From the
Hypochlorous acid is the major strong oxidant generated by human
neutrophils, and it has the potential to cause much of the tissue
damage that these inflammatory cells promote. It is produced from
hydrogen peroxide and chloride by the heme enzyme myeloperoxidase. To
unequivocally establish that hypochlorous acid contributes to
inflammation, a stable and unique marker for its reaction with
biomolecules needs to be identified. In this investigation we have
found that reagent hypochlorous acid reacts with tyrosyl residues in
small peptides and converts them to chlorotyrosine. Purified
myeloperoxidase in combination with hydrogen peroxide and chloride, as
well as stimulated human neutrophils, chlorinated tyrosine in the
peptide Gly-Gly-Tyr-Arg. Rather than reacting directly with the
aromatic ring of tyrosine, hypochlorous acid initially reacted with an
amine group of the peptide to form a chloramine. The chloramine then
underwent an intramolecular reaction with the tyrosyl residue to
convert it to chlorotyrosine. This indicates that tyrosyl residues in
proteins that are close to amine groups will be susceptible to
chlorination. Peroxidases are the only enzymes capable of chlorinating
an aromatic ring. Furthermore, myeloperoxidase is the only human enzyme
that produces hypochlorous acid under physiological conditions.
Therefore, chlorotyrosine will be a specific marker for the production
of hypochlorous acid in vivo and for the involvement of
myeloperoxidase in inflammatory tissue damage.
Neutrophils and monocytes are implicated in the tissue damage
that occurs in numerous inflammatory pathologies, such as
atherosclerosis, adult respiratory distress syndrome, rheumatoid
arthritis, ischemia reperfusion injury, and inflammatory bowel
disease(1, 2, 3) . They may also promote cancers
that are associated with inflammation(4, 5) . In
numerous studies it has been proposed that oxidants generated by
neutrophils and monocytes cause the damage that these phagocytic cells
perpetrate(6) . However, to date there is a lack of convincing
evidence that oxidants cause tissue injury in vivo. This
arises principally because of the difficulties in detecting short-lived
oxidants. Consequently, specific markers of oxidants need to be found
to either confirm or refute their role in inflammation. Ideally, these
should be stable and unique reaction products of oxidatively modified
biomolecules.
Stimulated phagocytes generate vast amounts of
superoxide that dismutates to form hydrogen peroxide(7) . These
two oxidants are relatively benign but can give rise to more reactive
secondary oxidants including hypochlorous acid, hydroxyl radical, and
singlet oxygen(7) . Hypochlorous acid is produced from hydrogen
peroxide and chloride by the heme enzyme myeloperoxidase(8) ,
and it is the major strong oxidant generated by
neutrophils(9, 10, 11) . It is also produced in
appreciable amounts by monocytes(12) . Hypochlorous acid reacts
most rapidly with thiols and thioethers, but the products are not
unique to this oxidant (13, 14). Hypochlorous acid also reacts readily
with amines to form chloramines(15) ; however, they are unstable
and decay to form aldehydes or nitriles(16) .
For a useful
marker to be derived from hypochlorous acid, a stable
carbon-chlorine bond must be generated. Reagent hypochlorous acid
and that derived from myeloperoxidase, hydrogen peroxide, and chloride,
chlorinate unsaturated fatty acids and cholesterol to form
chlorohydrins(17) . The chlorine atom of hypochlorous acid is
also incorporated into proteins(18) . The amino acids that are
most likely to be chlorinated are tyrosine, tryptophan, and
histidine(19) . There has been extensive work on the iodination
of tyrosine, mainly involving thyroid peroxidase in the production of
thyroid hormones(20) . Peroxidases, including myeloperoxidase,
also brominate tyrosyl residues of proteins(21) . Chlorination
of tyrosine has received little attention. At nonphysiological pH,
chloroperoxidase chlorinates free tyrosine (22) and tyrosyl
residues in barley
This study was undertaken to determine the products formed when
hypochlorous acid reacts with tyrosine in peptides. The ability of
purified myeloperoxidase and stimulated neutrophils to chlorinate
tyrosyl residues was also investigated. We show that reagent
hypochlorous acid and that generated by myeloperoxidase and stimulated
neutrophils chlorinate tyrosyl residues to produce chlorotyrosine. We
propose that chlorotyrosine should prove to be a specific marker for
hypochlorous acid production during inflammation.
Myeloperoxidase was purified from human leukocytes as
described previously(25) . Its purity index
(A
Neutrophils were isolated from the blood of healthy donors by
Ficoll-Hypaque centrifugation, dextran sedimentation, and hypotonic
lysis of red cells(30) . Cell preparations contained
95-97% neutrophils. After isolation, neutrophils were resuspended
in phosphate-buffered saline (PBS)
Production of superoxide by neutrophils stimulated with
phorbol myristate acetate (100 ng/ml) was determined by measuring
superoxide dismutase-inhibitable cytochrome c reduction(32) . To determine hypochlorous acid production,
cells were stimulated in the presence of 15 mM taurine, and
the formation of taurine chloramine was measured by its ability to
oxidize 2-nitro-5-thiobenzoate(27) .
Peptides were dissolved in PBS, and hypochlorous acid was
added while vortexing the solution. Reacted peptides were normally left
at room temperature for at least 2 hours before analysis. To follow the
time course of chlorination, samples were removed at intervals, added
to 200 µM dithiothreitol to scavenge any chloramines, and
then subjected to amino acid analysis. Gly-Gly-Tyr-Arg was also
chlorinated with myeloperoxidase, hydrogen peroxide, and chloride.
Reactions were run at 20 °C and started by adding hydrogen peroxide
to PBS containing myeloperoxidase and peptide. To prevent inactivation
of myeloperoxidase, hydrogen peroxide was added in 50-µM aliquots at 10-min intervals. After an hour of incubation,
peptides were subjected to amino acid analysis.
Neutrophils (2
Chloramines of taurine, glycine and lysine were formed by
adding 100 µM hypochlorous acid to an equivalent
concentration of the amines in PBS. After 5 min, Gly-Gly-Tyr-Arg was
added to each chloramine, and the mixture was incubated at room
temperature for 60 min. Residual chloramines and amino acid composition
were then determined.
Peptides were hydrolyzed under vacuum for 12 h at 100 °C
in the presence of rapidly boiling hydrochloric acid with 1% phenol and
then subjected to amino acid analysis by the method outlined by Cohen
and Strydom(33) . Concentrations of tyrosine and chlorotyrosine
in hydrolyzed peptides were calculated by comparing their peak heights
in HPLC chromatograms with those obtained for a series of standards of
known concentration. Free tyrosine and chlorotyrosine were analyzed by
reverse phase HPLC with diode array detection. Analysis was performed
using a 220
Chloramines were determined by adding 2-nitro-5-thiobenzoate
and measuring the loss in absorbance at 412 nm (
The peptides were also examined
by electrospray mass spectrometry. The [M+H]
With the doubly charged
[M+2H]
Using amino acid analysis and mass spectrometry to identify
products, we have shown that reagent hypochlorous acid converts tyrosyl
residues in several peptides to chlorotyrosine. It is likely that the
isomer formed is 3-chlorotyrosine, because the hydroxyl group on the
aromatic ring of tyrosine activates the ortho positions for
substitution. The para position is also activated, but it is blocked
for substitution. With each peptide, chlorotyrosine was the major
product and accounted for at least 50% of the reacted tyrosine. In
contrast, free tyrosine was not converted to chlorotyrosine, which is
in agreement with the findings of an earlier study(24) .
Conversion of tyrosyl residues to chlorotyrosine is likely to be
physiological possible, because purified myeloperoxidase and that
released by stimulated neutrophils catalyzed chlorination of tyrosine
in Gly-Gly-Tyr-Arg. Our evidence for the requirement of myeloperoxidase
in chlorination by neutrophils was that the reaction was blocked by
catalase, which scavenges hydrogen peroxide, and by the heme poison,
azide. In addition, neutrophils from a myeloperoxidase-deficient
individual did not chlorinate the tetrapeptide. Based on our finding
that several unrelated peptides were chlorinated (),
formation of chlorotyrosyl residues is unlikely to be restricted to a
few peptides and proteins that are uniquely susceptible to
chlorination. Rather, it is likely that numerous peptides and proteins
could be chlorinated by myeloperoxidase, so that chlorotyrosine may
prove to be an ideal marker for inflammatory reactions of neutrophils.
In support of this proposal, hypochlorous acid chlorinates tyrosine in
globin.
Hypochlorous acid initially
converted an amine group on Gly-Gly-Tyr-Arg to a chloramine, which
decayed considerably faster than glycine chloramine or tyrosine
chloramine. As the chloramine of the tetrapeptide decayed, there was a
corresponding loss of tyrosine and formation of chlorotyrosine. From
these results we conclude that the chloramine on the tetrapeptide
chlorinates the tyrosine ring.
Chlorination appears to involve an
intramolecular rearrangement, because the chloramines of lysine,
glycine, or taurine were unable to chlorinate Gly-Gly-Tyr-Arg. A
similar rearrangement involving a chloramine intermediate occurs when
hypochlorous acid reacts with procainamide to form
3-chloroprocainamide(36) . It is also known that aromatic
chloramines rearrange in the presence of hydrochloric acid to give ring
chlorinated products(37) . These Orton rearrangments are
intermolecular reactions in which molecular chlorine is liberated from
the chloramine and then reacts with activated aromatic
rings(37) . It is unlikely that this type of rearrangement
explains the chlorination of the tetrapeptide, because the reaction
conditions used would not favor release of molecular chlorine.
Molecular modelling of Gly-Gly-Tyr-Arg revealed that in the
energy-minimized structure, the terminal amino group can come within 3
Å of the tyrosine ring. Thus, a possible mechanism could involve
homolysis of the nitrogen-chlorine bond of the terminal glycine
chloramine, facilitated by addition of the chlorine atom to the
juxtaposed tyrosine ring. In support of this proposal, homolysis of the
nitrogen-chlorine bond has been shown to occur in the
decomposition of N-chloroamides and adenosine
chloramine(38, 39) . It has also been suggested to occur
when hypochlorous acid reacts with aminopyrine to form a chloramine,
which subsequently gives rise to an aminopyrine radical(40) .
In a related reaction to what we report here, the tyrosyl residue in
the active site of D-amino acid oxidase is chlorinated by
chloramines(41) . The reaction is highly specific in that N-chloro derivatives of D-leucine, D-isoleucine, and D-norvaline are reactive, whereas
hypochlorous acid, monochloramine, N-chloro-D-alanine, and N-chloro-D-valine do not chlorinate the tyrosyl
residue. It was proposed that, given the structural specificity of this
reaction and that N-chloro-D-leucine is a relatively
unreactive chlorine donor, chlorination is likely to involve an
orientated intramolecular electrophilic substitution between the bound
chloramine and the tyrosyl residue at the active site. The possibility
of a free radical process, as we have suggested, was not excluded.
At high levels of hypochlorous acid, formation of chlorotyrosine
declined. This could result from conversion of chloramines to more
unstable dichloramines(16) , further ring chlorination to
dichlorotyrosine, or hypochlorous acid reacting with amide groups to
promote peptide cleavage(42) .
Chloramine formation from
amino groups is one of the more favored reactions of hypochlorous acid (13, 14) and has been shown to occur when neutrophils
are stimulated(15) . Amines are readily regenerated by reducing
agents such as thiols, ascorbate, and methionine, so that the
relatively slow rearrangement to form chlorotyrosine would be in
competition with these faster reactions. This is evident from the
ability of dithi- othreitol to stop chlorination of tyrosyl residues
when it was added to the chloramine of Gly-Gly-Tyr-Arg (Fig. 5).
However, stimulated neutrophils and monocytes are likely to deplete
reducing species in their immediate environment, allowing the
chlorination of tyrosine to occur.
Halogenation of aromatic rings is
an enzymatic activity that is unique to peroxidases(43) , and
myeloperoxidase is the only human enzyme capable of producing
hypochlorous acid in vivo(43) . Eosinophil peroxidase
does not oxidize chloride to hypochlorous acid under physiological
conditions(44, 45) . This ensures that chlorotyrosine
will be a specific marker for production of hypochlorous acid by
myeloperoxidase. The detection of chlorotyrosine in biological samples
would establish that myeloperoxidase produces hypochlorous acid in
vivo and enable assessment of the contribution that this potent
oxidant makes to the tissue injury of various inflammatory pathologies.
Peptides and tyrosine were dissolved in PBS, and
100 µM hypochlorous was added. The data are representative
of two experiments. ND, not detected.
The untreated peptide Gly-Gly-Tyr-Arg (GGYR), or that
treated with equimolar hypochlorous acid, was analyzed by mass
spectrometry as outlined under ``Experimental Procedures.''
The measured values are the result of one or two injections of the
sample. When two values were obtained, the values were averaged. All
measured values were within ±0.4 of the calculated mass.
[Y]
We thank Dr. Maurice Owen for assistance with the
amino acid analysis.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-amylase(23) . Hypochlorous acid reacts
with free tyrosine to produce a ring chlorinated nitrile
derivative(24) , but, as far as we are aware, its reaction with
tyrosyl residues in peptides and proteins has not been investigated.
Materials
/A
) was greater than 0.72 and its
concentration was determined using
= 91,000 M
cm
/heme(26) .
Peptides, amino acids, phorbol myristate acetate, bovine liver
catalase, bovine erythrocyte superoxide dismutase,
5,5`-dithiobis(2-nitrobenzoic acid), and monochlorodimedon were
purchased from the Sigma. 5-Thio-2-nitrobenzoic acid was prepared from
5,5-dithiobis(2-nitrobenzoic acid) as described previously(27) .
Enzymes used for peptide hydrolysis were obtained from Boehringer
Mannheim (Sydney, Australia). Hydrogen peroxide solutions were prepared
daily by diluting a 30% stock and calculating its concentration using
= 43.6 M
cm
(28) . Hypochlorous acid was
purchased from Reckitt and Colman (NZ) Ltd. (Auckland, NZ). Its
concentration was determined by measuring the loss in absorbance at 290
nm after reacting it with monochlorodimedon (
= 19,000 M
cm
)(29) .
Isolation of Human Neutrophils
(
)(10.0 mM sodium phosphate buffer, pH 7.4, with 140 mM sodium
chloride) containing 1.0 mM calcium chloride, 0.5 mM
magnesium chloride, and 1 mg/ml of glucose. Neutrophils were also
isolated from the blood of a myeloperoxidase-deficient individual,
whose cells have been shown to have approximately 5% of normal
peroxidase activity and produce hypochlorous acid at less than 5% of
the levels generated by normal neutrophils(31) .
Oxidant Production by Neutrophils
Chlorination of Peptides in Cell Free Systems
Chlorination of Gly-Gly-Tyr-Arg by Neutrophils
10
/ml) were suspended in
PBS containing 1.0 mM calcium chloride, 0.5 mM magnesium chloride, 1 mg/ml of glucose, and 300 µM Gly-Gly-Tyr-Arg and stimulated with 100 ng/ml of phorbol myristate
acetate. After 1 h at 37 °C, cells were pelleted by centrifugation,
and the supernatant was removed for amino acid analysis.
Reaction of Chloramines with Gly-Gly-Tyr-Arg
Amino Acid Analysis
4.6 mm Spheri 5 octadecylsilane 5-µm column and
an isocratic mobile phase of water/methanol/acetic acid (60:40:1).
Analysis of Chloramine Formation
= 14,100 M
cm
)(27) . To confirm that oxidation of
2-nitro-5-thiobenzoate was due the chloramine of Gly-Gly-Tyr-Arg and
not unreacted hypochlorous acid, monochlorodimedon, which reacts
rapidly with hypochlorous acid but not with chloramines(34) ,
was added to hypochlorous acid 1 min after the peptide. There was no
loss of monochlorodimedon, indicating that all the hypochlorous acid
had already reacted (data not shown).
Mass Spectrometry
MALDI-tof Mass Spectrometry
Molecular weights of
the untreated peptide and the peptide treated with reagent hypochlorous
acid were determined by MALDI-tof mass spectrometry on a Lasermat
(Finnigan MAT, San Jose, CA). The matrix for the thermolytic digest was
2,5-dihydroxybenzoic acid (10 mg/ml), and the matrix for the
chymotryptic A4 digest was -cyano-4-cinnamic acid (5 mg/ml). Both
matrices were prepared in acetonitrile/water/trifluoroacetic acid
(35:65:0.1) (v/v). A two-point internal calibration using a matrix ion
and the [M+H]
ion for metenkephalin was
used to improve mass accuracy for the thermolysin digests. All spectra
are the averages of five laser shots/slide. Samples were prepared by
adding the matrix solution (
0.5 µl) and a solution of the
peptide sample (
0.1 µl) to stainless steel Lasermat targets.
Samples were dried at room temperature and then placed in the
instrument.
Enzyme Digests
For the thermolysin digest, the
untreated peptide (4.4 nmol) and the peptide treated (
2.1
nmol) with reagent hypochlorous acid were dried, reconstituted in 10
mM Tris-HCl buffer, pH 8.0, containing 10 mM calcium
chloride, and incubated with thermolysin (0.022 nmol) in a final volume
of 4.5 µl at 37 °C for 30 min. Samples were stored at 4 °C
for 2 days before analysis. For the chymotrypsin A4 digest, the
untreated (
8.8 nmol) and treated (
4.2 nmol) peptides were
reconstituted in 10 mM Tris-HCl buffer, pH 8.2, and incubated
with chymotrypsin (0.04 nmol) in a final volume of 3.5 µl at 37
°C for 14 h. For both digests, control samples that contained the
appropriate enzyme and buffer but no peptide were also analyzed.
Electrospray Ionization Mass
Spectrometry
Electrospray mass spectrometry was performed on a
TSQ 7000 mass spectrometer (Finnigan MAT). Untreated peptide (4.4
nmol) and peptide treated with reagent hypochlorous acid (
2.1
nmol) were introduced by injection into a fixed loop (5 µl). The
mobile phase was methanol/water/acetic acid (50:50:1) (v/v) pumped by a
Series II 1090 liquid chromatograph (Hewlett-Packard Co., Palo Alto,
CA) at 200 µl/min. Mass spectra were recorded for the treated and
untreated peptides, and mass spectrometry/mass spectrometry product ion
spectra of [M+H]
(m/z 50-500) and [M+2H]
(m/z 50-280) were also recorded. The
collision gas was methane (4 millitorr), and the collision energy was
-100 eV. All mass spectra were averaged across the peak, and the
background was subtracted.
Reaction of Hypochlorous Acid with Peptides Containing
Tyrosine
When the peptide Gly-Gly-Tyr-Arg was reacted with
equimolar reagent hypochlorous acid, there was a loss in its tyrosine
content, whereas the amounts of glycine and arginine present were
unaffected (Fig. 1, A and B). Concomitant with
the loss in tyrosine, there was production of a novel compound that
coeluted with 3-chlorotyrosine on HPLC analysis (Fig. 1B). Addition of hypochlorous acid to three other
peptides caused a similar loss in tyrosine and formation of
chlorotyrosine (). The yield of chlorotyrosine ranged from
30 to 55% of the hypochlorous acid added, and 50-75% of the
reacted tyrosine was accounted for as chlorotyrosine. When hypochlorous
acid was added to free tyrosine, there was no detectable formation of
chlorotyrosine, although 60% of the amino acid had reacted (). Addition of hypochlorous acid to Gly-Gly-Tyr-Arg up to
a concentration of 150 µM resulted in increasing formation
of chlorotyrosine (Fig. 2). The yield of chlorotyrosine was
approximately 60%. At greater than 150 µM hypochlorous
acid, there was a progressive decline in the concentration of
chlorotyrosine detected.
Figure 1:
Amino acid analysis of hydrolyzed
Gly-Gly-Tyr-Arg before and after reaction with hypochlorous acid. A, the untreated peptide Gly-Gly-Tyr-Arg (100
µM). B, peptide treated with 50 µM hypochlorous acid in PBS. Cl Tyr,
chlorotyrosine.
Figure 2:
Reaction of Gly-Gly-Tyr-Arg with reagent
hypochlorous acid or myeloperoxidase/hydrogen peroxide/chloride.
Gly-Gly-Tyr-Arg (100 µM) was either reacted with varying
concentrations of hypochlorous acid in PBS () or incubated in PBS
at 20 °C with 20 nM myeloperoxidase, and chlorination was
started by adding varying concentrations of hydrogen peroxide (
).
After 2 h the peptide was hydrolyzed, and the production of
chlorotyrosine was determined by amino acid analysis. Data are
representative of two experiments.
Gly-Gly-Tyr-Arg was also chlorinated with
purified myeloperoxidase, hydrogen peroxide, and chloride. There was no
chlorination in the absence of enzyme, hydrogen peroxide, or chloride.
Production of chlorotyrosine increased with increasing concentrations
of hydrogen peroxide up to a maximum of 200 µM (Fig. 2). With 100 µM hydrogen peroxide, about
45% of the hydrogen peroxide was accounted for by the formation of
chlorotyrosine. Above 200 µM hydrogen peroxide, the
production of chlorotyrosine declined.
Analysis of Chlorinated Gly-Gly-Tyr-Arg by Mass
Spectrometry
Direct MALDI-tof analysis of the untreated and
treated Gly-Gly-Tyr-Arg confirmed the presence of one chlorine atom in
the treated sample (). To locate the site of substitution,
fragments of the peptide were generated by the addition of thermolysin,
which should cleave the Gly-Tyr bond, and the masses were
determined by MALDI-tof. Ion abundances for the intact peptides
decreased over time, and new species appeared (Fig. 3). For the
untreated peptide m/z 338.8 corresponds to
[YR+H], and for the treated peptide m/z 373.3 corresponds to
[YR-Cl+H]
(Fig. 3). The mass
difference of 34.5 Da is accounted for by the substitution of a proton
for a chlorine atom. All other ions in the spectra were derived from
either the matrix, the buffer, or the internal calibrant, metenkephalin (Fig. 3, m, b, and me, respectively).
Figure 3:
MALDI-tof mass spectra of thermolysin
digests of untreated Gly-Gly-Tyr-Arg (A) or peptide treated
with equimolar hypochlorous acid (B). For details see
``Experimental Procedures.'' b, buffer; m,
matrix; me, metenkephalin.
To establish whether the site of chlorine substitution was on the
tyrosine or arginine residue, the peptides were subjected to digestion
with chymotrypsin A4 to remove the C-terminal arginine residue.
Following digestion, the untreated and treated peptides showed the
presence of a species at m/z 175. This corresponds to
arginine ([R+H] calculated mass,
175.2). From these data it is apparent that both peptides contain an
unmodified C-terminal arginine residue. Spectra from control slides
confirmed that the m/z 175 ions were derived from the
peptides. From these results it is apparent that chlorine was
substituted on the tyrosyl residue.
and [M+2H]
ions were selected as
parent ions (at Q1), and the product ion spectra (Q3) were recorded.
The fragmentation pattern was entirely consistent with the presence of
a single chlorine substitution on the tyrosine residue. With
[M+H]
ions selected at Q1, the most
abundant ions observed were at m/z 136.0 for the
untreated sample and m/z 170.0 for the treated sample (I). These molecular masses are probably derived from the
immonium ion of the tyrosyl residue, and the mass difference of 34.0 is
consistent with its chlorination(35) . Arginine,
[R+H]
, was detected at m/z 175 in both the treated and untreated samples with similar
relative abundances. No ions characteristic of chlorinated arginine
were detected.
ion at Q1, in addition to the
immonium ions, two other ions consistent with the fragmentation of the
tyrosyl residue were detected (I). In the untreated sample m/z 107.0 is probably derived from the p-methylphenoxy ion of tyrosine. The corresponding chlorinated
ion, m/z 140.8, was detected in the treated sample.
In the untreated sample the ion m/z 91.1 arising from the benzyl moiety
of tyrosine was observed. The corresponding chlorinated ion, m/z 124.8, was detected in the treated sample.
The Time Course of Gly-Gly-Tyr-Arg
Chlorination
Maximum formation of chlorotyrosine and loss of
tyrosine occurred 2 h after adding hypochlorous acid to Gly-Gly-Tyr-Arg (Fig. 4). This was preceded by formation of peptide chloramine,
which occurred in the first minute of the reaction and accounted for
all the hypochlorous acid (Fig. 5). The chloramine was unstable,
and its decay mirrored the loss in the tyrosine content of the
tetrapeptide. Hypochlorous acid also reacted quantitatively with
tyrosine and glycine to convert them to chloramines (Fig. 5).
Although tyrosine chloramine was also relatively unstable, it was not
converted to chlorotyrosine (). Glycine chloramine, in
contrast to the other chloramines, was very stable, and only 20% of it
decayed over 2 h. From these results it is apparent that hypochlorous
acid initially reacts with an amine group of Gly-Gly-Tyr-Arg to form a
chloramine that then chlorinates the tyrosine ring.
Figure 4:
The time course for the reaction of
Gly-Gly-Tyr-Arg with hypochlorous acid. Gly-Gly-Tyr-Arg (100
µM) was dissolved in PBS at 20 °C and reacted with
HOCl (100 µM). The reaction was terminated at the
indicated times by adding 200 µM dithiothreitol, and the
peptide was then hydrolyzed for analysis of tyrosine () and
chlorotyrosine (
). Data are representative of two
experiments.
Figure 5:
The time course for the decay of
chloramines. Hypochlorous acid (100 µM) was added to
either 100 µM of Gly-Gly-Tyr-Arg (), tyrosine
(
), or glycine (
). The concentration of chloramine present
was determined at the indicated time points by withdrawing an aliquot
and adding it to 5-thio-2-nitrobenzoic acid. Other reaction conditions
were as described in Fig. 4. Data are representative of two
experiments.
Chloramines of
taurine, glycine, and lysine were unable to chlorinate the tyrosyl
residue in Gly-Gly-Tyr-Arg. They lost 0, 6, and 15%, respectively, of
their reactivity toward 2-nitro-5-thiobenzoate when incubated alone for
1 h. For each chloramine, only about 20% of its reactivity was lost
after an hour when it was added to the peptide, and there was no
detectable formation of chlorotyrosine (results not shown). For
chloramine determinations, duplicate experiments agreed within 2
µM. From these results we conclude that the intermolecular
reaction of chloramines with tyrosyl residues is not favored.
Chlorination of Gly-Gly-Tyr-Arg by
Neutrophils
When human neutrophils were stimulated with phorbol
myristate acetate in the presence of Gly-Gly-Tyr-Arg, they produced
approximately 3 µM chlorotyrosine/10 cells in
the first 10 min of the reaction. Under the conditions of the assay,
neutrophils generate hydrogen peroxide at about 3
µM/min/10
cells(11) . Thus 10% of the
hydrogen peroxide was accounted for by the production of
chlorotyrosine. Formation of chlorotyrosine was enhanced by
myeloperoxidase and superoxide dismutase and blocked by catalase (Fig. 6). It was fully inhibited by azide and did not occur with
myeloperoxidase-deficient neutrophils, indicating that chlorination of
the tyrosyl residue was catalyzed by myeloperoxidase. Enhancement of
chlorination by superoxide dismutase is consistent with the ability of
this enzyme to cause about a 20% increase in hypochlorous acid
production by neutrophils(11) .
Figure 6:
Chlorination of Gly-Gly-Tyr-Arg by
stimulated human neutrophils. Neutrophils (2 10
/ml)
were suspended in PBS (pH 7.4) at 37 °C containing 300 µM Gly-Gly-Tyr-Arg, 0.5 mM MgCl
, 1 mM
CaCl
, and 1 mg/ml glucose. Cells were stimulated with 100
ng/ml of phorbol myristate acetate (PMA) in the presence or
absence of 20 µg/ml of superoxide dismutase (+ SOD),
100 nM myeloperoxidase (+ MPO), 1 mM azide (+ Azide), or 20 µg/ml of catalase (+ Catalase). After 1 h, cells were pelleted and the
supernatants were subjected to amino acid analysis. Results are
expressed as means and ranges of duplicate experiments and are
representative of those obtained from the cells of three individuals.
The results for the myeloperoxidase deficient neutrophils were obtained
once from a single individual.
(
)
Table: Reactivity of tyrosine and peptides with
hypochlorous acid
Table: 0p4in
Value for chymotrypsin
A4 digests is the mean of seven aliquots.
Table: Electrospray mass spectrometry of peptide
fragments
(imm), tyrosine immonium ion;
[Y]
(phen), p-methylphenoxy ion; and
[Y]
(benzyl), benzyl ion.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.