4 and 8.0 for NO2Y and 3ClY [19], respectively (see
Supplementary Material available online at http://
informahealthcare.com/doi/abs/10.3109/10715762.2014.
954110). The effect of 3-nitro and 3-chloro groups on pKa
values and reaction kinetics is consistent with an electro-
philic aromatic substitution reaction where phenolate is
more reactive than a phenol and a chlorine substituent is
less deactivating than a nitro substituent.
The rate of NO2Y loss due to chlorination in the pep-
tide Ac-HGNY(NO2)AE-NH2 is 700-fold faster than an
equivalent amino acid mix (Figure 3 and Supplementary
Material available online at http://informahealthcare.com/
doi/abs/10.3109/10715762.2014.954110). We argue that
this increased rate of chlorination is due to the close prox-
imity maintained between the histidine chloramine and
the NO2Y residues in the peptide context. The rapid inter-
molecular reaction of HOCl with a polypeptide amine to
form a chloramine followed by the slower intramolecular
chlorination of a nearby tyrosine analog predicts that the
kinetics of peptide tyrosine chlorination will follow first
order kinetics (see Supplementary Material available
online at http://informahealthcare.com/doi/abs/10.3109/
10715762.2014.954110). To our knowledge, this report
is the first to demonstrate that the kinetics of tyrosine
analog chlorination in a peptide or protein with a nearby
amine follows first order kinetics.
The relative kinetics of NO2Y chlorination by histi-
dine or lysine chloramines appear independent of reac-
tion order. The second order kinetics of AcTyr or
N-acetyl-3chlorotyrosine (Ac3ClTyr) chlorination by
lysine chloramines are 1025Ϯ275 times slower than
chlorination by histidine chloramines [19]. Likewise, the
first order kinetics of NO2Y chlorination in a peptide by
a lysine chloramine is 1024 times slower than a histidine
chloramine (Table II). However, tyrosine chlorination by
a HisCl is 9-fold faster in the peptide context (Table II)
whereas the expectation was 3-fold faster given the data
in Table I.
Figure 4. Determining the rate order of NO2Y chlorination by
chloramines within a peptide. Excess Ac-KGNY(NO2)AE-NH2
was reacted with HOCl at 37°C, pH 7.4. The initial reaction rate
was determined by using HPLC to measure the formation of
ClNO2Y at early time points where less than 20% of the reactant
peptide was consumed. The samples were quenched with excess
cysteine before HPLC analysis. The initial reaction rate was
calculated by linearly fitting the early time points. Because the
reaction of Lys with HOCl is significantly faster than the
chlorination of NO2Y, it was assumed that the concentration of
peptide containing lysine chloramine was equal to the amount of
HOCl added. The initial reaction rate was plotted to (A) the
concentration of Ac-K(Cl)GNY(NO )AE-NH2 to model a first-
order reaction and to (B) the square2of the concentration of this
peptide to model a second-order reaction. The linearity of the
first-order fit indicates that NO2Y is chlorinated through an
intramolecular reaction with the lysine chloramine.
Measurements of 3ClY or NO2Y from biological
tissues usually measure these surrogate markers in the
context of proteins instead of the free amino acid. The
observation that tyrosine residues with histidine or lysine
residues nearby are preferentially chlorinated is better
explained by the intramolecular reaction with a nearby
chloramine than the direct intermolecular chlorination by
HOCl to form a protein-bound 3ClY [18,19,24,26,29].
The fast reaction of HOCl with protein amines (∼105
calculated using reaction times where the loss of ClNO2Y
was not observed.
M
Ϫ1 sϪ1 ) better competes with the antioxidant glutathione
(∼108 MϪ1 sϪ1 ) than the direct chlorination of tyrosine
analogs (∼102 MϪ1 sϪ1 ) and explains the preferential chlo-
rination of some protein-bound tyrosine residues, provided
the intramolecular chlorination kinetics are sufficiently
fast [20,30].
The data in this report as well as our previous report
[19] allow us to compare the chlorinating potential of
HOCl and the chloramines of lysine or histidine reacting
with tyrosine analogs (Table I). The chlorination of
tyrosine analogs in Table I by HOCl is 23Ϯ1 and
23,750Ϯ6,000 times faster than HisCl and LysCl, respec-
tively. The kinetics of 3ClY reacting with HOCl or a his-
tidine chloramine are approximately 3 times faster than
tyrosine chlorination while the kinetics of NO2Y chlorina-
tion are 3 times slower. The kinetics of chlorination are
affected by pH, with maximum rates at pH values of
The loss of NO2Y will depend on the quantity of
HOCl although the likelihood of NO2Y loss in biological
tissues is still uncertain, given that most reports do not
measure both NO2Y and 3ClY. We expect that the forma-
tion of 3ClNO2Y in biological tissues is less likely than
the formation of Cl2Y, given that HOCl reacts 9-fold

Nitrotyrosine chlorination 1361
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Declaration of interest
[19] Curtis MP, Hicks AJ, Neidigh JW. Kinetics of 3-Chlorotyrosine
formation and loss due to hypochlorous acid and chloramines.
Chem Res Toxicol 2011;24:418–428.
The authors report no declarations of interest. The authors
alone are responsible for the content and writing of the
paper.
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Supplementary material available online
Supplementary Material and Figures 1–5.