Lysine Residues Direct the Chlorination of Tyrosines in YxxK Motifs
of Apolipoprotein A-I when Hypochlorous Acid Oxidizes HDL
Constanze Bergt1, Xiaoyun Fu1, Nabiha P. Huq2, Jeff Kao2, Jay W. Heinecke1
1Department of Medicine, University of Washington, Seattle, WA 98195
2Department of Chemistry, Washington University, St. Louis, MO 63110
Address correspondence to: Jay Heinecke, Division of Metabolism, Endocrinology and
Nutrition, Box 356426, University of Washington, Seattle, WA 98195, email:
Abbreviations: DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; ESI, electrospray
ionization; HDL, high density lipoprotein; MALDI, matrix assisted laser desorption ionization;
MS, mass spectrometry; PBS, phosphate buffered saline; TFA, trifluoroacetic acid; TOF, time-
of-flight; TOCSY, total correlation spectroscopy.
Running title: Regiospecific chlorination of tyrosine residues
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Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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SUMMARY
Oxidized lipoproteins may play an important role in the pathogenesis of atherosclerosis. Elevated
levels of 3-chlorotyrosine, a specific end product of the reaction between hypochlorous acid
(HOCl) and tyrosine residues of proteins, have been detected in atherosclerotic tissue. Thus,
HOCl generated by the phagocyte enzyme myeloperoxidase represents one pathway for protein
oxidation in humans. One important target of the myeloperoxidase pathway may be high density
lipoprotein (HDL), which mobilizes cholesterol from artery wall cells. To determine whether
activated phagocytes preferentially chlorinate specific sites in HDL, we used tandem mass
spectrometry (MS/MS) to analyze apolipoprotein A-I that had been oxidized by HOCl. The
major site of chlorination was a single tyrosine residue located in one of the protein’s YxxK
motifs (Y, tyrosine; K, lysine; x, non-reactive amino acid). To investigate the mechanism of
chlorination, we exposed synthetic peptides to HOCl. The peptides encompassed the amino acid
sequences YKxxY, YxxKY, or YxxxY. MS/MS analysis demonstrated that chlorination of
tyrosine in the peptides that contained lysine was regioselective and occurred in high yield if the
substrate was KxxY or YxxK. NMR and MS analyses revealed that the Nε amino group of lysine
was initially chlorinated, which suggests that chloramine formation is the first step in tyrosine
chlorination. Molecular modeling of the YxxK motif in apolipoprotein A-I demonstrated that
these tyrosine and lysine residues are adjacent on the same face of an amphipathic α-helix. Our
observations suggest that HOCl selectively targets tyrosine residues that are suitably juxtaposed
to primary amino groups in proteins. This mechanism might enable phagocytes to efficiently
damage proteins when they destroy microbial proteins during infection or damage host tissue
during inflammation.
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INTRODUCTION
Protein oxidation has been implicated in the pathogenesis of diseases ranging from
ischemia-reperfusion injury to atherosclerosis as well as in the aging process itself (1). However,
most studies of protein oxidation have focused on the vulnerability of individual amino acid side
chains, including the phenolic group of tyrosine and the thiol groups of methionine and cysteine.
Remarkably little is known about the influence of nearby residues or of specific sequence motifs
on the susceptibility of protein-bound amino acid chains to oxidation.
One example of such effects is the oxidation of specific cysteine residues in tyrosine
phosphatases by hydrogen peroxide (2). This reaction, which affects phosphatase activity, is
thought to rely on a high local concentration of positively charged amino acid side chains to
stabilize the reactive anionic form of the thiol. Another example is the apparent effect of acidic
and basic amino acid residues on protein nitrosation (3). However, an extensive study of model
proteins oxidized by peroxynitrite revealed few obvious common features among the tyrosine
residues that were targeted for nitration (4). Recently, histidine and cysteine residues at the metal
binding site have been identified as primary targets during metal catalyzed oxidation of ß-
amyloid peptides and iron regulatory protein 2 (5,6).
Neutrophils, monocytes and some population of macrophages use the heme enzyme
myeloperoxidase (7-9) to produce hypochlorous acid (HOCl), a potent cytotoxic oxidant (10,11).
Cl- + H2O2 + H+ → HOCl + H2O (Equation 1)
HOCl plays a critical role in destroying microbial pathogens (12,13). However, it can also react
with proteins, lipids, and nucleic acids in host tissues, contributing to inflammatory damage
(14,15).
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Because proteins are a major target for HOCl, the acid’s reactions with amino acids and
peptides have been widely studied (16-21). HOCl readily oxidizes the sulfur-containing amino
acids cysteine and methionine, producing disulfides, oxyacids, sulfoxides, and compounds in
which sulfur is cross-linked to nitrogen (22-24). However, other reactive species, such as
hydroxyl radical and peroxynitrite, can also generate oxygenated sulfur products (25,26), which
therefore cannot serve as definitive markers for HOCl-induced damage. In contrast, in vitro and
in vivo studies have shown that 3-chlorotyrosine is a specific product of myeloperoxidase
(13,27,28).
Chlorination of the phenolic ring of tyrosine may have physiological relevance because both
active myeloperoxidase, the enzyme that generates HOCl, and elevated levels of 3-
chlorotyrosine have been detected in human atherosclerotic lesions (8,9,14,29). One important
target might be high density lipoprotein (HDL). HDL is believed to inhibit atherosclerosis by
mobilizing excess cholesterol from cells of the artery wall, but oxidation of HDL has been
proposed to alter its biological properties, thereby contributing to the pathogenesis of
atherosclerosis (30,31). Previous studies have shown that methionine and phenylalanine residues
in apolipoprotein A-I, the major protein in HDL, are oxidized by reactive intermediates (32-35).
Tyrosine residues are converted to o,o’-dityrosine by tyrosyl radical (36). However, little is
known about the vulnerability to chlorination of specific tyrosine residues in apolipoprotein A-I.
HOCl also reacts with primary amino groups, producing primary (RNClH) and secondary
(RNCl2) chloramines (37,38).
HOCl + RNH2 → RNClH + H2O + HOCl RNCl2 + H2O (Equation 2)
Studies with free α-amino acids have shown that they form unstable α-amino chloramines,
which are deaminated and decarboxylated into aldehydes (39-41). In contrast, HOCl yields stable
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chloramines at the primary ε-amino group of lysine, which lacks an adjacent carboxylic acid
(19). It has been suggested that N-centered radicals deriving from chloramines could contribute
to protein fragmentation and tissue damage (42).
Both HOCl and chloramines react with aromatic and unsaturated compounds to form
chlorinated products (7,37,43). HOCl is in equilibrium with molecular chlorine, and this potent
electrophile has been proposed to mediate the chlorination of free tyrosine (28). It remains to be
established whether this reaction pathway is relevant to the chlorination of protein-bound
tyrosine residues. N-Terminal chloramines have been proposed to be intermediates in the
formation of 3-chlorotyrosine in synthetic peptides (27,44). However, little is known about the
factors that control the site-specific chlorination of tyrosine residues in proteins. In the current
study, we use HDL, synthetic peptides and tandem mass spectrometric analysis to investigate the
role of HOCl and chloramines in tyrosine chlorination.
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EXPERIMENTAL PROCEDURES
Materials
Sodium hypochlorite (NaOCl), trifluoroacetic acid (TFA), and HPLC grade CH3CN and
methanol were obtained from Fisher Scientific (Pittsburgh, PA). Phosphate buffered saline
(PBS), free and acetylated amino acids, and α-cyano-4-hydroxycinnamic acid were purchased
from Sigma (St. Louis, MO). Peptides AcGYKRAYE (YKxxY), AcGEYARKY (YxxKY), and
AcGEYAREY (YxxxY) were prepared by the Protein and Nucleic Acid Chemistry Laboratory,
Washington University (St. Louis, MO). Peptides AcPYSDELRQRLAARLE-NH2 (Yxxxxx),
AcPYSDELKQRLAARLE-NH2 (YxxxxK), AcPYSDEKLQRLAARLE-NH2 (YxxxKx),
AcPYSDKELQRLAARLE-NH2 (YxxKxx), AcPYSKDELQRLAARLE-NH2 (YxKxxx) and
AcPYKSDELQRLAARLE-NH2 (YKxxxx) were synthesized by GenScript Corporation (Scotch
Plains, NJ). Purity of the peptides was confirmed by HPLC and mass spectrometric analysis.
Methods
HDL isolation. Plasma was prepared from blood anticoagulated with EDTA collected
from healthy adults that had fasted overnight. HDL (density 1.125-1.210 g/mL) was prepared by
sequential ultracentrifugation from plasma and was depleted of apolipoprotein E and
apolipoprotein B100 by heparin-agarose chromatography (45).
Oxidation reactions. Reactions were carried out at 37 oC in PBS (10 mM sodium
phosphate, 138 mM NaCl, 2.7 mM KCl, pH 7.4) supplemented with 100 µM peptide or 1 mg/ml
HDL protein. Reactions were initiated by adding oxidant and were terminated by the addition of
10- to 50-fold molar excess of L-methionine. Concentrations of HOCl and H2O2 were
determined spectrophotometrically (ε292 = 350 M -1 cm-1 and ε240 = 39.4 M-1cm –1) (46,47). The
pH dependence of reactions was determined using buffers (100 mM) composed of phosphoric
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acid, monobasic sodium phosphate and dibasic sodium phosphate and 100 mM NaCl. Protein
was determined using the Lowry assay (BioRad; Hercules, CA).
HPLC analysis of peptides. Peptides were separated at a flow rate of 1 mL/min on a
reverse-phase column (Beckman ODS, 4.6 x 250 mm) using a Beckman HPLC system
(Fullerton, CA) with UV detection at 215 nm. The peptides were eluted using a gradient of
solvent A (0.1% TFA in H2O) and solvent B (0.1% TFA in 90% CH3CN, 10% H2O). Solvent B
was increased from 10% to 50% over 25 min.
Isolation of peptide-bound chloramines. AcGEYARKY (100 µM) was incubated with
HOCl (100 µM) in PBS (pH 7.4) for 2 min at room temperature and the reaction mixture was
subjected immediately to HPLC without methionine addition. The methionine-sensitive
oxidation product was collected and stored on ice for further analysis.
Oxidation of lipid-associated peptides. 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine
(DPPC) associated peptides were prepared using a peptide to DPPC ratio of 1:20 (mol/mol).
DPPC was dissolved in ethanol and dried under nitrogen. PBS was added and the mixture was
vortexed vigorously and incubated for 15 min at 37 ºC. Vesicles were aliquoted and incubated
with 25 µM peptides for 20 min at 37 ºC. Peptides were oxidized with HOCl in PBS at 37 ºC
(0.5 and 1, mol oxidant/mol peptide, lipid-free and DPPC-associated, respectively). The reaction
was stopped with excess methionine after 30 min.
Proteolytic digestion of proteins. Native or HOCl modified HDL was incubated overnight
at 37 oC with sequencing grade modified trypsin (Promega, Madison, WI) at a ratio of 25:1
(w/w) protein:trypsin in 50 mM NH4HCO3, pH 8. Digestion was halted by acidification (pH 2-3)
with TFA.
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Electrospray ionization mass spectrometry (ESI-MS). LC-ESI-MS analyses were
performed in the positive ion mode with a Finnigan Mat LCQ ion trap instrument (San Jose, CA)
coupled to a Waters 2690 HPLC system (Milford, MA)(48). Synthetic peptides were separated at
a flow rate of 0.2 mL/min on a reverse-phase column (Beckman ODS; 2.1 x 250 mm) using a
gradient of solvent A (0.2% HCOOH in H2O) and solvent B (0.2% HCOOH in 80% CH3CN,
20% H2O). Solvent B was increased from 10% to 50% over 25 min. Tryptic digests of HDL
were separated at a flow rate of 0.2 mL/min on a reverse-phase column (Vydac C18 MS; 2.1 x
250 mm). The electrospray needle was held at 4500 V. Nitrogen, the sheath gas, was set at 80
units. The collision gas was helium. The temperature of the heated capillary was 220oC.
Matrix assisted laser desorption ionization (MALDI) time of flight (TOF) MS. MALDI
TOF-MS analyses were performed on a Voyager DE-STR (PerSeptive Biosystems, Foster City,
CA) in the reflectron mode using delayed ion extraction (49).The accelerating voltage was 2.5
kV, the delay time was 300 nsec, the mirror to accelerating voltage ratio was 1.12, and the low
mass gate was 400 Da. Spectra (100 shots) were acquired with a laser power of 1850. α-Cyano-
4-hydroxycinnamic acid (10 mg/mL) in 0.1% TFA:CH3CN (1:1, v/v) was used as matrix.
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NMR Spectroscopy. NMR spectra were obtained on the peptide and the chloramine
intermediate (3.6 mM and 0.5 mM, respectively) in 0.5 mL of PBS (10% D2O, 90% H2O, pH
7.4). The spectra were acquired on a Varian Unity INOVA-600 spectrometer (599.749 MHz for
1H) and processed using VNMR software (Varian, Palo Alto, CA). 1-D NMR experiments were
carried out at 4 °C using presaturation or hard-pulse WATERGATE sequences for water
suppression (50). Spectra were obtained under the following conditions: pre-acquisition delay =
0.5 sec, acquisition time = 1.416 sec (16k complex data points), pulse width = 7.8 µsec, spectral
width = 5650.1 Hz and 2 msec 15G/cm field-gradient pulses.
Total correlation spectroscopy (TOCSY) experiments for peptides and peptide-bound
chloramines were recorded using an MELV-17 mixing sequence of 100 ms flanked by two 2
msec trim pulses with 256 t1 and 2048 t2 data points. After two-dimensional Fourier
transformation, the spectra resulted in 2048 x 2048 data points that were phase and baseline
corrected in both dimensions.
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RESULTS
HOCl Preferentially Chlorinates a Single Tyrosine Residue in Apolipoprotein A-I.
The 10 amphipathic helices in apolipoprotein A-I, HDL’s major protein, are thought to
play essential roles in lipid binding, lipoprotein stability, and reverse cholesterol transport
(51,52). Five of the 7 tyrosine residues in apolipoprotein A-I are located in amphipathic helices
(Fig. 1A). It is noteworthy that 3 of those tyrosines reside in a YxxK motif (Y29xxK32,
Y192xxK195, and Y236xxK239; Y = tyrosine, K = lysine, x = other amino acids). Importantly, the
helical wheel representation of amphipathic helices predicts that tyrosine and lysine residues in
this motif will lie next to each other on the same face of the α-helix (Fig. 1B) (51,52). We
therefore used HDL to determine whether HOCl preferentially chlorinates specific tyrosine
residues in proteins.
LC-ESI-MS analysis of the trypsin digest of apolipoprotein A-I detected peptides that
collectively covered ~ 80% of the protein’s sequence and included all 7 peptides predicted to
contain tyrosine. To determine which tyrosine residues can be chlorinated, we oxidized HDL
with HOCl and used reconstructed ion chromatograms to detect: i) each of the peptides that
contained tyrosine; and ii) any tyrosine-containing peptides that had gained 34 amu (addition of
1 chlorine and loss of 1 hydrogen).
We exposed HDL to HOCl (80:1, mol/mol, oxidant:HDL particle) in a physiological
buffer (138 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate) at neutral pH for 120 min at
37°C and then terminated the reaction with a 20-fold molar excess (relative to oxidant) of
methionine. Because the average HDL3 particle contains 2 mol of apolipoprotein A-I (7 tyrosine
residues, 243 amino acids) and 1 mol of apolipoprotein A-II (8 tyrosine residues, 154 amino
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acids), the ratio of oxidant to substrate (mol:mol) was ~ 30:1 for apolipoproteins A-I and A-II,
3:1 for tyrosine residues, and 1:8 for total amino acids.
After digesting unmodified and oxidized HDL with trypsin, we analyzed the resulting
peptides. LC-ESI-MS and MS/MS analysis detected 3 tryptic peptides whose mass corresponded
to the mass of the precursor peptide plus 34 amu, which suggests the formation of 3-
chlorotyrosine (Table 1). The 3 peptides came from residues 189-195 ([LAEYHAK + 34 amu +
2H]2+, m/z 433.2), residues 227-238 ([VSFLSALEEYTK + 34 amu + 2H]2+, m/z 710.9), and
residues 28-40 ([DYVSQFEGSALGK + 34 amu + 2H]2+, m/z 717.9). Using LC-ESI-MS/MS
analysis, we confirmed each peptide’s sequence and showed that its tyrosine had been targeted
for chlorination (data not shown). Quantification of the ion current of each precursor and product
peptide using reconstructed ion chromatograms (Table 1) indicated that the major tyrosine
oxidation product was LAE(ClY)HAK (Fig. 2). Importantly, the tyrosine residue in this region of
apolipoprotein A-I is located in a YxxK motif. The other chlorinated peptides were present at
much lower levels. These findings indicate that HOCl chlorinates 3 of the 7 tyrosines in
apolipoprotein A-I (Table1) and that the major oxidation product (~ 50% 3-chlorotyrosine) was
located in the YxxK motif of LAEYHAK. The other 2 tyrosines were chlorinated in much lower
yield (< 6% 3-chlorotyrosine); one of these tyrosine residues was also located in a YxxK motif
(VSFLSALEEYTK[K], where [K] represents a lysine residue removed by trypsin). These
observations suggest that the amino group of lysine might direct protein oxidation to specific
sites.
Lysine Residues Direct the Regiospecific Chlorination of Tyrosine in Peptides.
To explore the influence of lysine residues on the chlorination of nearby tyrosine
residues, we investigated the reaction of HOCl with 3 model peptides: AcGYKRAYE,
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AcGEYARKY, and AcGEYAREY (YKxxY, YxxKY, and YxxxY). We included lysine in 2 of
the peptides because it has a primary ε-amino group that forms a long-lived chloramine when
exposed to HOCl (19), and chloramines can react with phenolic groups (37) such as the one in
tyrosine. The 3 peptides contained the same amino acids (acetyl-G, 2Y, K, R, A, E), including
glutamic acid (E), but the latter replaced lysine (K) in YxxxY. Glutamic acid was included
because its negative charge ensures aqueous solubility. Arginine was included because its
positive charge promotes peptide ionization during mass spectrometric analysis. The acetyl
group on the N-terminus prevented the primary amine from forming a chloramine.
We exposed each peptide to HOCl (1:1, mol/mol) in a physiological buffer for 30 min at
37°C, terminated the reaction with methionine (which reacts rapidly with HOCl), and analyzed
the reaction products by HPLC with UV detection, LC-ESI-MS, and LC-ESI-MS/MS. When the
substrate was YKxxY or YxxKY, HPLC with UV detection revealed a high yield (~ 55%, mol
product/mol oxidant) of one major product and smaller yields of a minor product (Fig. 3A,B;
Table 2). In contrast, oxidation of YxxxY generated four minor products that individually
accounted for only ~ 11% of the total oxidant (Fig. 3C, peaks 1-4; Table 2). LC-ESI-MS analysis
of the peptides YKxxY and YxxKY incubated in buffer alone demonstrated doubly charged ions
at mass-to-charge ratio (m/z) 464.8, the anticipated m/z of the doubly protonated unmodified
peptide [peptide + 2H]+2. LC-ESI-MS analysis of the two reaction products derived from YKxxY
and the two derived from YxxKY revealed ions of m/z 481.8, corresponding to the masses of the
doubly protonated peptides plus 34 amu [peptide + 34 amu + 2H]+2. These observations suggest
that each peptide loses a hydrogen atom and gains a chlorine atom when it is exposed to HOCl (–
1 amu, + 35 amu), which is consistent with chlorination of the phenolic ring of one of each
peptide’s two tyrosine residues. LC-ESI-MS analysis of YxxxY incubated in buffer alone
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demonstrated a singly charged ion of m/z 929.3, the anticipated m/z of the unmodified peptide
[peptide + H]+. Two of its oxidation products (Fig. 3C, peak 1 and peak 2) exhibited ions at m/z
963.3 [peptide + 34 amu + H]+, suggesting that each contained a single chlorine atom. In
contrast, the other two (Fig. 3C, peak 3 and peak 4) exhibited ions of m/z 997.3 [precursor
peptide + 68 amu + H]+, suggesting that HOCl oxidizes YxxxY into a mixture of peptides that
have 3-chlorotyrosine at either the first or last position or into a peptide that contains 3,5-
dichlorotyrosine residue at one position.
We used tandem mass spectrometry (MS/MS) to confirm the sequence of each precursor
peptide and to identify the amino acid residue that was amenable to chlorination (Fig. 4). MS/MS
analysis of YxxKY revealed a series of b-ions (b2, m/z 228.8; b5, m/z 619.2; b6, m/z 747.3) and y-
ions (y1, m/z 181.9; y4, m/z 537.2; y5, m/z 700.3) consistent with the predicted sequence
AcGEYARKY (Fig. 4A). MS/MS analysis of the major oxidation product of YxxKY (Peak 1;
Fig. 3B) demonstrated a series of b-ions and y-ions that had gained 34 amu (b5 + 34, m/z 653.1;
b6 + 34, m/z 781.3; y5 + 34, m/z 734.2; y6 + 34, m/z 863.4). However, the masses of ions b2 (m/z
228.9), y1 (m/z 182.0), and y4 (m/z 537.2) were unchanged (Fig. 4B). These observations indicate
that exposing YxxKY to HOCl generates a major oxidation product that contains a chlorine atom
on its first tyrosine residue (AcGEClYARKY; ClYxxKY). MS/MS analysis of peak 2 (Fig. 3B),
the minor product of YxxKY oxidation, demonstrated y1, y4, and y5 ions that had gained 34 amu
(y1 + 34, m/z 216.0; y4 + 34, m/z 571.2; y5 + 34, m/z 734.3) and ions that were unaffected (b4, b5,
and b6). These observations indicate that AcGEYARKClY (YxxKClY) is the minor product
when YxxKY is oxidized with HOCl. The sequences of the YxxKY peptide and its major and
minor oxidation product were confirmed using MALDI-TOF-MS analysis with post source
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decay (data not shown). These observations indicate that HOCl converts YxxKY into a single
major oxidation product, ClYxxKY.
We used this analytical approach to confirm the sequences of YKxxY and YxxxY and to
identify the residues that are chlorinated when these peptides are exposed to HOCl (data not
shown). MS/MS analysis revealed that the major and minor product of YKxxY oxidation were
respectively AcGYKRAClY (YKxxClY) and AcGClYKRAY (ClYKxxY). Oxidation of YxxxY
produced a different pattern of products: peaks 1 and 2 were monochlorinated on a single
tyrosine residue (AcGEClYAREY and AcGEYAREClY), whereas peaks 3 and peak 4 had two
chlorine atoms on a single tyrosine residue (AcGECl2YAREY and AcGEYARECl2Y).
Our results indicate that HOCl chlorinates tyrosine residues in peptides containing the motif
KxxY or YxxK with high yield and that chlorination is regiospecific for the tyrosine residue two
residues away from a lysine residue (Table 2). In contrast, only small amounts of
monochlorotyrosine and dichlorotyrosine form in YxxxY, which lacks a lysine residue (Table 2).
Moreover, we found no evidence for chlorination of both tyrosines in YxxxY. Instead, each
tyrosine residue was oxidized to either the monochlorinated or dichlorinated derivative (and the
other was left unchlorinated). These findings suggest that lysine plays a regiospecific role in the
chlorination of tyrosine in peptides.
YxxKY Reacts with HOCl to Produce a High Yield of Chlorotyrosine.
To characterize the reaction of HOCl with peptides, we incubated YxxKY with HOCl
(1:1, mol/mol) in a physiological buffer at neutral pH and 37ºC, and identified the reaction
products by HPLC. Peptide chlorination (monitored as production of both ClYxxKY and
YxxKClY) was complete by 60 min. At an equimolar ratio of oxidant and peptide, the product
yield exceeded 85% (Fig. 5A). It was maximal when the ratio of oxidant to peptide was ≤1, and
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it decreased at higher ratios (Fig. 5B). Chlorination of tyrosine in the peptides was optimal under
alkaline conditions, suggesting that hypochlorite (ClO-, the conjugate base of HOCl, pKa 7.5)
was the chlorinating agent or that deprotonation of the lysine ε amino group (pKa 10.0) favored
the reaction (Fig. 5C).
We also investigated the effects of proposed antioxidants on the chlorination of YxxKY
(Fig. 5D). At equimolar concentrations, both vitamin C and methionine completely blocked 3-
chlorotyrosine formation. In contrast, trolox, a water-soluble form of vitamin E (53), failed to
inhibit peptide chlorination. These findings indicate that compounds that react rapidly with
HOCl (54) but not radical scavengers, might be able to inhibit protein chlorination by HOCl.
Regiospecific Chlorination of Tyrosine Requires Peptide-bound Lysine.
To assess whether lysine can direct tyrosine chlorination when it is free in solution rather
than incorporated into a peptide, we incubated HOCl with N-acetyl-tyrosine alone (1:1, mol/mol)
or with mixtures of free amino acids representing the residues in YKxxY, YxxKY, or YxxxY.
The amino acids were acetylated on the Nα-amino group to mimic a peptide bond. Oxidation of
N-acetyl-tyrosine alone with HOCl generated N-acetyl-3-chlorotyrosine and N-acetyl-3,5-
dichlorotyrosine, as determined by HPLC with UV detection (Fig. 6A) and ESI-MS analysis
(data not shown). However, the yield of 3-chlorotyrosine was much lower (~ 10%, mol/mol,
oxidized amino acid/oxidant) than that obtained when HOCl oxidized the peptides YKxxY and
YxxKY (~ 55%). When a mixture of N-acetylated (Ac) amino acids identical to that found in
YxxxY (Ac-Tyr, Ac-Gly, Ac-Glu, Ac-Ala, Ac-Arg) was oxidized with HOCl, we observed the
same low product yields of N-acetyl-chlorotyrosine and N-acetyl-dichlorotyrosine that were
obtained by oxidizing YxxxY (Fig. 3C) or N-acetyl-tyrosine alone (Fig. 6B). When the mixture
of N-acetylated amino acids representing those in YxxKY and YKxxY (Ac-Tyr, Ac-Gly, NαAc-
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Lys, Ac-Arg, Ac-Ala and Ac-Glu) was exposed to HOCl, neither N-acetyl-3-chlorotyrosine nor
N-acetyl-3,5-dichlorotyrosine were detected (Fig. 6C). These observations strongly suggest that
lysine and tyrosine must be incorporated into a peptide for chlorination of the tyrosine to be
efficient.
Chloramines Mediate the Regiospecific Chlorination of Peptide-bound Tyrosine.
To determine whether the formation of lysine chloramine is a necessary step in the
regiospecific chlorination of peptides, we incubated YxxKY with an equimolar amount of HOCl
and immediately analyzed the reaction mixture. For these studies, we omitted methionine from
the mixture because alkylated thiols rapidly reduce chloramines (37,55). HPLC analysis revealed
a single peak of material (Fig. 7A, retention time 20 min) that was absent in the reaction
mixtures exposed to HOCl and then methionine (Fig. 3B). This product could be collected and
detected after it was again subjected to HPLC (Fig. 7B), indicating that it was reasonably stable
under our analytical conditions. Moreover, methionine converted it back to its precursor peptide
(Fig. 7C). These observations indicate that HOCl converts YxxKY into a stable intermediate that
can be converted back to the precursor peptide by methionine.
To investigate the nature of the reaction intermediate, we subjected the unmodified
peptide and the methionine-sensitive oxidation product to 1-D and 2-D NMR analyses (Fig. 8).
High-resolution 1H-NMR analysis of the unmodified peptide revealed the lysine ε NH2 and ε
CH2 proton resonances were at 7.5 ppm and 2.85 ppm, respectively (Fig. 7A, left panel). The
assignment of these protons was confirmed by TOCSY (Fig. 8A, right panel). The methionine-
sensitive oxidation product lacked the ε NH2 protons and exhibited a marked downfield shift of
the ε CH2 resonance (3.55 ppm; Fig. 8B, right and left panels). These observations strongly
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suggest that the ε NH2 group in the methionine-sensitive oxidation product had been modified by
the addition of a strongly electron-withdrawing group(s).
We used MALDI TOF-MS analysis to confirm the identity of the methionine-sensitive
intermediate derived from YxxKY. The HPLC-purified intermediate displayed three major ions
of m/z 628.3, m/z 962.2, and m/z 996.2, corresponding to the masses of the singly protonated
precursor peptide, the protonated peptide plus 34 amu, and the protonated peptide plus 68 amu
(Fig. 9A). The peaks of material that had gained 34 amu and 68 amu demonstrated the isotopic
clusters characteristic of monochlorinated and dichlorinated compounds, respectively. Post
source decay analysis of the protonated precursor peptide (m/z 628.3) showed an unmodified
lysine residue (K; Fig. 9B). In contrast post source decay analysis of the ions at m/z 962.2 and
m/z 996.2 demonstrated that the lysine residue had gained 34 amu (K + 34 amu; Fig. 9C) and 68
amu (K + 68 amu; Fig. 9D). These observations strongly suggest that HOCl converts the Nε
amino group of YxxKY into a monochlorinated and/or dichlorinated intermediate. The mixture
of chlorinated and non-chlorinated species detected by MALDI TOF-MS likely reflects a loss
and/or exchange of chlorine atoms during ionization.
To determine whether lysine Nε chloramine is an intermediate in the reaction pathway
that generates peptide-bound 3-chlorotyrosine, we oxidized the YxxKY peptide with an
equimolar amount of HOCl and used HPLC to monitor the progress curve of the reaction (Fig.
10). Immediately following the addition of oxidant, the reaction mixture contained
approximately equal amounts of the unmodified peptide and its chloramine. As the reaction time
increased, both the unmodified peptide and the lysine Nε-chloramine disappeared in concert with
the appearance of peptide-bound 3-chlorotyrosine. The stoichiometry of the reaction was ~ 1 mol
of product peptide per 0.5 mol chloramine intermediate. Collectively, these observations provide
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strong evidence that chloramines derived from the Nε-amino group of lysine are crucial
intermediates in the regioselective chlorination of peptide-bound tyrosine.
Lysine Residues in YxxK and YxxxK Motifs Promote the Chlorination of Tyrosine in Lipid-free
and Lipid-associated Peptides.
Molecular modeling indicates that tyrosine and lysine residues separated by 3 other
amino acids (YxxxK) reside on the same side of an α-helix (51,52), raising the possibility that
the phenolic group of tyrosine and the Nε amino group of lysine could come close enough to
interact chemically. Because apolipoprotein A-I contains only one tyrosine residue in a YxxxK
motif, it is difficult to assess whether lysine directs tyrosine chlorination by studying this
molecule. We therefore synthesized the model peptide AcPYSDELRQRLAARLE-NH2
(Yxxxxx), which duplicates helix 7 of apolipoprotein A-I. This helix contains tyrosine 166,
which was not chlorinated by HOCl under our experimental conditions (Table 1; peptide 161-
171) and does not reside in a YxxK/KxxY motif (Fig. 1). AcPYSDELRQRLAARLE-NH2 is
predicted to adopt an α-helical conformation ~ 40% of the time (56), suggesting that it should
approximate the secondary structure of the intact apolipoprotein.
After acetylating the peptide’s N-terminus to prevent the primary amine from forming a
chloramine, we exposed it to HOCl and quantified its chlorination by LC-ESI-MS. In parallel
studies, we attempted to mimic the lipid environment that envelops apolipoprotein A-I in HDL
by incubating the peptide with DPPC vesicles before exposing it to HOCl. When we exposed
Yxxxxx to HOCl in a physiological buffer for 30 min at 37 °C, we detected very little
chlorination of either the lipid-free or lipid-associated peptide (Fig. 11).
To explore the role of the YxxxK motif in promoting tyrosine chlorination, we prepared
variants of the Yxxxxx peptide. In this case, the lysine residue was separated from the tyrosine
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residue by 0, 1, 2, 3, or 4 amino acids (YKxxxx, YxKxxx, YxxKxx, YxxxKx, YxxxxK). After
we exposed these peptides to HOCl, LC-ESI-MS analysis revealed that YKxxxx, YxKxxx,
YxxKxx, and YxxxKx yielded progressively greater amounts of chlorinated peptide (Fig. 11).
The level of chlorination declined when the tyrosine and lysine residues were separated by 4
amino acids (YxxxxK). MS/MS analysis confirmed that tyrosine was always the site of
chlorination. When the peptides were lipid-associated, those containing YxxK or YxxxK motifs
were most susceptible to chlorination, perhaps because of increased α-helical structure. These
observations indicate that lysine residues permit the chlorination of tyrosine in synthetic peptides
that mimic a region of apolipoprotein A-I that is normally resistant to chlorination. Chlorination
of lipid-associated peptides was maximal when the lysine residue was 2 or 3 amino acid residues
away from the tyrosine residue.
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DISCUSSION
Our results provide strong evidence that lysine residues are involved in the regiospecific
formation of 3-chlorotyrosine when peptides or proteins containing tyrosine are exposed to
HOCl. They also indicate that chloramines are central to the reaction pathway. Thus, specific
amino acid sequences—YxxK and KxxY motifs—directed the regioselective chlorination of
synthetic peptides by HOCl in high yield. Moreover, the single major site of 3-chlorotyrosine
formation in apolipoprotein A-I of HDL resided in a YxxK motif. Remarkably, 50% of the
tyrosine residues in this YxxK motif were chlorinated when the mole ratio of HOCl to protein-
bound amino acid residues in HDL was 0.125, indicating that this region of the protein was
targeted selectively for oxidation. Collectively, these observations indicate that the proximity of
tyrosine to lysine, which is able to form chloramines, is likely to be one important factor that
determines the sites at which HOCl can chlorinate tyrosine residues in proteins.
We used synthetic peptides containing tyrosine to explore the mechanism by which
specific sequence motifs direct chlorination by HOCl. Because of our results with apolipoprotein
A-I and because primary amino groups form relatively stable chloramines, which can be potent
chlorinating reagents (37,38), we also included lysine residues in some of the peptides. When we
used the lysine-containing peptides YKxxY and YxxKY, the yield of 3-chlorotyrosine (relative
to HOCl) exceeded 65%. Moreover, a single tyrosine residue was preferentially chlorinated.
Significantly, a tyrosine residue located two amino acids away from a lysine residue was
chlorinated in much higher yield than a tyrosine immediately adjacent to a lysine. In contrast,
chlorination of tyrosine was not regiospecific in the peptide YxxxY, which lacked a lysine and
therefore a primary Nε amino group. Moreover, the product yield for chlorination of each
tyrosine residue in this peptide was <15%, demonstrating that YxxxY was a much poorer
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substrate for chlorination than the lysine-containing peptides. It is noteworthy that equal amounts
of 3-chlorotyrosine and 3,5-dichlorotyrosine formed in YxxxY (ClYxxxY, Cl2YxxxY, YxxxClY,
YxxxCl2Y) but that chlorination did not modify two different tyrosine residues in the same
molecule (ClYxxxClY). Also, we failed to detect tyrosine chlorination when we added HOCl to
a mixture of N-acetylated amino acids that mimicked the composition of the lysine-containing
YKxxY and YxxKY peptides. This suggests that lysine must be an integral part of a peptide to
direct the chlorination of tyrosine in high yield.
Primary amino groups react rapidly with HOCl to form chloramines (37,38,57).
Chloramines located on carbon atoms that lack a carboxylic acid group are long-lived and so
could be biologically relevant oxidizing intermediates (19,27,28,57). Indeed, our HPLC
procedure isolated a relatively stable oxidation product from the reaction of HOCl with peptide
YxxKY that was converted back to the precursor peptide by methionine. MALDI TOF-MS
analysis revealed the presence of three species in the methionine-sensitive oxidation product—
precursor peptide, monochloramine, and dichloramine. This suggests that molecular
decomposition and rearrangement during ionization by MALDI allow chlorine to be exchanged
among different species during the analysis (37,58). NMR analysis confirmed that the oxidized
intermediate was chlorinated on the Nε amino group of its lysine residue. Analysis of the
progress curve of the reaction of YxxKY with HOCl revealed that the initial step in the reaction
generated a high yield of peptide-bound chloramine, which disappeared as products containing
chlorinated tyrosine appeared. These observations provide strong evidence that the reaction
pathway for tyrosine chlorination involves the rapid initial formation of a chloramine on the ε
amino group of the lysine residue followed by a slower reaction in which the chloramine attacks
the aromatic ring of tyrosine. It is noteworthy that the stoichiometry of peptide chlorination
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implicates a dichloramine as the intermediate in the reaction pathway and that our NMR and MS
analyses detected dichloramines in the methionine-sensitive oxidation product. These
observations support the proposal that a dichloramine is the chlorinating intermediate in the
reaction pathway (58). Because the rate constant for the reaction of HOCl with lysine is
relatively high (K2 = 5 × 103 M-1s-1), the ε amino group of lysine (and the free amino group at the
N-terminus of proteins) together with the thiol residues of cysteine and methionine (K2 = 3.0
×107 M-1s-1 and 3.8 ×107 M-1s-1, respectively) might be initial primary targets when HOCl
oxidizes proteins (18,59).
It is noteworthy that 50% of the Tyr 192 residues in apolipoprotein A-I were chlorinated
when the mole ratio of HOCl to protein-bound amino acid residues of HDL was 1 to 8. The
reason why Tyr 192 is so much more amenable to chlorination than the two other tyrosine
residues (Tyr 115 and Tyr 236) that also reside in a YxxK domain might relate to differences in
spatial orientation, solvent accessibility, and the local amino acid environment. Importantly Tyr
115 lies in close proximity to Met 112. The alkylated thiol of methionine is extremely reactive
with HOCl, and protein-bound methionine residues have been proposed to act as local
antioxidants (60). Thus, Met 112 might scavenge HOCl in this region of apolipoprotein A-I.
Indeed, Met 112 is one of the primary targets for oxidation by HOCl in apolipoprotein A-I (34).
Another key factor controlling the reactivity of protein-bound amino acids is local
secondary structure. Apolipoprotein A-I contains 10 antiparallel amphipathic α-helices that
promote lipid association and are critical for removing cholesterol from cells (51,52). The helical
wheel representation of amphipathic helices predicts that tyrosine and lysine residues in the
YxxK (and KxxY) motif will lie next to each other on the same face of the α-helix (51). Both the
helical wheel representation (Fig. 1B) and the crystal structure of lipid-free apolipoprotein A-I
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(61) indicate that LAEYHAK, which includes the single tyrosine residue in apolipoprotein A-I
that was chlorinated in high yield (50%), contain the tyrosine and lysine residues in close
proximity. Importantly, the α-carbons of the two amino acid residues are separated by ~ 4.5 Å,
which suggests that the phenolic ring and primary amino groups of the tyrosine and lysine
residues in the YxxK/KxxY motif can interact chemically. The crystal structure of lipid-free
apolipoprotein A-I reveals that Tyr 192 lies in the middle of an amphipathic α-helix whereas Tyr
236 is located at the end of an amphipathic α-helix (61), where a less orderly structure might
hamper the interaction of the lysine and tyrosine residues and the production of 3-chlorotyrosine.
Molecular modeling indicates that tyrosine and lysine residues that are separated by 3
other amino acids also lie next to one another on the same face of an α-helix. Because their α-
carbons are separated by ~ 6 Å, their side chains might be able to interact chemically in
YxxxK/KxxxY motifs. However, only one tyrosine in apolipoprotein A-I resides in such a motif.
Therefore, it is difficult to use the intact molecule to explore the role of YxxxK/KxxxY in
tyrosine chlorination. Instead, we synthesized the model peptide AcPYSDELRQRLAARLE-NH2
(Yxxxxx) and exposed it to HOCl. In parallel studies, we determined whether lipid association
makes this peptide more amenable to chlorination. We selected this particular sequence because
it mimics a region of apolipoprotein A-I that contains a tyrosine residue that is resistant to
chlorination. When we exposed the peptide to HOCl, it was chlorinated in low yield.
When we introduced a lysine residue into the peptide at a position that was 2 or 3 amino acids
away from the tyrosine residue (YxxKxx and YxxxKx), the lipid-free and lipid-associated
peptides were chlorinated in high yield. These observations suggest that the YxxxK/KxxxY
motif might also promote the chlorination of tyrosine residues in α-helices. We also observed a
significant level of chlorination when the lysine and tyrosine residues were separated by 4 amino
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acids (YxxxxK) and therefore would be expected to lie on opposite sides of an α-helix. It is
possible that chlorination was feasible because the side chains were long enough to permit the
phenolic ring of tyrosine and the primary amino groups of lysine to interact. Alternatively, a
conformational change might have brought the two groups together—synthetic peptides are very
flexible, and the ones we studied are predicted to reside in an α-helix only ~ 40% of the time
(56). In future studies, it will be important to determine whether lysine residues promote the
chlorination of tyrosine residues when the two are separated by 3 or 4 amino acids in a well-
defined α-helical structure.
Primary sequence and secondary structure alone do not determine the local structure of a
protein, however, and many other factors could correctly orient the Nε-chloramine of lysine (or
an Nα chloramine of the N terminus of a protein) to the phenolic ring of tyrosine. For example,
the tertiary structure might juxtapose residues that lie far apart in the primary sequence or
secondary structure. It is even possible that lysine residues in one protein could facilitate the
chlorination of tyrosine residues in a different but closely associated protein. Features of tertiary
structure might also explain why Tyr 29 of apolipoprotein A-I was chlorinated even though it
does not reside in a YxxK motif and why Tyr 115, which does lie in a YxxK motif, was not
chlorinated. For example, Tyr 29 lies in a globular region of apolipoprotein A-I and might
interact with a primary amino group in another region of the protein. In addition, there might be
a difference in solvent accessibility or an association of the side chains of Tyr 115 or its adjacent
lysine with lipid.
Tyrosine192 and 236 are located in amphipathic helices 8 and 10 of the C-terminal of
apolipoprotein A-I; these helices are essential for interactions with lipid (52,62). It will be
interesting to determine whether chlorination of these tyrosines alters the biochemical or
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biophysical properties of apolipoprotein A-I and its ability to mobilize cholesterol from cells. It
also will be important to determine whether chlorination of specific protein residues by
phagocytes is important for killing bacteria or damaging host tissue even though only ~ 1 in
1,000-5,000 tyrosine residues is modified (13,14,63,64). Presumably, loss of specific tyrosine
residues that are critical to protein integrity or function could be deleterious to bacteria or host
tissues despite the small absolute level of tyrosine chlorination.
Based on our findings, we propose that the primary ε amino group of lysine facilitates the
regioselective chlorination of tyrosine residues in proteins by a pathway involving a chloramine
intermediate. Modeling and structural studies indicate that tyrosine and lysine residues separated
by two amino acids are adjacent on the same face of an α-helix, suggesting that the YxxK motif
could direct protein chlorination if it resided in a α-helix. Consistent with this proposal, we
found that a single tyrosine residue in the 8th amphipathic α-helix of apolipoprotein A-I was the
major site of chlorination by HOCl and that this tyrosine resided in the YxxK motif. Moreover,
additional structural features might also juxtapose primary amino groups and tyrosine residues in
proteins to permit site-specific chlorination of tyrosine residues. It will be of great interest to
determine whether regiospecific chlorination can affect protein function and to investigate the
possible role of this reaction mechanism in the chlorination of proteins in vivo.
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ACKNOWLEDGMENTS
This work was supported by grants AG021191 and HL64344 from the National Institutes of
Health.
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FIGURE LEGENDS
Figure 1. Apolipoprotein A-I. (A) Primary sequence of apolipoprotein A-I and its 10 proposed
amphipathic helices (65).(B) Helical wheel representation of tryptic fragment 189-195
LAEYHAK in apolipoprotein A-I. Note that the tyrosine and lysine residue in the YxxK motif
are located on the same face of the α-helix. Modeling studies and the crystal structure of
apolipoprotein A-I indicate that the α-carbons of tyrosine and lysine are located ~ 4.5 Å from
each other in the motif (51,61).
Figure 2. MS/MS identification of the major site of tyrosine chlorination in apolipoprotein
A-I exposed to HOCl. MS/MS analysis of [LAEYHAK + H]+ (m/z 831.4) and [LAEClYHAK +
H] + (m/z 865.4) in HDL oxidized with HOCl. HDL was exposed to HOCl (80:1, mol/mol,
oxidant:HDL particle) for 120 min at 37°C in PBS (pH 7.4). After terminating the reaction with
L-methionine, HDL proteins were digested with trypsin, and the tryptic digest peptides were
subjected to analysis by LC-ESI-MS/MS.
Figure 3. HPLC analysis of YKxxY, YxxKY, and YxxxY exposed to HOCl. Peptide (100
µM) was incubated for 30 min at 37oC in PBS (A) or PBS supplemented with 100 µM HOCl
(B). Reactions were initiated by adding oxidant and terminated by adding L-methionine. The
reaction mixture was analyzed by reverse-phase HPLC and UV detection at 215 nm.
Figure 4. MS/MS analysis of YxxKY peptide (AcGEYARKY) and its reaction products.
The peptide was oxidized with HOCl as described in the legend to Fig. 3 and analyzed by LC-
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ESI-MS/MS. A, MS/MS spectrum of precursor YxxKY. B, MS/MS spectrum of product Peak 1
(Fig. 3B). C, MS/MS spectrum of product Peak 2 (Fig. 3B).
Figure 5. Reaction requirements for tyrosine chlorination by HOCl. YxxKY (100 µM) was
oxidized by incubating the peptide with 100 µM HOCl in PBS for 60 min at 37 °C. At the end of
the incubation, the reaction was terminated with methionine, and the reaction products were
quantified using reverse-phase HPCL with monitoring of products at 215 nm. Where indicated,
the time of the reaction (A), the concentration of HOCl (B), or the pH of the reaction mixture (C)
were varied, or the indicated concentration of antioxidant (D) was included in the reaction
mixture prior to the addition of HOCl.
Figure 6. HPLC analysis of N-acetyl-tyrosine exposed to HOCl. N-acetyl-tyrosine (Ac-Tyr)
alone or Nα-acetylated amino acids (Ac-amino acid) mimicking the amino acid composition of
the YxxKY and YKxxY or YxxxY peptides were incubated with 100 µM HOCl in PBS for 60
min at 37oC. The reaction was terminated with L-methionine and the mixture was subjected to
HPLC analysis on a reverse-phase column with monitoring of products at 280 nm. (A) Ac-Tyr
alone (200 µM). (B) Amino acid composition of YxxxY (200 µM Ac-Tyr, 200 µM Ac-Glu, 100
µM Ac-Ala, 100 µM Ac-Gly, 100 µM Ac-Arg). (C) Amino acid composition of YKxxY and
YxxKY (200 µM Ac-Tyr, 100 µM Ac-Ala, 100 µM Ac-Glu, 100 µM Ac-Gly, 100 µM Ac-Lys,
100 µM Arg).
Figure 7. HPLC analysis of the methionine-sensitive reaction product formed in YxxKY
exposed to HOCl. (A) YxxKY peptide was oxidized with HOCl as described in the legend to
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Fig. 3 without the addition of methionine, and immediately subjected to HPLC analysis on a
reverse-phase column. The oxidized peptide eluting at 20 min was collected and reanalyzed by
reverse phase HPLC without further treatment (B) or was reduced with excess methionine and
then reanalyzed (C).
Figure 8. High-resolution 1H-NMR (A) and TOCSY (B) analysis of the methionine-sensitive
oxidation product formed in YxxKY exposed to HOCl. YxxKY peptide was oxidized with
HOCl as described in the legend to Fig.3, and its methionine-sensitive oxidation product was
isolated by HPLC on a reverse-phase column as described in Methods. Spectra were obtained on
precursor peptide or oxidized peptide solubilized in PBS (prepared with 10% D2O and 90% H2O,
pH 7.4).
Figure 9. MALDI TOF-MS and MS/MS analysis of the methionine-sensitive product
formed in YxxKY exposed to HOCl. YxxKY peptide was oxidized with HOCl as described in
the legend to Fig. 3, and its methionine-sensitive oxidation product was isolated by HPLC as
described in Methods. (A) The oxidation product was immediately analyzed by MALDI TOF-
MS using α-cyano-4-hydroxycinnamic acid as the matrix. MS/MS spectrum of (B) protonated
precursor peptide (ion of m/z 928.3, M + H), (C) protonated and chlorinated peptide (ion of m/z
962.2, M + H + 34 amu), (D) protonated and dichlorinated peptide (ion of m/z 996.2, M + H + 68
amu). Note that MS/MS analysis demonstrates the addition of 34 and 68 amu to the lysine
residue in the peptide.
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Figure 10. Progress curve of YxxKY oxidation by HOCl. YxxKY peptide was oxidized with
HOCl as described in the legend to Fig. 3 without the addition of methionine. The reaction
mixture was analyzed by reverse-phase HPLC and UV detection at 215 nm.
Figure 11. MS analysis of lipid-free and lipid-associated Yxxxxx, YxxxxK, YxxxKx,
YxxKxx, YxKxxx and YKxxxx exposed to HOCl. Peptide AcPYSDELRQRLAARLE-NH2
(Yxxxxx) mimics a region of apolipoprotein A-I that contains a tyrosine residue that is resistant
to chlorination (Table 1; peptide 161-171). Variants of the Yxxxxx peptide contained lysine
residues that were separated from the tyrosine residue by 0, 1, 2, 3, or 4 amino acids (YKxxxx,
YxKxxx, YxxKxx, YxxxKx, YxxxxK). Lipid-associated peptides were prepared by incubation
with DPPC vesicles (peptide:DPPC, 1:20, mol/mol). Peptides (25 µM) were exposed to HOCl
for 30 min at 37 oC in PBS. Reactions were initiated by adding oxidant and terminated by adding
L-methionine. The mol/mol ratios of peptide/oxidant were 0.5 and 1 for lipid-free and lipid-
associated peptides, respectively. The reaction mixture was analyzed by LC-ESI-MS. Peak areas
of the reconstructed ion chromatograms of ions at m/z corresponding to the native peptides [M +
2H]2+ and the chlorinated products [M + 34 + H]2+ were used for quantification. Results
represent the mean of two independent experiments.
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Table 1. LC-ESI-MS detection of peptides containing 3-chlorotyrosine in a tryptic digest of
HDL exposed to HOCl.
Residue Sequence Precursor, m/z Product, m/z
[M + H]+ [M + 2H]2+ [M + 34 + H]+ [M + 34 + 2H]2+ %Yield1
13-23 DLATVYVDVLK 1235.7 618.3 ud2 ud -
28-40 DYVSQFEGSALGK 1400.7 700.8 1434.7 717.8 2
97-106 VQPYLDDFQK 1252.6 626.8 ud ud -
108-116 WQEEMELYR* 1283.6 642.3 ud ud -
108-116 WQEEM(O)ELYR* 1299.6 650.3 ud ud -
161-171 THLAPYSDELR 1301.6 651.3 ud ud -
189-195 LAEYHAK 831.4 416.2 865.4 433.2 52
227-238 VSFLSALEEYTK 1386.7 693.9 1420.7 710.9 6
HDL was exposed to HOCl (80:1, mol/mol, oxidant:HDL particle) for 120 min at 37°C in PBS
(pH 7.4). A tryptic digest of HDL proteins was analyzed by LC-ESI-MS and MS/MS.
Chlorinated peptides were detected and quantified using reconstructed ion chromatograms of
precursor and product peptides. Peptide sequences were confirmed using MS/MS. Results are
representative of those found in three independent experiments.
1 (mol product peptide/mol precursor peptide) x 100
2 ud, undetectable
* HOCl oxidizes methionine (M) to methionine sulfoxide (34).
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Table 2. Formation of 3-chlorotyrosine and 3,5-dichlorotyrosine in YKxxY, YxxKY, and
YxxxY exposed to HOCl.
Motif Peptide Product Sequence % Product Yield1
YKxxY AcGYKRAYE Peak 1 AcGClYKRAYE 11
Peak 2 AcGYKRAClYE 60
YxxKY AcGEYARKY Peak 1 AcGEClYARKY 49
Peak 2 AcGEYARKClY 19
YxxxY AcGEYAREY Peak 1 AcGEClYAREY 10
Peak 2 AcGEYAREClY 11
Peak 3 AcGECl2YAREY 8
Peak 4 AcGEYARECl2Y 6
Peptides YKxxY, YxxKY, or YxxxY (100 µM) were incubated for 30 min at 37oC in PBS
supplemented with 100 µM HOCl. Reactions were initiated by adding oxidant and terminated by
adding methionine. Product yield was determined by HPLC with monitoring of A215. Peptides
were sequenced using LC-ESI-MS/MS.
1 (mol product peptide/mol HOCl) x 100
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0 25
Yxxxxx
YxxxxK
YxxxKx
YxxKxx
YxKxxx
YKxxxxM
otif
Chlorinated Product [%]
Lipid-freeLipid-associated
Fig. 11
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Constanze Bergt, Xiaoyun Fu, Nabiha P. Huq, Jeff L. Kao and Jay W. HeineckeA-I when hypochlorous acid oxidizes HDL
Lysine residues direct the chlorination of tyrosines in YxxK motifs of apolipoprotein
published online December 3, 2003J. Biol. Chem.
10.1074/jbc.M309046200Access the most updated version of this article at doi:
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