Site-specific hypochlorous acid-induced oxidation of recombinant human myoglobin affects specific amino acid residues and the rate of cytochrome b5-mediated heme reduction

Article abstract of DOI:10.1016/j.freeradbiomed.2009.09.023

Myeloperoxidase catalyzes the reaction of chloride ions with H₂O₂ to yield hypochlorous acid (HOCl), which can damage proteins. Human myoglobin (HMb) differs from other Mbs by the presence of a cysteine residue at position 110 (Cys110). This study has (i) compared wild-type and a Cys110Ala variant of HMb to assess the influence of Cys110 on HOCl-induced amino acid modification and (ii) determined whether HOCl oxidation of HMb affects the rate of ferric heme reduction by cytochrome b₅. For wild-type HMb (HOCl:Mb ratio of 5:1?mol:mol), Cys110 was preferentially oxidized to a homodimeric or cysteic acid product-sulfenic/sulfinic acids were not detected. At a HOCl:Mb ratio 10:1?mol:mol, methionine (Met) oxidation was detected, and this was enhanced in the Cys110Ala variant. Tryptophan (Trp) oxidation was detected only in the Cys110Ala variant at the highest HOCl dose tested, with oxidation susceptibility following the order Cys > Met > Trp. Tyrosine chlorination was evident only in reactions between HOCl and the Cys110Ala variant and at a longer incubation time (24?h), consistent with the formation via chlorine-transfer reactions from preformed chloramines. HOCl-mediated oxidation of wild-type HMb resulted in a dose-dependent decrease in the observed rate constant for ferric heme reduction (approx two-fold at HOCl:Mb of 10:1?mol:mol). These data indicate that Cys110 influences the oxidation of HMb by HOCl and that oxidation of Cys, Met, and Trp residues is associated with a decrease in the one-electron reduction of ferric HMb by other proteins; such heme-Fe³⁺ reduction is critical to the maintenance of function as an oxygen storage protein in tissues. Crown Copyright

Full text of DOI:10.1016/j.freeradbiomed.2009.09.023

Free Radical Biology & Medicine 48 (2010) 35–46
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Free Radical Biology & Medicine
journal homepage: www.elsevier.com/locate/freeradbiomed
Original Contribution
Site-specific hypochlorous acid-induced oxidation of recombinant human myoglobin
affects specific amino acid residues and the rate of cytochrome b5-mediated
heme reduction
,1
Andrea J. Szuchman-Sapir a, David I. Pattison b, Michael J. Davies b,c, Paul K. Witting a,c,
⁎
a
Vascular Biology Group, ANZAC Research Institute, Concord Repatriation General Hospital, Concord, NSW 2139, Australia
The Heart Research Institute, Sydney, NSW 2050, Australia
b
c
The Faculty of Medicine, The University of Sydney, Sydney, NSW 2006, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Myeloperoxidase catalyzes the reaction of chloride ions with H2O2 to yield hypochlorous acid (HOCl), which
can damage proteins. Human myoglobin (HMb) differs from other Mbs by the presence of a cysteine residue
at position 110 (Cys110). This study has (i) compared wild-type and a Cys110Ala variant of HMb to assess
the influence of Cys110 on HOCl-induced amino acid modification and (ii) determined whether HOCl
oxidation of HMb affects the rate of ferric heme reduction by cytochrome b5. For wild-type HMb (HOCl:Mb
ratio of 5:1 mol:mol), Cys110 was preferentially oxidized to a homodimeric or cysteic acid product—
sulfenic/sulfinic acids were not detected. At a HOCl:Mb ratio 10:1 mol:mol, methionine (Met) oxidation was
detected, and this was enhanced in the Cys110Ala variant. Tryptophan (Trp) oxidation was detected only in
the Cys110Ala variant at the highest HOCl dose tested, with oxidation susceptibility following the order
CysNMetNTrp. Tyrosine chlorination was evident only in reactions between HOCl and the Cys110Ala variant
and at a longer incubation time (24 h), consistent with the formation via chlorine-transfer reactions from
preformed chloramines. HOCl-mediated oxidation of wild-type HMb resulted in a dose-dependent decrease
in the observed rate constant for ferric heme reduction (approx two-fold at HOCl:Mb of 10:1 mol:mol).
These data indicate that Cys110 influences the oxidation of HMb by HOCl and that oxidation of Cys, Met, and
Trp residues is associated with a decrease in the one-electron reduction of ferric HMb by other proteins; such
heme-Fe3+ reduction is critical to the maintenance of function as an oxygen storage protein in tissues.
Crown Copyright © 2009 Published by Elsevier Inc. All rights reserved.
Received 15 April 2009
Revised 12 August 2009
Accepted 23 September 2009
Available online 2 October 2009
Keywords:
Myoglobin
Amino acid modification
Hypochlorous acid
Protein oxidation
Heme reduction
MetMb reductase
Free radicals
Peroxidase enzymes play an important role in host defense against
infection [1], with these enzymes catalyzing the formation of a range
of oxidant species. For example, the potent two-electron oxidant
hypochlorous acid (HOCl) is generated enzymatically from hydrogen
peroxide (H2O2) and chloride ions by the action of myeloperoxidase
[2]. The rapid production of HOCl by immune cells [3] is implicated in
the bactericidal and cytotoxic responses [4] that form part of the host
defense system.
Proteins represent a major target for HOCl, resulting in modifications
to protein side chains and, to a lesser extent, backbone fragmentation and
covalent cross-linking [5,6]. Oxidative reactions of HOCl with amino acids
and peptides have been widely studied (e.g., [7–9]). Reagent HOCl readily
oxidizes sulfur-containing amino acids (cysteine (Cys), cystine, and
methionine (Met) [10]) as well as histidine (His), lysine (Lys), tryptophan
(Trp), and tyrosine (Tyr) residues, resulting in protein unfolding [11] and,
in some cases, diminished or altered protein function [12–14].
Reaction of HOCl with Met and Cys residues occurs rapidly with
second-order rate constants, k, of approximately 3.0×107 M−1 s−1
[5]. Met sulfoxide is a major product of Met oxidation, whereas
disulfides and cysteic acid are stable end products derived from Cys
oxidation [9]. These Cys-derived products are generated via unstable
intermediates, including the sulfenyl chloride, and sulfenic and
sulfinic acids (in the case of cysteic acid) [6]. These intermediates
can be reduced by biological reductants to regenerate the free thiol
group on the Cys residue. This reversible Cys oxidation has been
implicated as a key step in the regulation of enzyme catalysis [15].
Sulfenamide, sulfinamide, and sulfonamide cross-links have also been
reported as products of HOCl-mediated oxidation in some proteins
[10,16]. Unstable chloramines are known to be generated on reaction
of HOCl with Lys and His side chains [17,18] and decomposition of
these species can generate aldehydes and ketones [7] or nitrogen-
centered radicals [19,20] that can induce secondary damage.
Abbreviations: DTT, dithiothreitol; HMb, wild type human Mb; C110A, Cys110Ala
variant of human Mb; Mb, myoglobin; oxyMb, oxidized Mb; MPO, myeloperoxidase;
PBS, phosphate buffered saline.
⁎
Corresponding author. Fax: 61 2 9767 9101.
E-mail address: pwitting@med.usyd.edu.au (P.K. Witting).
Current address: Discipline of Pathology, Bosch Research Institute Faculty of
1
Oxidation of Trp side chains by HOCl may occur through at least
two mechanisms. The first involves direct HOCl-mediated oxidation of
Medicine, The University of Sydney, NSW 2006, Australia.
0891-5849/$ – see front matter. Crown Copyright © 2009 Published by Elsevier Inc. All rights reserved.
doi:10.1016/j.freeradbiomed.2009.09.023

36
A.J. Szuchman-Sapir et al. / Free Radical Biology & Medicine 48 (2010) 35–46
Trp to give 2-hydroxyindole [6]. In contrast, decay of reactive
chloramines results in radical generation and the potential formation
of N-formylkynurenine and kynurenine [6]. Chlorination of Tyr
residues, which yields the stable products 3-chloro-Tyr and 3,5-
dichloro-Tyr, can occur via both HOCl- and chloramine-mediated
reactions; these chlorinated phenols are widely used as specific
markers for HOCl-mediated protein damage in vivo [21–23].
dIII fragment from the amplified DNA, which also contained the
mutant Mb coding, was ligated to the BamHI–HindIII fragment from
the vector pMb3 to yield the final expression vector [29]. The circular
plasmid was then transformed to Escherichia coli for protein
expression as described [30,31]. Where relevant, Mb (isolated as a
fusion product) was purified by anion-exchange chromatography
(Whatman DE52 resin) [24,29]. The fusion protein was reconstituted
with excess hemin (heme:protein ∼1.5) (Porphyrin Products, Logan,
UT, USA), treated with trypsin (18 mg/g protein), incubated at 25°C to
cleave the fusion segment, and purified further. Under these
conditions, recombinant Mb was isolated as metMb. Each batch of
The reactions of horse heart Mb with HOCl have been studied
recently, with specific modifications to Met and Trp residues
characterized by mass spectrometry [8]. Although human and horse
Mb shows considerable sequence homology, the former has a surface-
exposed Cys110 residue and a greater number of Met residues (three
instead of two). Both of these differences and particularly the
presence of the Cys110 residue might be expected to modulate
HOCl-mediated damage to the human protein compared to the horse
heart isoform. Previous studies have shown that this Cys residue
strongly influences the chemistry of globin-centered radicals derived
from the reaction of HMb with peroxides [24,25]. In this paper the
reactions of HOCl with HMb and its C110A variant have been studied
using electrospray mass spectroscopy and peptide mass analyses to
evaluate the role of the Cys110 residue. It is shown that this residue is
the major site of oxidation particularly at low oxidant excesses. In the
absence of Cys110, or after consumption of this residue, Met is the
major target, followed by Trp. Formation of chloro-Tyr, probably
mediated by chloramines, has also been detected, though only in the
absence of Cys110 and as a late event after oxidation of Met and Trp
residues. HOCl-mediated oxidation of Cys and Met residues decreased
the rate of ferric Mb reduction by cytochrome b5, which is believed to
be the intracellular protein responsible for maintaining Mb in the
ferrous state for binding of molecular oxygen. Oxidation of Mb by
HOCl may, therefore, critically affect Mb function in muscle tissues.
purified protein exhibited an ASoret/A280
ratio N5, with different
nm
batches of protein used for independent experiments. The proteins
were snap frozen in liquid nitrogen and then stored at −80°C. Where
required, samples were thawed on ice immediately before use.
Reaction conditions
All reactions were carried out at 37°C for 15 min in 250 mM PBS
(pH 7.4) containing 50 μM HMb. All reagents and protein solutions
were freshly prepared, and oxidation was initiated by addition of
HOCl (0- to 10-fold mol excess) to the protein solution with rapid
mixing. Final HOCl concentrations are indicated in the figure legends.
The addition of HOCl did not alter the reaction pH (data not shown).
Proteolytic digestion
Mb (50 μM) was precipitated by the addition of trichloroacetic acid
(5% v/v), pelleted by centrifugation (2 min at 1739 g), washed twice
with ice-cold acetone, and dried under N2(g). Pellets were reduced
with 45 mM DTT and incubated at 50°C for 15 min followed by
alkylation with 100 mM iodoacetamide at 20°C for 15 min. Sequenc-
ing grade trypsin (Promega, Madison, WI, USA) was added at a ratio of
68:1 (w/w) Mb:trypsin in digestion buffer (PBS containing 2 M urea
and 0.1 M NH4HCO3) and incubated overnight at 37°C. Digestion was
terminated by addition of 0.1% trifluoroacetic acid (TFA) solution and
freezing.
Experimental procedures
Materials
Trypsin (Type III, 10,200 unit mg−1 protein), urea, EDTA, diethy-
lenetriaminepentaacetic acid, B-nicotinamide adenine dinucleotide,
tryptone, and yeast extract were from Sigma (Sydney, Australia).
Dithiothreitol and NaCl were obtained from Fisher Scientific (Fair
Lawn, NJ, USA). Preswollen anion-exchange diethylaminoethyl cellu-
lose (DE52) was from Whatman (Millipore, Australia). Cytochrome b5
and human NADPH-p450 reductase were purchase from Invitrogen.
Buffers were prepared using distilled water purified by Nanopure
filtration. Buffers were stored over Chelex-100 resin (Bio-Rad) at 4°C
for 24 h to remove contaminating transition metal ions. Solutions of
HOCl were standardized by using ɛ292 (OCl−)=350 M−1 cm−1 and
ɛ235 (HOCl)=100 M−1 cm−1 [26]. The stock was diluted into
phosphate-buffered saline (PBS; 250 mM, pH 7.4) before use. Solutions
of HMb were prepared immediately before use in 250 mM PBS (pH 7.4)
and standardized using ɛ409 =188,500 M−1 cm−1 [27]. This buffer
system was selected to avoid potentially rapid secondary reactions of
HOCl with cell medium components that may interfere with the
reactions between HOCl and HMb. All other chemicals employed were
analytical grade reagents.
Liquid chromatography (LC) electrospray ionization mass spectrometry
(ESI-MS)
LC/MS analyses were performed in the positive ion mode with a
Finnigan LCQ Deca XP ion trap instrument (San Jose, CA, USA) coupled
to a Finnigan Surveyor HPLC system. Peptides were separated on a
reverse-phase column (Zorbax C18 MS column; 3 μm, 25 cm, flow rate
0.4 ml min−1) at 30°C using solvent A (0.1% v/v TFA in water) and
solvent B (0.1% v/v TFA in CH3CN). Peptides were eluted using the
following gradient: 5 to 10% B over 5 min, 10 to 20% B over 10 min, 20
to 50% B over 15 min, then 50 to 100% B over 5 min with a 15-min
wash at 100% B before 10 min reequilibration to 5% B. The electrospray
needle was maintained at 4.5 kV. Nitrogen, the sheath gas, was set at
80 units and the collision gas was helium. The temperature of the
heated capillary was set to 250°C and the peptides were identified by
searching the NCBI database (http://www.ncbi.nlm.nih.gov) using
the Sequest search engine. Where required, MS/MS analyses were
performed on selected peptides eluting from the column under
identical conditions. Comparisons of the spectra (containing y-ion and
b-ion daughter fragments that corresponded to the systematic
cleavage along the peptide backbone) were performed with the
Sequest search engine and afforded the assignment(s) of the modified
amino acid within the product peptide.
Preparation of recombinant wild-type and variant human myoglobin
DNA manipulations were performed as described previously [28].
Complementary oligomers were synthesized and DNA sequencing
was performed at SUPAMAC (University of Sydney, Australia). Site-
directed mutagenesis was performed with the pBluescript II KS (
)
vector (Stratagene, La Jolla, CA, USA). DNA was amplified by the
polymerase chain reaction with a PfuTurbo DNA polymerase
(Stratagene). Point mutations were confirmed by DNA sequence
analysis before protein expression in bacteria. Next, the BamHI–Hin-
Quantification of protein thiols
The concentration of protein thiol groups on recombinant HMb
was determined by reaction with 5,5′-dithio-2-nitrobenzoic acid

A.J. Szuchman-Sapir et al. / Free Radical Biology & Medicine 48 (2010) 35–46
37
Table 1
(DTNB) [32]. DTNB (0.5 mM) was added to wild-type HMb (50 μM) and
incubated for 30 min in the dark at 20°C. Initially, thiol content was
measured at 412 nm and calculated using ɛ412 =13,600 M−1 cm−1
[32]. Overall, native HMb contained 0.93 0.1 mol/mol free thiol.
Human Mb (50 μM) was then exposed to reagent HOCl (0, 50, or
250 μM) and incubated at 37°C. After 15 min, the reaction was
quenched on ice by adding L-Met (10 times the oxidant concentration).
Samples were then diluted 0.75-fold in PBS containing 0.5 mM DTNB
and incubated for 30 min in the dark at 20°C. For wild-type HMb, the
absorbance of the DTNB adduct (412 nm) was near coincident with the
heme absorption at 408 nm. In addition, some heme bleaching
occurred after exposure to HOCl (data not shown). To avoid this
interference, TNB− anion formation was measured at 446 nm after
treatment with HOCl. In this case, the changes in the thiol content in
wild-type HMb were calculated using ɛ446=8273 M−1 cm−1 [33].
Peptide sequences from complete digestion of native forms of the wild-type and
recombinant human Mba
AA position
Fragment sequence
Mass
Retention time
(min)
(m/z ([M+H]+
)
1–16
17–31
35–42
48–56
57–62
64–77
80–96
GLSDGEWQLVLNVWGK
VEADIPGHGQEVLIR
GHPETLEK
HLKSEDEMK
ASEDLK
HGATVLTALGGILK
GHHEAELKPLAQSHATK
YLEFISECIIQVLQSK
YLEFISEAIIQVLQSK
HPGDFGADAQGAMNK
ALELFR
1800.8
1633.8
910.9
1116.7
662.3
1350.8
1853.7
1970.4
1880.8
1515.5
748.6
32.6
24.1
13.7
14.2
10.3
31.4
18.9
34.4
37.0
19.4
26.9
13.0
20.18
103–118b
103–118c
119–133
134–139
141–147
148–153
DMASNYK
ELGFQG
828.4
650.3
a
SDS–PAGE and Western blot
Samples of HMb (50 μM) or the Cys110Ala variant were exposed to 0- to 5-fold
molar excess of HOCl and the protein was then digested with trypsin (HMb/trypsin 68/
1 v/v) to afford a complete digestion of the heme protein. The peptides were separated
by LC/MS and analyzed by MS/MS. Peptide sequences were confirmed using MS/MS
and searching the NCBI peptide database with Sequest.
A discontinuous minigel system, consisting of 12% (w/v) acrylamide
separating gel and 3% (w/v) stacker gel, was used to separate human
Mb. Protein bands were electroblotted onto nitrocellulose (1 h, 70 V),
and the membrane was blocked with 5% w/v milk powder solution in
Tris-buffered saline (TBS-T; 1 h, 20°C), washed thoroughly with TBS-T,
and then exposed to anti-human Mb antibody (1:1000 v/v; Sigma,
Australia) and incubated overnight at 4°C. The membrane was then
washed three times (TBS-T), anti-mouse horseradish peroxidase was
added (final dilution 1:2500 v/v; Invitrogen, Australia), and the
membrane was incubated further at 20°C. After 1 h, the antigen–
antibody complexes were detected using enhanced chemilumines-
cence (Kodak Image Station 2000, Integrated Bioscience, Australia).
b
Carboxymethylated cysteine peptide formed after reaction of the unoxidized Cys
with iodoacetamide (+57 mass units).
c
Peptide detected in the Cys110Ala variant human Mb, where Cys has been replaced
by Ala. Results are representative of those from n=4 independent experiments.
Overall, the peptide recovery covered 92.8% of the predicted sequence of HMb.
Consistent with the known sequence, wild-type HMb contained
one Cys residue (peptide YLEFISECIIQVLQSK at positions 103–118,
with a mass-to-charge (m/z) ratio of 1970.4), which was detected in
its carboxymethylated form (yielding a parent ion M+57 amu) as a
result of alkylation with iodoacetamide before trypsin digestion.
Three Met residues were detected within the peptide sequences:
HLKSEDEMK (positions 48–56; m/z 1116.7), HPGDFGADAQGAMNK
(positions 119–133; m/z 1515.5), and DMASNYK (positions 141–147;
m/z 828.4). Two Trp residues were identified in the peptide
GLSDGEWQLVLNVWGK (positions 1–16; m/z 1800.8), and Tyr
residues were present in the peptide sequences YLEFISECIIQVLQSK
(positions 103–118; m/z 1970.4) and DMASNYK (positions 141–147;
m/z 828.4). The Cys110Ala variant of HMb yielded the anticipated
peptide YLEFISEAIIQVLQSK (positions 103–118; m/z 1880.8). All the
other peptides from this variant were identical to those of the wild-
type protein (not shown).
Reduction of myoglobin by cytochrome b5
Oxidation reactions of wild-type ferric HMb (final concentration
50 μM in heme) were carried out at 37°C for 15 min in 250 mM PBS
(pH 7.4) and initiated by adding HOCl (0-, 5-, and 10-fold mol excess).
Reactions were terminated by adding 12.5 mM L-Met on ice. The one-
electron reduction of native and HOCl-oxidized ferric HMb by
recombinant human cytochrome b5 was investigated as described
previously [34,35]. Briefly, a sample (10 μl) of the Mb preparation
(final concentration 2 μM) was added to a reaction mixture containing
0.12 μM human recombinant cytochrome b5, 0.05 μM human NADPH-
P450 reductase, glucose 6-phosphate (50 U), catalase (375 U), 20 mM
glucose, 0.2 mM EDTA, and 2 mM NADP+ in 100 mM phosphate buffer
(pH 7.4). The reaction was carried out under air in a quartz cuvette
and spectra (350 to 600 nm) were recorded every 1 min against a
reference sample containing the reaction mixture without Mb, using a
Shimadzu UV-1601 spectrophotometer at 25°C.
HOCl oxidizes Cys residues in wild-type human Mb
To investigate HOCl modification of HMb, the wild-type or
Cys110Ala variant proteins (50 μM) were exposed to 1 or 5 mol
equivalents of HOCl, or the identical volume of PBS (control), for
15 min at 37°C. After the reactions were quenched on ice, the products
were digested with trypsin and the resulting peptides were identified
as described above.
HOCl-treated HMb yielded a product peak in the LC chromatogram
at 31.3 min (Fig. 1A). MS analysis of the peptide that gives rise to this
peak yielded an m/z ratio of 1961.5 consistent with a mass increase of
48 amu to the YLEFISECIIQVLQSK peptide (positions 103–118).
Exposure of the wild-type HMb to increasing concentrations of
HOCl led to a dose-dependent increase in the peak area of this
modified peptide (Fig. 1B), whereas the carboxymethylated deriva-
tive of the corresponding parent peptide (m/z 1970.4, eluted at
34.4 min) did not show a significant decrease in peak area (Fig. 1C),
suggesting that oxidation of this peptide to give the modified peptide
with m/z 1961.5 is a minor pathway. MS/MS analysis of the m/z
1970.4 peptide (Fig. 2A) confirmed the presence of the alkylated
Cys110 residue (+57 atomic mass units, y9+57, Fig. 2A) in the
YLEFISECIIQVLQSK sequence. Tandem MS/MS analysis (Fig. 2B) of the
Statistical analyses
All analyses were performed with Prism software (v3.0; GraphPad,
San Diego, CA, USA). Differences between groups were assessed with
one-way ANOVA and Tukey's or Dunnet's multiple comparison tests
or Bonferroni's post hoc test for specific comparisons, where
appropriate. A value of Pb0.05 was considered significant.
Results
Native peptides from wild-type and C110A variant human Mbs,
generated by trypsin digestion, were identified by LC/MS and tandem
MS/MS. Twelve peptides that covered ∼93% of the predicted
sequence of the wild-type and the C110A variant proteins were
identified (Table 1). Peptides containing Cys, Met, Trp, and Tyr
residues were investigated further, as these are considered potential
sites of oxidation by HOCl [8].

38
A.J. Szuchman-Sapir et al. / Free Radical Biology & Medicine 48 (2010) 35–46
Fig. 1. Liquid chromatographic analyses of specific oxidative modification of Cys110 in wild-type human Mb after exposure to reagent HOCl. (A) Protein samples (50 μM) were
treated with vehicle (control, 1:0) or reagent HOCl (1- or 5-fold mol excess relative to protein) and incubated at 37°C. After 15 min, the protein fraction was digested by trypsin
overnight at 37°C. The resulting peptides were separated by reverse-phase HPLC with gradient elution as described under Experimental procedures. With oxidant treatment a new
peak appears that elutes at 31.3 min. (B and C) Quantitative ESI-MS/MS analyses of the relative abundance of the peaks at 31.3 and 34.4 min, respectively, corresponding to ions with
m/z 1961.5 (B, peptide containing Cys110+48 amu, assigned to the oxidized peptide containing cysteic acid) and the parent YLEFISECIIQVLQSK peptide after derivatization with
iodoacetamide (IAA) (C; m/z 1970.4). The peak areas of the eluted peptides were expressed relative to the corresponding maximum peak areas for the control and oxidized (cysteic
acid-containing) samples. Data are the means SD of three independent experiments using different batches of Mb. ⁎⁎ Significant differences compared to the control at the Pb0.01
level as determined by one-way ANOVA with Tukey's multiple comparison test.
corresponding modified peptide obtained from the HOCl-treated HMb
(m/z 1961.5, [M+48+H]+) verified that the Cys110 residue was the
site of modification (b8 ion).
HMb (Fig. 3). In the absence of HOCl, the recombinant HMb
contained trace quantities of a protein band with a molecular mass
corresponding to that of the homodimer (∼34 kDa) (Fig. 3A).
Exposure to increasing concentrations of HOCl yielded a dose-
dependent increase in the intensity of the homodimer band (cf.
bands at 34 kDa in Figs. 3A and 3B) together with a corresponding
loss of the native Mb band at 17 kDa. Addition of DTT significantly
decreased the extent of dimer formation when a HOCl:HMb molar
ratio of 5:1 was employed (Figs. 3A and 3B), suggesting that the
disulfide was a major product generated by HOCl, with this not being
detected in the LC/MS/MS studies because of the reduction process
employed in the sample preparation. Sulfenic and/or sulfinic acid
products may not have been detected for similar reasons. The extent
of thiol modification on HMb induced by HOCl was also examined by
quantifying the concentration of the remaining thiol groups on the
protein after exposure to varying concentrations of HOCl (Fig. 3C). As
anticipated the thiol content of the native protein was rapidly
depleted upon addition of HOCl (final concentration 1 mol/mol),
Examination of the series of daughter y-ions and b-ions cleaved
after the Cys110 residue (fragments y11+48, y12+48, y13+48,
b9+48, b12+48, and b15+48; Fig. 2B) confirmed that Cys110
gained 48 amu. This modification is consistent with the conversion
of Cys110 to cysteic acid by HOCl. No further modifications were
detected on these product peptides despite the presence of amino
acid residues such as Tyr and Lys that are potential targets for HOCl.
No evidence was obtained for alternative modifications of the Cys
residues such as sulfenic or sulfinic acids [6]. The extent of conversion
of Cys110 to cysteic acid by HOCl was assessed by determining the
relative peak abundance as ∼10% of the total Cys present in the native
peptide.
To explore whether other Cys-derived products were formed and
not detected because of the workup conditions employed, Western
blot analyses were performed on native and HOCl-treated wild-type

A.J. Szuchman-Sapir et al. / Free Radical Biology & Medicine 48 (2010) 35–46
39
Fig. 2. MS/MS analyses of the Cys110-modified peptide formed in wild-type human Mb exposed to reagent HOCl. Samples of human Mb were oxidized with HOCl as described in the
Fig. 1 legend. Products were separated by HPLC and analyzed by MS/MS and their identities were confirmed by searching the NCBI peptide database with the Sequest search engine.
(A) MS/MS spectrum of the Cys110-containing peptide (m/z 1970.4) with the addition of 57 amu corresponding to the carboxymethylated Cys (y9, see arrow) formed by the
addition of iodoacetamide to the unoxidized Cys, thereby confirming the sequence of the native peptide YLEFISECIIQVLQSK. (B) MS/MS spectrum showing the fragment ions
generated from the oxidized Cys110-containing peptide (m/z 1961.5, [M+48+H]+). Peptide fragment ions formed from bond cleavage after the Cys110 residue (b9+48, b10+48
from the N-terminus and y11+48, y12+48, y13+48 from the C-terminus). By contrast, daughter fragments cleaved before Cys110 in the sequence of the peptide residue remained
unchanged (b6, y7, y8). b⁎ and y⁎ ions refer to b and y fragmentations that have additionally lost ammonia (NH3). These data confirm the conversion of Cys110 to cysteic acid
(i.e., gain of three oxygen atoms). Data are representative of three independent experiments using different batches of Mb.
indicating that the reaction between the free thiol and HOCl occurs
rapidly and is near stoichiometric in nature (data not shown)—no
further change in absorbance was detected at higher HOCl:Mb ratios.
Taken together, these data indicate that the reaction of HMb with
HOCl generates a range of thiol oxidation products with formation of
disulfide and cysteic acid derivatives being the only stable modifica-
tions detected.
MS/MS analysis of these peptides confirmed the modification of the
Met residues to Met-sulfoxide (not shown). These modifications were
verified using the Sequest search engine.
Steady-state levels of these oxidized products derived from the
wild-type HMb increased concomitant with the consumption of the
corresponding native peptide (Figs. 4A–4F; filled bars). A comparison
of the extent of oxidation of Met55, Met142, and Met131 at the same
HOCl:HMb ratio indicated differential extents of oxidation of these
residues (Table 2). Thus, the percentage yield of Met55 sulfoxide was
significantly greater than that obtained for sulfoxides of Met131 and
Met142 at a HOCl:HMb ratio of 1 mol:mol (Table 2). Maximum
percentage yields (assigned as 100%) were obtained for reactions of
HMb with a five-fold molar excess of HOCl. These data indicate that
HOCl exhibits a preference for oxidation of Met55 over Met131 and
Met142 at the oxidant concentrations studied.
The same Met-sulfoxide peptides were detected by LC/MS/MS on
oxidation of the Cys110Ala-variant Mb by HOCl (Figs. 4A–4F; open
bars). The slight preference for oxidation of Met55 over other Met
residues identified for the wild-type protein was still evident in
Cys110Ala HMb at the HOCl:Mb ratio of 1 mol:mol (Table 2).
HOCl-mediated oxidation of Met and Trp in wild-type and
Cys110Ala-variant HMbs
The extent of conversion of Met to Met-sulfoxide was determined
by LC/MS/MS by quantifying the loss of the Met-containing native
peptides and the generation of their corresponding modified forms,
on treatment with increasing concentrations of HOCl. HOCl-treated
HMb yielded three new peaks in the liquid chromatogram with
retention times of 8.2, 9.7, and 16.4 min. Subsequent MS analysis of
the peptides that give rise to these peaks yielded m/z ratios of 844.6,
1132.4, and 1531.6, consistent with a mass increase of 16 amu to the
Met-containing peptides at positions 142, 55, and 131, respectively.

40
A.J. Szuchman-Sapir et al. / Free Radical Biology & Medicine 48 (2010) 35–46
that corresponded to an increase of 4, 16, 32, and 64 amu, respectively,
compared to the vehicle-treated control (data not shown). No loss of
this peptide was detected with the wild-type protein with comparable
oxidant treatments (Fig. 5B). These data are consistent with the
formation of kynurenine (+4 amu) and the incorporation of one, two,
or four oxygen atoms into the Trp-containing peptide.
Tyrosine chlorination
Potential modification of Tyr-containing peptides was investigated
by LC/MS/MS (as above) using wild-type and Cys110Ala-variant HMb
exposed to HOCl after short (15 min) or long (24 h) incubation
periods (Fig. 6). For wild-type HMb, no change in the parent peptide
containing Tyr103 was evident irrespective of the incubation period;
however, the peptide containing Tyr146 (DMASNYK, positions
141–147; m/z 828.4) diminished significantly (not shown). MS/MS
analysis of the product peptide (eluting at 8.2 min) indicated that Met
modification was primarily responsible for the decrease in this
peptide, with no modification to Tyr146 detected (not shown).
Thus, no significant chlorination to Tyr146 was detected with the
wild-type protein under any of the reaction conditions examined.
Although Tyr chlorination may occur to a greater extent at much
higher HOCl:Mb ratios this was not examined further, as low molar
excesses of HOCl are likely to be most pathophysiologically relevant.
In contrast, a new peak (eluting at 37.8 min, Fig. 6C) was detected in
the reactions between the Cys110Ala variant of HMb 24 h after
treatment with 5 mol excess of HOCl (Fig. 6, inset). The formation of
this modified peptide occurred concomitant with the consumption
of the corresponding Tyr103-containing parent peptide (YLEFI-
SEAIIQVLQSK, positions 103–118; m/z 1880.8), whereas the Tyr146-
containing peptide (DMASNYK, positions 141–147; m/z 828.4) was
unaffected after 24 h.
Subsequent MS analyses of the new peak revealed two species
with m/z 1914.6 and 1916.6. Tandem MS/MS confirmed the addition
of 34 atomic mass units to the Tyr103-containing peptide
corresponding to chlorination of the Tyr103 residue (Fig. 6D). This
product was not observed at the shorter reaction times. Similar to the
wild-type protein, the loss of the other Tyr-containing peptide
(DMASNYK, positions 141–147; m/z 828.4) was attributed to
modification of Met142, with no modification to Tyr146 detected
(not shown). As a role for chloramines in the formation of chlorinated
Tyr residues is well established [5,22,36], the formation of this
chlorinated product in the Cys110Ala variant of HMb is assigned to
chlorine transfer from preformed chloramines generated on the
protein (cf. chloramine formation in HOCl-modified horse Mb [8]).
Fig. 3. Reactivity of Cys110 in human wild-type Mb with HOCl. Human Mb (50 μM) was
treated with equimolar HOCl at 37°C and pH 7.4. After 15 min the reaction was
quenched on ice with the addition of 10-fold mol excess of L-Met. (A) A sample of the
mixture was separated on SDS–PAGE and transferred to a nitrocellulose membrane
followed by incubation with a monoclonal anti-Mb antibody, and (B) the dimeric bands
were quantified using densitometry (ImageJ 1.4). Lane 1, control wild-type human Mb
in the absence of HOCl. Lanes 2 and 3, wild-type human Mb treated with 1 and 5 mol
HOCl/mol protein, respectively. Lane 4, as lane 3 except after treatment with excess
DTT (3.5 mM). (C) The extent of thiol loss was determined by measuring changes to
total thiol as described under Experimental procedures. Significant differences
compared to the controls are indicated (*Pb0.05 and **Pb0.01) as determined by
one-way ANOVA with Bonferroni's multiple comparison test. Data represents mean
values SD, n=3 separate experiments. The gel shown is representative of five
independent experiments with different preparations of human wild-type Mb.
Reduction of wild-type and HOCl-oxidized human Mb by cytochrome b5
To test whether HOCl-induced modification of ferric HMb protein
affects the rate or extent of one-electron reduction by cytochrome b5,
we monitored the spectral changes in reaction mixtures containing
wild-type HMb or HOCl-oxidized HMb and recombinant cytochrome
b5 in the presence of cytochrome P450 reductase and an NADPH-
regenerating system (see Experimental procedures). Monitoring
changes in wild-type ferric HMb by absorbance spectroscopy
(Fig. 7) showed a time-dependent decrease in absorbance at
410 nm and a corresponding increase in the peak at 420 nm. This
was accompanied by an increase in absorbance maxima at 542 and
580 nm (Fig. 7A, inset). These changes resemble the spectral changes
occurring during the reduction of methemoglobin to oxyhemoglobin
by cytochrome b5 [34].
To ascertain whether Trp was also a target for HOCl in HMb (cf.
previous data for the horse heart form [8]), both native and modified
forms of the peptide containing both Trp residues of wild-type HMb
and Cys110Ala-variant HMb (GLSDGEWQLVLNVWGK; m/z 1800.8)
were quantified (Fig. 5). For the Cys110Ala variant, a 1:1 molarexcess of
HOCl did not affect this peptide, whereas treatment of the protein with
5 mol:mol HOCl significantly decreased the native peptide (Figs. 5A and
5B). Exposure of the Cys110Ala-variant HMb to increasing concentra-
tions of HOCl also led to the appearance of four new peaks in the LC
chromatograms with m/z ratios 1804.6, 1816.6, 1832.6, and 1864.7
A comparison of the time-dependent changes in absorbance for
the native and HOCl-oxidized wild-type HMb indicated a decrease in
the observed rate constants for the cytochrome b5-mediated
reduction (Fig. 7B). This decrease in rate constant was significant for
HMb exposed to a 10-fold molar excess of HOCl (Table 3).

A.J. Szuchman-Sapir et al. / Free Radical Biology & Medicine 48 (2010) 35–46
41
Fig. 4. Relative contents of native and oxidized Met142-, Met55-, and Met131-containing peptides from wild-type and the Cys110Ala-variant human Mb exposed to reagent HOCl.
Samples of human Mb (50 μM) were exposed to 0-, 1-, or 5-fold mol excess of HOCl and subsequently digested with trypsin. The resultant peptides were separated by LC/MS.
Modifications to Met142-, Met55-, and Met 131-containing peptides (m/z 828.4, m/z 1116.7, and m/z 1515.5, respectively) are shown for both the wild type (black bars) and the
Cys110Ala variant (open bars) of human Mb. Finally, the extent of modification was expressed as a fraction of the peak area in the corresponding control (native) peptides.
Statistically significant differences compared to the controls are indicated (*Pb0.05 and **Pb0.01 levels), respectively, as determined by one-way ANOVA with Tukey's multiple
comparison test. Data represent the means SD from five or six independent experiments using different batches of Mb.
Discussion
Exposing recombinant HMb to reagent HOCl (1 or 5 mol:mol)
Met-sulfoxide-containing peptides (as detected by LC/MS/MS) on
exposure to HOCl; however, the extent of oxidation of each of these
Met residues was increased as judged by the relative increases in the
corresponding parent ions formed at the same HOCl:HMb ratio.
Modifications to the peptide containing Trp7 and Trp14 in the
Cys110Ala variant include four new species with m/z ratios indicative
of 4, 16, 32, and 64 amu increases; these m/z values are consistent
with the formation of kynurenine and the incorporation of one, two,
or four oxygen atoms in to the Trp-containing peptide. Chlorination of
tyrosine was also detected at the highest HOCl:HMb ratio 24 h after
treatment. Our data indicate that the Cys110 residue of HMb plays a
role in determining the site and extent of oxidative modification of
amino acids in reactions between HOCl and human Mb.
results in modifications to the Cys110 residue, yielding primarily
disulfides (as evidenced by homodimer formation) and cysteic acid to
a lesser extent as confirmed by LC/MS/MS analyses. Concurrent with
Cys110 modification, exposure of Mb to HOCl generates Met-sulfoxide
at Met55, Met131, and Met142 in the wild-type protein. Tandem MS
analyses of the tryptic peptide digests indicate that Met55 is
preferentially oxidized, relative to Met131 and Met142, at low
HOCl/Mb ratios. One likely reason for this enhanced extent of
oxidation is that Met55 has a greater solvent accessibility to HOCl in
the bulk phase compared to the other two residues (cf. data for horse
heart Mb [8]). In contrast, the Trp- and Tyr-containing peptides
remained unaffected by HOCl at the protein/HOCl ratios investigated
here. The Cys110Ala variant of human Mb yielded the same series of
The inflammatory response subsequent to heart attack involves
recruitment and activation of mononuclear cells and, therefore, local
concentrations of HOCl are probably elevated in the affected myo-

42
A.J. Szuchman-Sapir et al. / Free Radical Biology & Medicine 48 (2010) 35–46
Table 2
lipids and some antioxidants [9,38]. One potential target for HOCl in
the myocardium is myoglobin because of its relatively high abundance
in cardiomyocytes (0.3–0.5 mM) [39] and skeletal and smooth muscle
fibers (∼0.1 mM) [40]. Human Mb possesses a single Cys110 residue,
which is not present in most other Mb isoforms [24,41]. Under aerobic
conditions, the Cys110 residue of HMb is known to react, via a complex
mechanism, with ·NO to yield S-nitrosylated HMb (S–NO Mb) [42], by
analogy to S-nitroso hemoglobin [43,44]. However, unlike hemoglo-
bin, for which nitrosation is dependent on the allosteric (R–T) transi-
tion [44], the degree of Mb oxygen saturation does not affect the
accessibility of the Cys110 for S-nitrosation, as judged by comparable
X-ray crystal structures of ferric and ferrous Mb bound to a diatomic
Percentage yields of oxidized products from methionine-containing peptides derived
from recombinant wild type and Cys110Ala variant of human Mba
HOCl:Mb ratio (mol:mol)
Wild-type HMb
Cys110Ala variant of HMb
Met142+16 (m/z 844.6)
4.1 (2.8)
9.6 (2.1)
0
1
5
2.2 (0.8)
14.1 (3.7)⁎⁎
100 (9.5)⁎⁎
100 (12)⁎⁎
Met55+16 (m/z 1132.4)
3.2 (1.4)
36.3 (18.7)⁎⁎
100 (21)⁎⁎
0
1
5
2.6 (2.1)
34.2 (11.7)⁎
100 (28)⁎⁎
·
ligand (see Fig. 5 in Ref. [45]). Importantly, NO released from S–NO
Met131+16 (m/z 1531.6)
oxyMb remains biologically active and readily dilates preconstricted
vessels in a manner similar to that of authentic endothelium-derived
relaxant factor [46]. The studies reported here provide evidence that
thiol depletion and both reversible and irreversible oxidation of
Cys110 (yielding disulfide and cysteic acid, respectively) by low con-
centrations of HOCl may have an impact on the ability of Mb to
participate in S-nitrosylation reactions, and potentially limit the capa-
city of the protein to store vasoactive ·NO within the walls of aortic
vessels or cardiac tissues, particularly under conditions of chronic
inflammation under which HOCl is known to be formed [47–49].
The oxidation of Met residues has been suggested as a regulator of
cellular function through impairing enzyme function [50–53] and by
acting as an endogenous antioxidant that protects other oxidant-
susceptible amino acids within proteins [54]. The possibility that
Met can be regenerated from the corresponding sulfoxide through
the action of Met-sulfoxide reductase suggests the potential for Met
0
1
5
3.1 (1.6)
8.8 (4.6)⁎⁎
100 (11)⁎⁎
2.0 (0.3)
11.3 (3.2)⁎⁎
100 (6)⁎⁎
a
Samples of HMb (50 μM) or the Cys110Ala variant were exposed to 0- to 5-fold
molar excess of HOCl and the protein was then digested with trypsin (HMb/trypsin
68/1 v/v) to afford a complete digestion of the heme protein. The peptides were
separated by LC/MS and analyzed by MS/MS and the extent of oxidation was expressed
as a percentage of the maximal level of oxidation for the particular Met-containing
peptide. Results are presented as means
experiments. Statistically significant differences compared to the corresponding control
were determined by one-way ANOVA with Tukey's multiple comparison test.
⁎
(
SD) from n=5 or 6 independent
Pb0.05.
Pb0.01.
⁎⁎
cardium [37]. Proteins are the major component of most biological
systems, and the rate constants for reactions of amino acid side chains
with HOCl are more than 10-fold higher than those for reactions with
Fig. 5. Relative quantification of the peptide containing Trp7 and Trp14 in the Cys110Ala-variant and wild-type human Mb after exposure to reagent HOCl. Samples of (A, B) the
Cys110Ala-variant and (C) wild-type human Mb were exposed to 0 (open bar), 1-fold (gray bar), or 5-fold (black bar) mol excess of HOCl for 15 min as described under Experimental
procedures, followed by trypsin digestion. The resultant peptides were separated by LC/MS. (A) Chromatogram of the Trp7/Trp14-containing peptide (m/z 1800.8) from Cys110Ala-
variant human Mb after exposure to 0, onefold, and fivefold molar excess of HOCl. (B and C) Quantified chromatograms of Cys110Ala-variant human Mb and the wild-type Mb,
respectively, expressed relative to the corresponding peak areas in control samples in the absence of HOCl. Data are the means SD from three independent experiments using
different batches of Mb. ⁎⁎Significantly different compared to the controls, Pb0.01, as determined by one-way ANOVA with Tukey's multiple comparison test.

A.J. Szuchman-Sapir et al. / Free Radical Biology & Medicine 48 (2010) 35–46
43
Fig. 6. LC/MS analyses of the specific modifications to the Tyr103-containing peptide from the Cys110Ala variant of human Mb exposed to reagent HOCl for up to 24 h. Protein
samples (50 μM) were treated with (A) vehicle (control) or reagent HOCl (5-fold mol excess) and incubated at 37°C for either (B) 15 min or (C) 24 h before trypsin digestion for 18 h
at 37°C. Representative chromatograms show the decrease in the Tyr103 peptide (peak eluting at 37.1 min), with the concomitant appearance of a new peak (eluting at 37.8 min)
24 h after the initial treatment with HOCl, which was further analyzed by ESI-MS/MS. Inset: Relative quantification of the parent Tyr103 peptide peak expressed as the proportion of
the corresponding peak areas in control samples at the times indicated. (D) Corresponding MS/MS spectra for the m/z 1914.6 modified peptide (eluting at 37.8 min). Note that all
the b ions have the additional 34 amu corresponding to the chlorination of the Tyr at the N-terminus (b1–16 fragment) of the peptide, whereas the y1–15 ions are the same as in the
parent peptide. Data are the means SD from three independent experiments using different batches of Mb. ⁎Significantly different compared to the control, Pb0.05, as determined
by one-way ANOVA with Tukey's multiple comparison test.
residues to act as an in situ antioxidant [55]. The data obtained here
support the important role of Met residues in the scavenging of HOCl
and suggest that solvent-exposed Met residues can protect proteins
from environmentally proximate oxidizing agents [54].
Tryptophan is a key structural and functional amino acid residue
that is sensitive to oxidation [56–59], with Trp modification being
implicated in the loss of activity of a number of proteins [11,13].
Elevated levels of oxidized Trp, Tyr, and Met residues have been
detected in cardiac proteins of diabetic rats [60] and skeletal muscle
myosin from porcine longissimus dorsi muscle [61]. In other studies
[62] the Trp oxidation product N-formylkynurenine (corresponding to
the addition of two oxygen atoms) has been detected throughout the
mitochondrial proteome of normal human heart tissue, with evidence
presented for selective oxidation of a subset of proteins associated
predominantly with redox metabolism. For example, Trp oxidation in
the human mitochondrial complex I subunit seems to occur with
considerable specificity relative to other subunits, whereas Met
oxidation was completely random [63]. Mass increases of +16, +32,
and +64 amu in Trp-containing peptides [57,63] have been attributed
to the formation of 3-hydroxtryptophan (II) and N-formylkynurenine
(III) (Scheme 1). The Trp-derived products with increases in mass of
+4 may arise from further transformations of these derivatives to

44
A.J. Szuchman-Sapir et al. / Free Radical Biology & Medicine 48 (2010) 35–46
Formation of 3-chlorotyrosine was detected at Tyr103 in the
Cys110Ala variant of human Mb after reaction with 5 molar excess of
HOCl for 24 h. Both HOCl and chloramines have been shown to
chlorinate Tyr residues in proteins [22,47,64], and chloro-Tyr has been
widely employed as a marker of HOCl production in vivo [23].
Interestingly, in this study, Tyr103 chlorination was evident only 24 h
after exposure of the Cys110Ala variant of human Mb to HOCl and
probably involves slow secondary chlorination induced by protein
chloramines [9]. By contrast, no modification to Tyr146 was detected
in the short (141–147) peptide, in which only Met142 oxidation was
detected in the early phase of monitoring. This may suggest that
Met142 protected Tyr from modification, either through scavenging
oxidizing HOCl and/or chloramines or through some steric effect that
blocks the direct interaction of HOCl with Tyr residues. These
observations are also consistent with the slow reaction of HOCl with
Tyr (k=44 M−1 s−1) compared to Met (k=3.8×107 M−1 s−1),
Cys (k=3.0×107 M−1 s−1), Trp (k=1.1×104 M−1 s−1), and
amine-containing sites (His and α-amino k=1.0×105 M−1 s−1, Lys
k=5.0×103 M−1 s−1) [5,65]. As expected on the basis of these rate
constants Trp and Tyr oxidation were detected, at the molar ratios
examined, only in the absence of Cys and after significant Met
oxidation. These results confirm, as predicted on kinetic grounds, that
Cys and Met residues are the preferred targets for HOCl-mediated
oxidation on proteins even when these residues are sterically
hindered. Thus, Met residues are able to minimize oxidation of
other susceptible amino acids in the protein structure.
The primary role for myoglobin is as an oxygen-binding protein
that maintains oxygen homeostasis in muscle tissues. The ferrous
form of Mb is required for efficient reversible oxygenation to occur
and, therefore, levels of ferric Mb are strictly regulated in muscle
tissues [35]. However, both deoxy- and oxyMb can undergo
autoxidation and stimulated oxidation to generate the inactive ferric
(metMb) form. Ferric Mb can also participate in pro-oxidative
reactions that damage a wide range of biological targets [24,27,31].
Cytochrome b5 can mediate the one-electron reduction of ferric Mb
[35,66]. The results reported here show that HOCl-induced oxidation
of Mb is associated with a decrease in the rate constant for
cytochrome b5-mediated ferric Mb reduction, potentially affecting
the ability of the reductase system to maintain the active ferrous
form. If HOCl-mediated oxidation of Mb were to occur in cardiac
muscle, for example, during an inflammatory response after a heart
attack [37], then perturbation of the Mb redox state may affect
oxygen homeostasis and have a negative impact on mitochondrial
function (i.e., oxidative phosphorylation to yield ATP) in the ischemic
tissue.
Fig. 7. Reduction of native and HOCl-modified wild-type HMb by cytochrome b5. Samples
of wild-type HMb or HOCl-oxidized HMb (final HOCl:Mb ratio 5 or 10 mol:mol; final
concentration of Mb 2 μM) were added to a reaction mixture containing 0.12 μM human
recombinant cytochrome b5, 0.05 μM human NADPH-P450 reductase, and a reductase
system in a quartz cuvette at 25°C as outlined under Experimental procedures. Spectra
were recorded every 1 min over the range 350 to 600 nm against a reference sample
containing the reaction mixture without HMb. (A) Spectral changes within 10 min.
Arrows indicate increases (up) and decreases (down) in absorbance over time,
respectively. Inset shows the corresponding spectral region between 500 and 590 nm.
(B) The time-dependent change in log A(410 nm) for HMb reduction-mediated
cytochrome b5, curves represent wild-type HMb treated with 0 (circle), 5 (square), or
10 (triangle) mol excess of HOCl. Data are themeans SD from at least three independent
experiments using different batches of Mb and fresh solutions of cytochrome b5.
One limitation on the likely occurrence of these reactions during an
inflammatory response in vivo is the competition between MPO and
Mb for H2O2. The rate constant for the reaction between MPO and H2O2
is ∼107 M−1 s−1, whereas that for reaction of Mb depends on the
redox state of the protein and whether dioxygen is bound. Ferric Mb is
more reactive than oxyMb with rate constant k ∼5.6×102 M−1 s−1
[67], compared to k ∼ 21 M−1 s−1 [68], respectively; deoxyMb reacts
with k ∼3.6×103 M−1 s−1 [69]. The concentration of ferric Mb is
controlled by an associated metMb reductase [35,66] and is on the
order of 10% of the total protein in muscle cells at any given time [39].
Thus, the concentrations of ferric Mb in skeletal and cardiac muscle
would be expected to be ∼10 and 30 μM, respectively. The
concentration of circulating MPO varies with the level of inflammation.
In patients with coronary artery disease, plasma MPO concentrations
range 0.36–0.52 μM [70]. Therefore, based purely on these kinetic
values and concentrations, it would be expected that MPO will be the
more effective peroxidase and outcompete HMb for any H2O2 formed
during the inflammatory response.
kynurenine (IV) (e.g., ion at m/z 1804.6 shown in Fig. 6B). These
products may account for the observed HOCl-induced modifications to
both Trp7 and Trp14 detected in the Cys110Ala human Mb.
Table 3
Rate constants for the one-electron reduction of wild-type Mb with Cyt b5 before and
after exposure to HOCla
HOCl:Mb ratio
kobs (×102)
SD
0
5
10
0.87
0.68
0.39⁎
0.0007
0.0017
0.0003
a
Samples of HMb (50 μM) were exposed to 0- to 10-fold molar excess of HOCl. After
solution containing
the reaction was quenched, an aliquot was mixed with
a
cytochrome b5 with an NADPH-regenerating system as described in detail under
Experimental procedures. Spectra were recorded every minute against a reference
containing the reaction mixture in the absence of the Mb preparation. Rate constants
were calculated from the slope estimated by regression over the initial linear phase for
the time-dependent decay of the Soret band for ferric Mb (A410 nm). Values represent
means SD; n=3–5 experiments.
In summary, we show that reaction of HOCl with human Mb occurs
with considerable selectivity at the Cys110 residue. Oxidation at
Cys110 results in disulfide bond formation (i.e., protein homodimers)
⁎
Pb0.01; statistically lower than in the absence of HOCl, compared with control
using one-way ANOVA with Dunnet's post-hoc test.

A.J. Szuchman-Sapir et al. / Free Radical Biology & Medicine 48 (2010) 35–46
45
Scheme 1. Products of tryptophan oxidation.
[11] Hawkins, C. L.; Davies, M. J. Inactivation of protease inhibitors and lysozyme by
hypochlorous acid: role of side-chain oxidation and protein unfolding in loss of
biological function. Chem. Res. Toxicol. 18:1600–1610; 2005.
[12] Bonini, M. G.; Siraki, A. G.; Atanassov, B. S.; Mason, R. P. Immunolocalization of
hypochlorite-induced, catalase-bound free radical formation in mouse hepato-
cytes. Free Radic. Biol. Med. 42:530–540; 2007.
[13] Chetyrkin, S. V.; Mathis, M. E.; Ham, A. J.; Hachey, D. L.; Hudson, B. G.; Voziyan, P. A.
Propagation of protein glycation damage involves modification of tryptophan
residues via reactive oxygen species: inhibition by pyridoxamine. Free Radic. Biol.
Med. 44:1276–1285; 2008.
[14] Schoneich, C.; Sharov, V. S. Mass spectrometry of protein modifications by reactive
oxygen and nitrogen species. Free Radic. Biol. Med. 41:1507–1520; 2006.
[15] Claiborne, A.; Miller, H.; Parsonage, D.; Ross, R. P. Protein-sulfenic acid stabilization
and function in enzyme catalysis and gene regulation. FASEB J. 7:1483–1490; 1993.
[16] Raftery, M. J.; Yang, Z.; Valenzuela, S. M.; Geczy, C. L. Novel intra- and inter-
molecular sulfinamide bonds in S100A8 produced by hypochlorite oxidation.
J. Biol. Chem. 276:33393–33401; 2001.
and the generation of cysteic acid. Oxidation of Met and Trp residues
also occurs to a limited extent, with these reactions becoming more
prevalent at higher HOCl mol excesses. Studies with the Cys110Ala
variant of human Mb demonstrate that in the absence of Cys110, the
majority of damage is directed toward Met residues, with increasing
oxidation of both Trp residues and Tyr103 noted at higher HOCl:Mb
ratios. Overall, the kinetically favored reaction between Cys and HOCl
can play a role in determining the sites and extent of oxidative
damage to amino acid residues in proteins. These amino acid
modifications on HMb are associated with a decreased ability of the
protein to regulate intracellular oxygen homeostasis.
Acknowledgments
[17] Thomas, E. L.; Grisham, M. B.; Jefferson, M. M. Preparation and characterization of
chloramines. Methods Enzymol. 132:569–585; 1986.
[18] Pattison, D. I.; Davies, M. J. Kinetic analysis of the role of histidine chloramines in
hypochlorous acid mediated protein oxidation. Biochemistry 44:7378–7387;
2005.
[19] Hawkins, C. L.; Davies, M. J. Hypochlorite-induced damage to proteins: formation
of nitrogen-centred radicals from lysine residues and their role in protein
fragmentation. Biochem. J. 332 (Pt 3):617–625; 1998.
The authors are grateful to the Australian Research Council
(through the ARC Centres of Excellence, Fellowships and Discovery
Projects Schemes awarded to M.J.D. and P.K.W.) and the National
Heart Foundation (Grant-in-Aid G 07S30435 to P.K.W.) for financial
support.
[20] Hazell, L. J.; Davies, M. J.; Stocker, R. Secondary radicals derived from chloramines
of apolipoprotein B-100 contribute to HOCl-induced lipid peroxidation of low-
density lipoproteins. Biochem. J. 339 (Pt 3):489–495; 1999.
References
[1] Hampton, M. B.; Kettle, A. J.; Winterbourn, C. C. Inside the neutrophil phagosome:
oxidants, myeloperoxidase, and bacterial killing. Blood 92:3007–3017; 1998.
[2] Harrison, J. E.; Schultz, J. Studies on the chlorinating activity of myeloperoxidase.
J. Biol. Chem. 251:1371–1374; 1976.
[3] Sugiyama, S.; Okada, Y.; Sukhova, G. K.; Virmani, R.; Heinecke, J. W.; Libby, P.
Macrophage myeloperoxidase regulation by granulocyte macrophage colony-
stimulating factor in human atherosclerosis and implications in acute coronary
syndromes. Am. J. Pathol. 158:879–891; 2001.
[4] Mutze, S.; Hebling, U.; Stremmel, W.; Wang, J.; Arnhold, J.; Pantopoulos, K.;
Mueller, S. Myeloperoxidase-derived hypochlorous acid antagonizes the oxida-
tive stress-mediated activation of iron regulatory protein 1. J. Biol. Chem. 278:
40542–40549; 2003.
[5] Pattison, D. I.; Davies, M. J. Absolute rate constants for the reaction of
hypochlorous acid with protein side chains and peptide bonds. Chem. Res. Toxicol.
14:1453–1464; 2001.
[6] Hawkins, C. L.; Pattison, D. I.; Davies, M. J. Hypochlorite-induced oxidation of
amino acids, peptides and proteins. Amino Acids 25:259–274; 2003.
[7] Hazell, L. J.; van den Berg, J. J.; Stocker, R. Oxidation of low-density lipoprotein by
hypochlorite causes aggregation that is mediated by modification of lysine
residues rather than lipid oxidation. Biochem. J. 302 (Pt 1):297–304; 1994.
[8] Szuchman-Sapir, A. J.; Pattison, D. I.; Ellis, N. A.; Hawkins, C. L.; Davies, M. J.;
Witting, P. K. Hypochlorous acid oxidizes methionine and tryptophan residues in
myoglobin. Free Radic. Biol. Med. 45:789–798; 2008.
[9] Pattison, D. I.; Davies, M. J. Reactions of myeloperoxidase-derived oxidants with
biological substrates: gaining chemical insight into human inflammatory diseases.
Curr. Med. Chem. 13:3271–3290; 2006.
[10] Fu, X.; Mueller, D. M.; Heinecke, J. W. Generation of intramolecular and
intermolecular sulfenamides, sulfinamides, and sulfonamides by hypochlorous
acid: a potential pathway for oxidative cross-linking of low-density lipoprotein by
myeloperoxidase. Biochemistry 41:1293–1301; 2002.
[21] Kettle, A. J. Neutrophils convert tyrosyl residues in albumin to chlorotyrosine.
FEBS Lett. 379:103–106; 1996.
[22] Domigan, N. M.; Charlton, T. S.; Duncan, M. W.; Winterbourn, C. C.; Kettle, A. J.
Chlorination of tyrosyl residues in peptides by myeloperoxidase and human
neutrophils. J. Biol. Chem. 270:16542–16548; 1995.
[23] Winterbourn, C. C.; Kettle, A. J. Biomarkers of myeloperoxidase-derived hypo-
chlorous acid. Free Radic. Biol. Med. 29:403–409; 2000.
[24] Witting, P. K.; Douglas, D. J.; Mauk, A. G. Reaction of human myoglobin and H2O2:
involvement of a thiyl radical produced at cysteine 110. J. Biol. Chem. 275:
20391–20398; 2000.
[25] Witting, P. K.; Mauk, A. G. Reaction of human myoglobin and H2O2: electron
transfer between tyrosine 103 phenoxyl radical and cysteine 110 yields a protein-
thiyl radical. J. Biol. Chem. 276:16540–16547; 2001.
[26] Witting, P. K.; Wu, B. J.; Raftery, M.; Southwell-Keely, P.; Stocker, R. Probucol
protects against hypochlorite-induced endothelial dysfunction: identification of a
novel pathway of probucol oxidation to a biologically active intermediate. J. Biol.
Chem. 280:15612–15618; 2005.
[27] Witting, P. K.; Willhite, C. A.; Davies, M. J.; Stocker, R. Lipid oxidation in human
low-density lipoprotein induced by metmyoglobin/H2O2: involvement of alpha-
tocopheroxyl and phosphatidylcholine alkoxyl radicals. Chem. Res. Toxicol. 12:
1173–1181; 1999.
[28] Zoller, M. J.; Smith, M. Oligonucleotide-directed mutagenesis: a simple method
using two oligonucleotide primers and a single-stranded DNA template. Methods
Enzymol. 154:329–350; 1987.
[29] Varadarajan, R.; Szabo, A.; Boxer, S. G. Cloning, expression in Escherichia coli, and
reconstitution of human myoglobin. Proc. Natl. Acad. Sci. USA 82:5681–5684;
1985.
[30] Witting, P. K.; Mauk, A. G.; Lay, P. A. Role of tyrosine-103 in myoglobin peroxidase
activity: kinetic and steady-state studies on the reaction of wild-type and variant
recombinant human myoglobins with H2O2. Biochemistry 41:11495–11503; 2002.

46
A.J. Szuchman-Sapir et al. / Free Radical Biology & Medicine 48 (2010) 35–46
[31] Rayner, B. S.; Stocker, R.; Lay, P. A.; Witting, P. K. Regio- and stereo-chemical
oxidation of linoleic acid by human myoglobin and hydrogen peroxide: Tyr(103)
affects rate and product distribution. Biochem. J. 381:365–372; 2004.
[32] Eyer, P.; Worek, F.; Kiderlen, D.; Sinko, G.; Stuglin, A.; Simeon-Rudolf, V.; Reiner, E.
Molar absorption coefficients for the reduced Ellman reagent: reassessment. Anal.
Biochem. 312:224–227; 2003.
impairs reverse cholesterol transport by apolipoprotein A-I. Proc. Natl. Acad. Sci.
USA 105:12224–12229; 2008.
[52] Garner, B.; Witting, P. K.; Waldeck, A. R.; Christison, J. K.; Raftery, M.; Stocker, R.
Oxidation of high density lipoproteins. I. Formation of methionine sulfoxide in
apolipoproteins AI and AII is an early event that accompanies lipid peroxidation
and can be enhanced by alpha-tocopherol. J. Biol. Chem 273:6080–6087; 1998.
[53] Panzenbock, U.; Stocker, R. Formation of methionine sulfoxide-containing specific
forms of oxidized high-density lipoproteins. Biochim. Biophys. Acta 1703:
171–181; 2005.
[54] Levine, R. L.; Mosoni, L.; Berlett, B. S.; Stadtman, E. R. Methionine residues as
endogenous antioxidants in proteins. Proc. Natl. Acad. Sci. USA 93:15036–15040;
1996.
[55] Oien, D. B.; Moskovitz, J. Substrates of the methionine sulfoxide reductase system
and their physiological relevance. Curr. Top. Dev. Biol. 80:93–133; 2008.
[56] Maskos, Z.; Rush, J. D.; Koppenol, W. H. The hydroxylation of tryptophan. Arch.
Biochem. Biophys. 296:514–520; 1992.
[57] Bienvenut, W. V.; Deon, C.; Pasquarello, C.; Campbell, J. M.; Sanchez, J. C.; Vestal,
M. L.; Hochstrasser, D. F. Matrix-assisted laser desorption/ionization-tandem
mass spectrometry with high resolution and sensitivity for identification and
characterization of proteins. Proteomics 2:868–876; 2002.
[33] Riddles, P. W.; Blakeley, R. L.; Zerner, B. Reassessment of Ellman's reagent.
Methods Enzymol. 91:49–60; 1983.
[34] Kuma, F. Properties of methemoglobin reductase and kinetic study of methemo-
globin reduction. J. Biol. Chem. 256:5518–5523; 1981.
[35] Livingston, D. J.; McLachlan, S. J.; La Mar, G. N.; Brown, W. D. Myoglobin: cytochrome
b5 interactions and the kinetic mechanism of metmyoglobin reductase. J. Biol.
Chem. 260:15699–15707; 1985.
[36] Bergt, C.; Fu, X.; Huq, N. P.; Kao, J.; Heinecke, J. W. Lysine residues direct the
chlorination of tyrosines in YXXK motifs of apolipoprotein A-I when hypochlorous
acid oxidizes high density lipoprotein. J. Biol. Chem. 279:7856–7866; 2004.
[37] Frangogiannis, N. G.; Smith, C. W.; Entman, M. L. The inflammatory response in
myocardial infarction. Cardiovasc. Res. 53:31–47; 2002.
[38] Davies, M. J.; Hawkins, C. L.; Pattison, D. I.; Rees, M. D. Mammalian heme
peroxidases: from molecular mechanisms to health implications. Antioxid. Redox
Signaling 10:1199–1234; 2008.
[58] Staniszewska, M.; Nagaraj, R. H. Detection of kynurenine modifications in proteins
using a monoclonal antibody. J. Immunol. Methods 324:63–73; 2007.
[59] Helland, R.; Fjellbirkeland, A.; Karlsen, O. A.; Ve, T.; Lillehaug, J. R.; Jensen, H. B. An
oxidized tryptophan facilitates copper binding in Methylococcus capsulatus-
secreted protein MopE. J. Biol. Chem. 283:13897–13904; 2008.
[39] Wittenberg, B. A.; Wittenberg, J. B. Transport of oxygen in muscle. Annu. Rev.
Physiol. 51:857–878; 1989.
[40] Qiu, Y.; Sutton, L.; Riggs, A. F. Identification of myoglobin in human smooth
muscle. J. Biol. Chem. 273:23426–23432; 1998.
[41] Hubbard, S. R.; Hendrickson, W. A.; Lambright, D. G.; Boxer, S. G. X-ray crystal
structure of a recombinant human myoglobin mutant at 2.8 A resolution. J. Mol.
Biol. 213:215–218; 1990.
[42] Witting, P. K.; Douglas, D. J.; Mauk, A. G. Reaction of human myoglobin and nitric
oxide: heme iron or protein sulfhydryl (s) nitrosation dependence on the absence
or presence of oxygen. J. Biol. Chem. 276:3991–3998; 2001.
[60] Hamblin, M.; Friedman, D. B.; Hill, S.; Caprioli, R. M.; Smith, H. M.; Hill, M. F.
Alterations in the diabetic myocardial proteome coupled with increased
myocardial oxidative stress underlie diabetic cardiomyopathy. J. Mol. Cell. Cardiol.
42:884–895; 2007.
[61] Lund, M. N.; Luxford, C.; Skibsted, L. H.; Davies, M. J. Oxidation of myosin by haem
proteins generates myosin radicals and protein cross-links. Biochem. J. 410:565–574;
2008.
[43] Gow, A. J.; Stamler, J. S. Reactions between nitric oxide and haemoglobin under
physiological conditions. Nature 391:169–173; 1998.
[62] Taylor, S. W.; Fahy, E.; Murray, J.; Capaldi, R. A.; Ghosh, S. S. Oxidative post-
translational modification of tryptophan residues in cardiac mitochondrial
proteins. J. Biol. Chem. 278:19587–19590; 2003.
[63] Manzanares, D.; Rodriguez-Capote, K.; Liu, S.; Haines, T.; Ramos, Y.; Zhao, L.;
Doherty-Kirby, A.; Lajoie, G.; Possmayer, F. Modification of tryptophan and
methionine residues is implicated in the oxidative inactivation of surfactant
protein B. Biochemistry 46:5604–5615; 2007.
[44] Patel, R. P.; Hogg, N.; Spencer, N. Y.; Kalyanaraman, B.; Matalon, S.; Darley-Usmar,
V. M. Biochemical characterization of human S-nitrosohemoglobin: effects on
oxygen binding and transnitrosation. J. Biol. Chem. 274:15487–15492; 1999.
[45] Yang, F.; Phillips Jr., G. N. Crystal structures of CO-, deoxy- and met-myoglobins at
various pH values. J. Mol. Biol. 256:762–774; 1996.
[46] Rayner, B. S.; Wu, B. J.; Raftery, M.; Stocker, R.; Witting, P. K. Human S-nitroso
oxymyoglobin is a store of vasoactive nitric oxide. J. Biol. Chem. 280:9985–9993;
2005.
[64] Winterbourn, C. C.; Kettle, A. J. In: Pryor, W.A., ed. Bioassays for Oxidative Stress
Status. New York: Elsevier; 2001.
[47] Zheng, L.; Nukuna, B.; Brennan, M. L.; Sun, M.; Goormastic, M.; Settle, M.; Schmitt,
D.; Fu, X.; Thomson, L.; Fox, P. L.; Ischiropoulos, H.; Smith, J. D.; Kinter, M.; Hazen,
[65] Winterbourn, C. C. Comparative reactivities of various biological compounds with
myeloperoxidase-hydrogen peroxide-chloride, and similarity of the oxidant to
hypochlorite. Biochim. Biophys. Acta 840:204–210; 1985.
S. L. Apolipoprotein A-I is
a selective target for myeloperoxidase-catalyzed
oxidation and functional impairment in subjects with cardiovascular disease.
J. Clin. Invest. 114:529–541; 2004.
[66] Hagler, L.; Coppes Jr., R. I.; Herman, R. H. Metmyoglobin reductase: identification
and purification of
a reduced nicotinamide adenine dinucleotide-dependent
[48] Bergt, C.; Pennathur, S.; Fu, X.; Byun, J.; O'Brien, K.; McDonald, T. O.; Singh, P.;
Anantharamaiah, G. M.; Chait, A.; Brunzell, J.; Geary, R. L.; Oram, J. F.; Heinecke,
J. W. The myeloperoxidase product hypochlorous acid oxidizes HDL in the human
artery wall and impairs ABCA1-dependent cholesterol transport. Proc. Natl. Acad.
Sci. USA 101:13032–13037; 2004.
enzyme from bovine heart which reduces metmyoglobin. J. Biol. Chem. 254:
6505–6514; 1979.
[67] Galaris, D.; Korantzopoulos, P. On the molecular mechanism of metmyoglobin-
catalyzed reduction of hydrogen peroxide by ascorbate. Free Radic. Biol. Med. 22:
657–667; 1997.
[49] Marsche, G.; Furtmuller, P. G.; Obinger, C.; Sattler, W.; Malle, E. Hypochlorite-
modified high-density lipoprotein acts as a sink for myeloperoxidase in vitro.
Cardiovasc. Res. 79:187–194; 2008.
[50] Shao, B.; Oda, M. N.; Bergt, C.; Fu, X.; Green, P. S.; Brot, N.; Oram, J. F.; Heinecke,
J. W. Myeloperoxidase impairs ABCA1-dependent cholesterol efflux through
methionine oxidation and site-specific tyrosine chlorination of apolipoprotein A-I.
J. Biol. Chem. 281:9001–9004; 2006.
[68] Yusa, K.; Shikama, K. Oxidation of oxymyoglobin to metmyoglobin with hydrogen
peroxide: involvement of ferryl intermediate. Biochemistry 26:6684–6688; 1987.
[69] Wazawa, T.; Matsuoka, A.; Tajima, G.; Sugawara, Y.; Nakamuram, K.; Shikama, K.
Hydrogen peroxide plays a key role in the oxidation reaction of myoglobin by
molecular oxygen: a computer simulation. Biophys. J. 63:544–550; 1992.
[70] Stefanescu, A.; Braun, S.; Ndrepepa, G.; Koppara, T.; Pavaci, H.; Mehilli, J.; Schömig,
A.; Kastrati, A. Prognostic value of plasma myeloperoxidase concentration in
patients with stable coronary artery disease. Am. Heart J. 155:356–360; 2008.
[51] Shao, B.; Cavigiolio, G.; Brot, N.; Oda, M. N.; Heinecke, J. W. Methionine oxidation