Hypochlorous acid oxidizes methionine and tryptophan residues in myoglobin

Free Radical Biology & Medicine 45 (2008) 789–798

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Free Radical Biology & Medicine j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f r e e r a d b i o m e d

Original Contribution

Hypochlorous acid oxidizes methionine and tryptophan residues in myoglobin Andrea J. Szuchman-Sapir a, David I. Pattison b, Natasha A. Ellis a, Clare L. Hawkins b, Michael J. Davies b, Paul K. Witting a,⁎ a b

Vascular Biology Group, ANZAC Research Institute, Concord Repatriation General Hospital, Concord, NSW 2139, Australia The Heart Research Institute, 114 Pyrmont Bridge Road, Sydney, NSW 2050, Australia

a r t i c l e

i n f o

Article history: Received 21 April 2008 Revised 14 May 2008 Accepted 3 June 2008 Available online 19 June 2008 Keywords: Hypochlorous acid Myoglobin Methionine oxidation Chloramines Myocardial inflammation Free radicals

a b s t r a c t After acute myocardial infarction (AMI), infiltrating proinflammatory cells generate two-electron oxidants such as hypochlorous acid (HOCl). Myoglobin (Mb) is present at ∼ 0.3 mM in cardiomyocytes and, therefore, represents a significant target for oxidation. Exposure of horse Mb (50 μM) to reagent HOCl (0–500 μM) or activated human neutrophils (4–40 × 106 cells/ml) yielded oxidized Mb (Mbox) as judged by amino acid analysis and peptide mass mapping. HOCl/Mb ratios of 1–5 mol/mol gave Mbox with up to four additional oxygen atoms. Hydrolysis of Mbox followed by amino acid analysis indicated that methionine (Met) and tryptophan (Trp) residues were modified by HOCl. Peptide mass mapping revealed that Met55 was oxidized at a lower HOCl/Mb ratio than Met131 and this preceded Trp7/14 modification (susceptibility Met55 N Met131 N Trp7 N Trp14). Incubation of Mb with activated neutrophils and physiological chloride anion yielded Mbox with a composition similar to that determined with HOCl/Mb ratios b 2 mol/mol, with oxidation of Met, but not Trp, detected. These data indicate that Mb undergoes site-specific oxidation depending on the HOCl/protein ratio. As Mb is released from necrotic cardiomyocytes into the vasculature after AMI, HOClmodified Mb may be a useful surrogate marker to gauge the extent of myocardial inflammation. © 2008 Elsevier Inc. All rights reserved.

The mechanisms underlying the progression of inflammation and its relationship to the development of heart failure after acute myocardial infarction (AMI)1 are poorly understood. Initially, ischemic insult to cardiac tissue stimulates oxidative stress, which manifests as an increased production of reactive oxygen species (ROS) in the affected myocardium. Reperfusion of the ischemic tissue leads to further uncontrolled ROS production that is linked to the influx of calcium and blockade of the potassium-ATP (KATP) channels in cardiomyocytes [1] that promote dysfunction in heart tissues. Interestingly, early reperfusion of the tissue limits the infarct size and expansion, whereas late reperfusion, which does not reduce infarct size, can assist in healing through eliciting an inflammatory response, which stimulates ventricular remodeling [2–4]. However, there is now considerable evidence indicating that inflammation also expands myocardial injury [5,6] and may be associated with the early development of congestive heart failure [7]. After AMI, mononuclear cells such as neutrophils and leukocytes are recruited to the site of inflammation [6]. These cell types, when activated, release the enzyme myeloperoxidase [8,9], which catalyzes the reaction of chloride ions and hydrogen peroxide, producing hypochlorous acid (HOCl), a potent oxidant that readily reacts with a

Abbreviations: AMI, acute myocardial infarction; DTT, dithiothreitol; hhMb, horse heart Mb; KATP, potassium-ATP; Mb, myoglobin; Mbox, oxidized Mb; PBS, phosphatebuffered saline; PMA, phorbol 12-myristate 13-acetate; ROS, reactive oxygen species. ⁎ Corresponding author. Fax: +61 2 9767 9101. E-mail address: [email protected] (P.K. Witting). 0891-5849/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2008.06.010

wide range of biological molecules [10–13]. Up to 80% of the H2O2 generated by inflammatory cells is utilized to form a range of reactive oxygen species, with HOCl generated at rates between 20 and 400 μM/h accounting for 40–50% of H2O2 consumed in the presence of physiologic chloride anion [14–16]. Hypochlorous acid is cell permeable and capable of promoting both oxidation and chlorination of proteins, lipids, and nucleic acids within host tissues [17–22]. Within proteins, the sulfur-containing residues, Cys and Met, react most rapidly with HOCl, but also the side chains of Lys, His, Trp, and Tyr and the α-amino groups of amino acids and peptides are oxidized and/or chlorinated [18,22,23]. Reaction of HOCl with Cys produces disulfides, cysteic acid, and sulfonamides, apparently via sulfenic acid intermediates, which may decompose upon reaction with amines [24–27]. Reaction of HOCl with Met it produces Met sulfoxide [28,29], which has been proposed to cause protein aggregation and dysfunction [30]. Reaction of HOCl with Tyr residues can yield chlorinated Tyr derivatives, which have been employed as specific markers of HOCl-induced reactions in vivo [31,32]. Additionally, Trp residues in proteins are susceptible to HOCl oxidation [33,34] and protein amines (N-terminal and Lys) are readily chlorinated by HOCl, to yield unstable chloramines, which can lead to the formation of reactive radical intermediates [35,36]. Proteins are the major component of most biological systems, and the rate constant for the reaction of amino acid side chains with HOCl is more than 10 times higher than that for reactions with lipids and some antioxidants [12]. One potential target for HOCl in myocardial tissues is myoglobin (Mb), a 17-kDa heme protein that is also found in skeletal

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and smooth muscle fibers [37,38]. Myoglobin is present in relatively high abundance in cardiac myocytes with concentrations ranging from 0.3 to 0.5 mM [38]. Cytosolic Mb is involved in the storage of molecular oxygen and facilitates its transport by shuttling between the sarcolemma and the mitochondria for oxidative phosphorylation and is capable of releasing oxygen during hypoxia [39,40]. Herein the effects of reagent HOCl and activated neutrophils on the most abundant protein in cardiomyocytes have been investigated, and it has been demonstrated that Mb is modified through site-specific addition of up to four oxygen atoms at the Met and Trp residues on the protein backbone.

1739 g), and the precipitate was washed twice with ice-cold acetone and then dried under N2(g). Protein pellets were reduced with 45 mM DTT and incubated at 50°C for 15 min followed by alkylation with 100 mM iodoacetamide at room temperature for 15 min. Sequencing-grade trypsin (Promega) was added at a ratio of 63:1 (w/w) Mb:trypsin in digestion buffer (PBS containing a final concentration of 2 M urea and 0.1 M NH4HCO3) and incubated overnight at 37°C. Digestion was terminated by acidifying the samples with trifluoroacetic acid (0.1% v/v) solution and rapidly freezing.

Experimental procedures

Neutrophils were isolated from the peripheral blood of at least three individual healthy human donors by centrifugation through Ficoll-Hypaque, dextran sedimentation, and hypotonic lysis of contaminating red cells [44]. Isolated neutrophils were washed twice in Hanks' balanced salt solution, pH 7.4, prepared without calcium chloride, magnesium chloride, magnesium sulfate, sodium bicarbonate, or phenol red (buffer A). Next, the neutrophils were resuspended in buffer A, adjusted to a final density of 2 × 106 or 10 × 106 cells/mL, equilibrated at 37°C for 10 min, and then immediately mixed with hhMb (50 μM) in the presence or absence of 1.25 μg/ml phorbol 12myristate 13-acetate (PMA). After 2 h, individual reactions were terminated by centrifugation (377 g) at 4°C for 10 min to remove the cell pellet. Supernatants were then digested with trypsin and stored at −20°C for mass analysis.

Materials All chemicals were obtained from Sigma/Aldrich/Fluka (Castle Hill, NSW, Australia) with the exception of trypsin (sequencing grade; Promega, Madison, WI, USA) and Ficoll-Hypaque (Amersham Bioscience, Uppsala, Sweden). HOCl stock solution was standardized by measuring the absorbance at 292 nm [ɛ292 (−OCl) = 350 M− 1 cm− 1] and 235 nm [ɛ235 (HOCl) = 100 M− 1 cm− 1] [41]. HOCl solutions were prepared immediately before use by diluting the concentrated stock into phosphate-buffered saline (PBS; 250 mM, pH 7.4) that had been pretreated with Chelex resin to remove contaminating trace metal ions. Ferric horse heart Mb (hhMb) solutions were prepared immediately before use in 250 mM PBS (pH 7.4) and standardized by determining the peak absorbance at 409 nm (ɛ402 = 188,500 M− 1 cm− 1) [42]. Reaction conditions Reactions were carried out at 37°C for 15 min in 250 mM PBS (pH 7.4) containing 50 μM hhMb. All reagents and protein solutions were prepared fresh, and oxidation was initiated by adding a bolus of reagent HOCl (0- to 5-fold mol excess) to yield a final concentration as indicated in the figure legends.

Human neutrophils

Chloramine determination Chloramines were quantified by reaction with 5-thio-2-nitrobenzoic acid (TNB) as described previously [45]. Briefly, hhMb (50 μM) was treated with HOCl (0- to 5-fold mol excess) and the concentration of TNB monitored (after reaction with protein chloramines) at A412 nm (ɛ412 nm = 14,100 M− 1 cm− 1) [45] over 5–180 min and after 24 h. Liquid chromatography (LC) electrospray ionization mass spectrometry (ESI-MS)

Methanesulfonic acid hydrolysis and amino acid analysis Where required hhMb was precipitated by the addition of sodium deoxycholate (0.015% w/v) and trichloroacetic acid (5% w/v), and the precipitate was collected by centrifugation (2 min at 2739 g). The protein pellets were washed twice with ice-cold acetone and dried under a stream of N2 gas before being resolubilized in 150 μl of 4 M methanesulfonic acid containing 0.2% (w/v) tryptamine [43]. The samples were transferred to Picotag hydrolysis vessels and back flushed three times with N2 before being placed under vacuum and heated at 110°C. After 17 h, the hydrolysates were neutralized with 4 M NaOH, filtered (0.45-μm pore size; Millipore), and diluted (50-fold) before analysis. Individual amino acids were separated by HPLC after derivatization with o-phthaldialdehyde reagent (Sigma) containing 0.5% (v/v) 2-mercaptoethanol for 1 min before injection onto a Beckman Ultrasphere ODS column (4.6 × 250 mm, 5-μm particle size; BeckmanCoulter) as described previously [34]. Amino acid derivatives were monitored by fluorescence detection (RF 10A-XL; Shimadzu, Rydalmere, NSW, Australia) with λex 340 nm and λem 440 nm. Finally, each amino acid was quantified by comparison with the corresponding amino acid standard (500 μM stock solution; Sigma) containing 5 μM Met sulfoxide. The peak areas were normalized to the corresponding content of valine (a residue that does not react significantly with HOCl [22]) and expressed as a percentage of the control (hhMb alone) in the absence of HOCl. Proteolytic digestion Where required hhMb (50 μM) reaction solutions were precipitated by the addition of trichloroacetic acid (5% v/v) and centrifuged (2 min at

LC/MS analyses were performed in 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 at a flow rate of 0.4 ml/min on a reverse-phase column (Zorbax C18 MS column, 3 μm, 25 cm) using solvent A (0.1% v/v trifluoroacetic acid in water) and solvent B (0.1% v/v trifluoroacetic acid in CH3CN). Peptides were eluted using the following gradient: 5 to 10% B over 20 min, 10 to 20% B over 10 min, 20 to 50% B over 10 min, then 50 to 90% B over 20 min. The electrospray needle was held at 4500 V. Nitrogen, the sheath gas, was set at 80 units. The collision gas was helium. The temperature of the heated capillary was 250°C. Statistical analyses Analyses were performed with Prism software (v3.0; GraphPad, Inc). Differences were assessed between groups with ANOVA employing the Tukey multiple comparison test or Bonferroni post hoc test for specific comparisons. A value of p b 0.05 was considered significant in all tests. Results HOCl modifies isolated hhMb To assess whether HOCl modifies hhMb in vitro, hhMb (50 μM) was exposed to 5 mol equivalents of reagent HOCl or the identical volume of PBS (control) for 15 min at 37°C. After the reactions were quenched on ice, the products were analyzed by mass spectrometry (Fig. 1). Deconvolution of the mass-to-charge data from the corresponding

A.J. Szuchman-Sapir et al. / Free Radical Biology & Medicine 45 (2008) 789–798

Fig. 1. Deconvoluted ESI-mass spectra of hhMb treated with or without reagent HOCl. Samples of hhMb (50 μM) were exposed to 5 mol excess of HOCl and incubated at 37°C in phosphate buffer (pH 7.4). After 15 min, residual HOCl and protein chloramines were quenched on ice and the protein fraction was analyzed by ESI-MS. Deconvoluted mass spectra are shown for (left) the vehicle-treated control and (right) the HOCl-reaction products.

control (Fig. 1, left) or HOCl-treated samples (Fig. 1, right) indicated oxidative modification to native hhMb with multiple species present, each differing by 16 mass units, corresponding to the addition of an oxygen atom. In total, the addition of up to four oxygen atoms was detected at the highest HOCl:Mb ratio tested (Fig. 1, right).

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After identification of the parent peptides of interest, hhMb samples treated with increasing concentrations of HOCl (0- to 5-fold molar excess) were investigated. After 15 min, reaction mixtures were digested with trypsin and the modified peptides were identified by LC/MS/MS using the parameters established for the analysis of peptides from the native protein. Increasing concentrations of HOCl led to loss of the peak corresponding to the peptide sequence HLKTEAEMK (1086.7 m/z) at 37.7 min (Fig. 3), with the concomitant appearance of a new peak eluting at 32.2 min with a mass-to-charge ratio of 1101.3 m/z (Fig. 3). Subsequently, MS/MS analysis of the eluted peptide combined with systematic mass-matching to the predicted sequence confirmed the HLKTEAEMK sequence. Furthermore, the Met55 residue (Figs. 4A and 4B) was clearly identified by mass difference (see arrow in Fig. 4B). Identical analysis of the modified peptide (mass-to-charge ratio 1101.3 m/z) indicated an increase of 16 atomic mass units ([M + 16 + H]+ 1, Fig. 4C). Additional, MS/MS characterization revealed that the mass-to-charge ratio for Met55 was shifted 16 atomic mass units (Fig. 4D), supporting the conclusion that Met55 was oxygenated to the corresponding Met sulfoxide after exposure to HOCl. Loss of the HPGNFGADAQGAMTK peptide (1502.4 m/z) from mixtures of hhMb exposed to increasing HOCl concentrations occurred in parallel with the appearance of a new peptide (1518.4 m/z) with retention time of 39.8 min (data not shown). Corresponding MS/MS

Quantification of specific amino acid side-chain loss and product formation To determine whether the oxidation of specific amino acid residues contributed to the oxidative modification of hhMb, the relative susceptibility of side chains of hhMb to damage by HOCl was investigated by total amino acid analysis (Fig. 2). Addition of HOCl stimulated a dose-dependent decrease in the content of Met together with a corresponding increase in the yield of Met sulfoxide, which reached significance at HOCl:Mb ratios of 2.5–5 mol/mol (Fig. 2A). In contrast to Met, Trp was initially resistant to oxidation; however, the content of Trp also decreased significantly at HOCl:Mb ratios of 2.5– 5 mol/mol. No change in the concentration of other susceptible amino acids was observed. For example, Tyr and Lys remained unaffected at the concentrations of HOCl employed (Fig. 2A). Interestingly, although isolated His is susceptible to HOCl oxidation [46], no modification of His residues was detected after exposing hhMb to HOCl (data not shown). Employing the amino acid data for Met and Met sulfoxide, the Met redox state (defined as the ratio Met/(Met + MetSO)) decreased significantly with increasing concentrations of HOCl (Fig. 2B). HOCl oxidizes specific residues in hhMb To investigate further the molecular basis for the oxidative modification of the hhMb, native hhMb was digested with trypsin and the resulting peptides were identified through analysis with LC/MS and tandem MS/MS. Overall, the trypsin digest contained 11 peptides that covered 81% of the predicted sequence of the native protein (Table 1). The peptides that included the amino acids Met and Trp were investigated further, as they were known to be susceptible to oxidation by HOCl as assessed in the earlier amino acid analysis. The peptide sequences HLKTEAEMK (position 48–56, m/z 1086.7), containing Met55; HPGNFGADAQGAMTK (position 119–133, m/z 1502.4), containing Met131; and GLSDGEWQQVLNVWGK (position 1–16, m/z 1816.5), containing Trp at positions 7 and 14, were detected at retention times of 37.7, 42.3, and 49.8 min, respectively. An additional low-molecular-weight peptide with sequence TEAEMK that contained Met55 (position 51–56) was not employed in further analyses due to the poor detection of the peptide fragment. Instead, an incomplete protein digestion method was optimized using a lower ratio of hhMb to trypsin (63:1 v/v) and Met 55 was detected in the larger sequence noted above (position 48–56, Table 1).

Fig. 2. Amino acid analyses indicate oxidative modification of Met and Trp residues on HOCl-modified hhMb. Protein samples (50 μM) were treated with reagent HOCl (at the final concentrations indicated) or vehicle (control) and incubated at 37°C. After 15 min, the reaction was quenched on ice and the protein hydrolyzed with methanesulfonic acid (4 M). (A) The consumption of Met, Trp, Tyr, and Lys and the formation of MetSO (dark bars) upon treatment of hhMb (50 μM) with 0- to 5-fold molar excess of HOCl. Amino acid data are normalized to the corresponding valine content of the corresponding samples (valine is the internal standard of choice as it is not modified by HOCl). (B) The Met redox state (defined as the ratio of total Met/total (Met + MetSO)). ⁎p b 0.05 or ⁎⁎p b 0.01, significantly different from the control as determined by one-way ANOVA with Tukey's multiple comparison test. Data are presented as the means ± SD of four experiments using independent preparations of Mb and HOCl stock solutions.

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Table 1 Peptide sequences from complete and partial digestion of native and HOCl-oxidized hhMba Position

Fragment sequence

Predicted m/z ([M + H]+)

Observed

1–16 1–16 1–16 17–31 32–42 51–56 48–56 48–56 64–77 80–96 103–118 119–133 119–133 134–139 140–145

GLSDGEWQQVLNVWGK GLSDGEWQQVLNVWGK GLSDGEWQQVLNVWGK VEADIAGHGQEVLIR LFTGHPETLEK TEAEMK ⁎HLKTEAEMK ⁎HLKTEAEMK HGTVVLTALGGILK GHHEAELKPLAQSHATK YLEFISDAIIHVLHSK HPGDFGADAQGAMTK HPGDFGADAQGAMTK ALELFR NDIAAK

1817.0 1833.0 1849.0 1607.8 1272.4 708.8 1086.5 1102.5 1379.6 1855.0 1886.2 1503.6 1519.6 748.8 631.7

1816.5, 909.1 (+ 2) 1832.7 1847.4 1606.9, 804.6 (+2) 1271.5, 637.1 (+2) 708.5 1086.3 1101.3 1378.8, 690.8 (+2) 1855.8, 927.7 (+2) 1885.6, 943.5 (+2) 1502.4, 752.5 (+2) 1518.4 748.6 631.49

Modification Trp7 + 16 Trp7 + 16, Trp14 + 16

Met55 + 16

Met131 + 16

a Samples of hhMb (50 μM) were exposed to 0- to 5-fold molar excess of HOCl and the protein was then digested with trypsin (hhMb:trypsin 25:1 v/v) to afford a complete digestion of the heme protein. In some cases samples were treated with hhMb:trypsin 63:1 v/v to afford a partial digestion. The asterisk indicates peptides formed from an incomplete digestion by trypsin. The peptides were separated by LC/MS and analyzed by MS/MS. Peptide sequences were confirmed using MS/MS and a peptide database (Sequest). Results are representative of four independent experiments. Overall, the peptide recovery covered 81% of the predicted sequence of hhMb.

analysis (Fig. 5A) of the peak confirmed the sequence of the native peptide, which contained Met131 (see arrow in Fig. 5B). The modified peptide (1518.4 m/z) showed an increase of 16 atomic mass units ([M + 16 + H]+ 1, Fig. 5C) as assessed by MS. Tandem MS/MS analysis of the modified peptide (Fig. 5D) was unable to detect a specific ion corresponding to the oxidation of Met131. However, the data revealed an addition of 16 atomic mass units to peptide ions formed from bond cleavage after the Met131 residue (that is, fragments b14 + 16, b15 + 16). Furthermore, examination of the series of y ions (y5 + 16, y6 + 16, and y7 + 16; Fig. 5D) confirmed that Met131 gained 16 atomic mass units. Thus, as with Met55, Met131 was also oxygenated to Met sulfoxide in the presence of HOCl. No further modifications to these peptides were observed, despite the presence of amino acid residues such as His and Lys that are known targets for HOCl. In addition, there was no evidence

Fig. 3. LC/MS analyses of specific oxidative modification of Met55 that is formed in hhMb exposed to reagent HOCl. Protein samples (50 μM) were treated with vehicle (control, trace A) or reagent HOCl (0.5- to 5-fold molar excess, traces B–E, respectively) and incubated at 37°C. After 15 min, the protein fraction was digested by incubating with trypsin overnight at 37°C. The resulting peptides were separated by reverse-phase HPLC with gradient elution as described in detail under Experimental Procedures. Representative chromatograms show the loss of the Met55 HLKTEAEMK peptide (peak eluting at 37.7 min) with the concomitant appearance of a new peak (eluting at 32.2 min), which was further analyzed by ESI-MS/MS. Data are representative of four experiments using independent preparations of hhMb and HOCl stock solutions.

for further oxidation of the Met residues to Met sulfone (unlike similar studies with the corresponding isolated amino acid [34]). Quantification of HOCl-mediated oxidation of Met and Trp in hhMb The loss of Met and concomitant formation of Met sulfoxide determined by amino acid analyses was verified by LC/MS/MS by measuring the loss of the Met-containing native peptides and the generation of their corresponding modified forms with increasing HOCl concentrations. Overall, the formation of the oxidized peptides occurred with a parallel consumption of the corresponding Met residue after exposure to HOCl (Figs. 6A–6D). A comparison of the extent of oxidation for Met55 and Met131 at the same HOCl:hhMb ratio indicated a differential extent of oxidation. Thus, the sulfoxide level of Met55 reached 45.7 ± 9.6 and 86.1 ± 21.6% (compared to the maximal levels observed with a fivefold molar excess of HOCl) at HOCl:hhMb ratios of 0.5 and 1 mol/mol, respectively. In contrast, sulfoxide levels on Met131 reached only 18.2 ± 5.1 and 41.7 ± 6.9% (of their maximal levels at HOCl:hhMb of 5) at the same molar ratios. Together these data indicate that HOCl exhibits a slight preference for oxidation of Met55 over Met131, within the dose range studied here. In addition to Met, the amino acid studies suggested that Trp was also susceptible to oxidation by HOCl. To corroborate these chromatographic results, the peptide containing both Trp residues of hhMB, GLSDGEWQQVLNVWGK (1816.5 m/z), was investigated to determine whether there was any modification in the absence or presence of HOCl (Fig. 7). Simple MS analyses of control (vehicle treated) samples confirmed the sequence of the peptide (Fig. 7A), whereas the ions relating to Trp7 and Trp14 are exemplified by the data shown in Fig. 7B. Increasing HOCl:hhMb ratios led to a decrease in the content of native peptide and the formation of two new species. The first product was detected in reactions of hhMb with a 0.5 molar excess of HOCl and exhibited a mass-to-charge ratio of 1832.5 m/z, which corresponded to an increase of 16 atomic mass units relative to the native peptide (cf. Figs. 7A and 7C). A second species was detected under conditions where the HOCl:hhMb ratio was N1 mol/mol. This second product exhibited a mass-to-charge ratio of 1847.5 m/z that corresponded to an increase of 32 atomic mass units compared with the control (cf. Figs. 7A and 7E). Subsequent MS/MS analyses of the 1832.5 m/z peptide product revealed the addition of 16 mass units to Trp7, whereas Trp14 remained unaffected (Fig. 7D). At HOCl:hhMb ratios N1 mol/mol oxidation of the hhMb occurred at both Trp7 and Trp14, as evidenced by the shift of 16 atomic mass units for both Trp residues to yield an overall increase of 32 atomic

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Fig. 4. LC/MS and MS/MS analyses of the Met55 modified peptide formed in hhMb exposed to reagent HOCl. Samples of hhMb were treated with or without HOCl under conditions identical to those described in the legend to Fig. 3. The products were isolated by HPLC and analyzed by (A, C) MS and (B, D) MS/MS and their identities were confirmed by comparison with a peptide database (Sequest). The protonated hhMb ion with m/z 1086.7 corresponded to the HLKTEAEMK peptide (peak eluting at 37.7 min in Fig. 3). Comparisons of the corresponding MS (A and C) and MS/MS (B and D) spectra indicated the addition of an oxygen atom to Met55 in the peptide HLKTEAEMK (peak eluting at 32.2 min, m/z 1101.3). Note that the mass of the b8 fragment ion in the native peptide with m/z 940.3 (B) has increased by 16 atomic mass units to yield a peptide with m/z 956.3 (D). Data are representative of four experiments using independent preparations of hhMb and HOCl stock solutions.

mass units for the protein fragment (Fig. 7F). No further incorporation of oxygen atoms into the oxidized Trp residues and no other modifications to the peptide were detected at the oxidant concentrations studied, suggesting that the Trp residues were the sole targets for HOCl in this protein fragment. Analogous to the amino acid data (Fig. 1A), the peak response for the peptide containing native Trp7 and Trp14 decreased at higher HOCl concentrations than that for the peptides containing Met. However, significant decreases in peak response were observed with HOCl:hhMb ratios between 2.5 and 5 mol/mol (Fig. 8A). Monitoring the product with mass-to-charge ratio of 1847.5 m/z indicated a progressive increase in the amount of peptide in which both Trp residues had been oxidized and this reached a maximum at a HOCl:hhMb ratio of 5 mol/mol (Fig. 8B).

Myoglobin oxidation induced by activated human neutrophils The potential biological relevance of HOCl modification of Mb was examined by investigating the pattern of hhMb oxidation after exposure of the protein to PMA-activated human neutrophils. Isolated neutrophils were employed at two different cell densities (4 × 106 and 20 × 106 cells/ml) while maintaining a fixed concentration of hhMb. Oxidized products were readily detected by sampling the extracellular milieu using the combined approach of LC/ESI-MS. Two modified peptides were detected in the supernatant of the activated cells, and their identities were confirmed by MS/MS analysis (data not shown). A comparison of native and neutrophil-treated protein revealed an addition of 16 atomic mass units to Met55 (Fig. 9A) forming the

Fig. 5. LC/MS and MS/MS analyses of the Met131 modified peptide formed in hhMb exposed to reagent HOCl. Samples of hhMb were oxidized with HOCl as described in the legend to Fig. 3. Products were isolated by HPLC and analyzed by (A, C) MS and (B, D) MS/MS and their identities were confirmed by comparison with a peptide database. Comparisons between the MS (A and C) and the MS/MS (B and D) spectra show the ion generated from the Met131-containing peptide HPGNFGADAQGAMTK (m/z 1502.4) and the corresponding ion from the oxidized Met131-containing peptide HPGNFGADAQGAMTK (m/z 1518.4), indicating an increase of 16 atomic mass units ([M + 16 + H]+. Subsequent MS/MS analysis of the m/z 1502.4 peak confirmed the sequence of the native peptide, which contained Met131 (see arrow in B), whereas MS/MS of the modified peptide m/z 1518.4 (D) revealed an addition of 16 atomic mass units within the peptide fragment ions formed from bond cleavage after the Met131 residue (b14 + 16, b15 + 16 from the N-terminus and y5 + 16, y6 + 16, y7 + 16 from the C-terminus). By contrast, daughter fragments cleaved before Met131 in the sequence of the peptide residue remained unchanged (b⁎8 and b⁎10). b⁎ ions refer to b fragmentations that have lost ammonia (NH3). Data are representative of four experiments using independent preparations of hhMb and HOCl stock solutions.

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Discussion

Fig. 6. Relative content of native and oxidized Met55- and Met131-containing peptides in hhMb exposed to reagent HOCl. Samples of hhMb (50 μM) were exposed to 0- to 5-fold molar excess of HOCl and digested with trypsin. The resultant peptides were then separated by LC/MS. (A) Met55- and (B) Met131-containing peptides (m/z 1086.7 and m/z 1502.4, respectively) and their corresponding oxidized products (m/z 1101.3 and m/z 1518.4, C and D, respectively) were quantified and expressed relative to the corresponding peak areas, either in control samples (for the native peptides) or in the fully oxidized samples (for the MetSO-containing peptides). ⁎p b 0.05 or ⁎⁎p b 0.01, significantly different from the control as determined by one-way ANOVA with Tukey's multiple comparison test. Data are the means ± SEM of four independent experiments.

modified product previously identified with mass-to-charge 1102 m/z (Fig. 4). The second product contained a modification to the peptide containing Met131 (Fig. 9B) previously identified with mass-to-charge 1518 m/z (Fig. 5). Quantitation of the modified peptides revealed that Met55 reached a near-maximal extent of oxidation (88.2 ± 43.2%) when incubated with 2 × 106 neutrophils/ml compared to the levels of oxidized peptide observed with the higher neutrophil density (Fig. 9A). In contrast, under identical conditions the peptide containing oxidized Met131 reached only 43.4 ± 19.9% of that observed in the samples exposed to the higher neutrophil density (Fig. 9B). In these experiments with activated neutrophils, no other modified peptides were observed, in particular the single peptide containing Trp7 and Trp14 remained unchanged in the presence or absence of activated neutrophils (even though the amount of HOCl produced by the activated neutrophils yielded an approximate molar ratio of 1:1 HOCl to hhMb; data not shown). Overall, these data suggest that under the conditions employed, the HOCl generated by the activated neutrophils is insufficient to modify Trp in hhMb as it may be consumed by alternate (protein) targets such as those released by the activated mononuclear cells [47]. Together these observations suggest that HOCl generated by activated neutrophils preferentially oxidizes Met55, similar to that observed with a bolus addition of HOCl. Quantification of chloramine intermediates Reactive chloramines formed through the reaction of HOCl with amine and amide groups on hhMb (HOCl/hhMb ratios ranging 0–5 mol/mol) were monitored closely over an initial 3-h period and then again after 24 h of incubation. Significant concentrations of chloramines were detected with 2.5- and 5-fold molar excess of HOCl (Fig. 10). Importantly, and irrespective of the HOCl/hhMb ratio, the yield of chloramines formed on hhMb remained stable over the first 15 min of monitoring. However, at times N60 min (up to 24 h) significant chloramine decomposition was detected.

Inflammation of the myocardium after AMI represents a significant clinical problem. Infiltrating neutrophils and other mononuclear cells can produce potent oxidants that damage targets within cardiac myocytes (e.g., myoglobin that is present at 0.3–0.5 mM [38]). Here it is demonstrated that hhMb undergoes site-specific modifications in the presence of HOCl and this depends on the oxidant/protein ratio. Amino acid analyses revealed the specific loss of Met and Trp residues with parallel formation of Met sulfoxide at HOCl/hhMb ratios of 0.5– 5 mol/mol. These modifications were accompanied by the formation of protein chloramines that remained stable in the time frame for oxidation of Met and Trp amino acids. No significant decrease in the concentration of other amino acids was detected. Tandem MS analyses of the tryptic peptide digests from HOCl-treated hhMb indicated that peptides containing Met and Trp residues contained a combined total of up to four oxygen atoms. Peptide mass mapping studies confirmed that Met55 is oxidized at a lower HOCl/Mb ratio than Met131 and that both these targets are modified before Trp in the intact protein. In addition, the peptide containing Trp7 and Trp14 was modified by the addition of 16 mass units at relatively low HOCl concentration (HOCl/ Mb ratio of 0.5) and by 32 mass units at HOCl/Mb ratios N1, suggesting the addition of up to two oxygen atoms to this peptide. Subsequent MS/MS analyses revealed that Trp residues were oxygenated, with Trp7 more susceptible to oxidation than Trp14. This selective oxidation of Met and Trp is generally consistent with the order of oxidation susceptibility predicted by a computational model that combines the known amino acid composition of hhMb with the rate constants for the reactions of the corresponding isolated amino acids and HOCl [23]. Furthermore, the extent of gross amino acid consumption observed at various HOCl concentrations in these experimental studies (Fig. 2) is closely predicted by the computational model. However, in the intact protein the extent of oxidation followed the order Met55 N Met131 N Trp7 N Trp14, indicating that protein structure plays a role in defining oxidation susceptibility. Activated human neutrophils were also capable of promoting oxidation to Met55 and Met131 in hhMb. The physiological relevance of HOClinduced Mb oxidation is not known. If the pattern of Met and Trp oxidation in Mb is specific to neutrophil-derived HOCl, then it is possible that oxidized Mb may be a useful surrogate to assess the extent of myocardial inflammation. Oxidation of the apoprotein form of hhMb by reagent HOCl has been studied previously [30]. In the absence of the heme prosthetic group, HOCl causes protein aggregation and yields hhMb oligomers at oxidant/hhMb ratios similar to those employed here. Cross-linking of hhMb was not attributed to the formation of bimolecular covalent bonds, although an increase in the protein mass of 16 and 32 atomic mass units was suggested to occur through the oxidation of Met residues. Treatment of apo-hhMb with HOCl also resulted in the loss of protein fluorescence consistent with a loss of Trp. By contrast, the detailed mass mapping studies using intact holo-hhMb reported here demonstrate an unambiguous assignment of Met and Trp oxidation and suggest a specific order of reactivity toward HOCl for these residues. The crystal structure of hhMb reveals that Met55 is located at the protein surface, whereas Met131, Trp7, and Trp14 are located toward the hydrophobic core of the α-helical structure (not shown). Interestingly, oxidation of Met residues at the protein surface in general does not affect protein tertiary structure [48]. By contrast, the oxidation of Met in other environments can result in significant structural changes that interfere with protein activity, with both decreases [13,28] and increases in enzyme activity reported [49]. This regio-comparison of the sites for Met and Trp in the structure of Mb probably explains the differences in susceptibility of Met55 to oxidation by HOCl, with the latter approaching the protein from the hydrophilic environment. However, an explanation for the subtle

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Fig. 7. LC/MS and MS/MS analyses of the Trp7/Trp14 peptide in hhMb exposed to reagent HOCl. Samples of hhMb were treated with HOCl as described in the legend to Fig. 3. The products were isolated by HPLC and analyzed by (A, C, and E) MS followed by (B, D, and F) MS/MS, and the identities of the segments confirmed by comparison with a peptide database. Comparisons between the MS (A and C) and the corresponding MS/MS (B and D) spectra show the ions generated from the native Trp7/Trp14-containing peptide GLSDGEWQQVLNVWGK (1816.5 m/z). The corresponding ion from the peptide containing oxidized Trp7, GLSDGEWQQVLNVWGK (1832.5 m/z), showed an increase of 16 atomic mass units ([M + 16 + H]+). A similar comparison between MS (A and E) and MS/MS data (B and F) showing the ions of the native and oxidized Trp7/Trp14-containing peptide GLSDGEWQQVLNVWGK (1847.5 m/z) indicated an increase of 32 atomic mass units ([M + 32 + H]+). The MS/MS analysis of the m/z 1816.5 peak confirmed the sequence of the native peptide that contained Trp7 and Trp14 (see arrows in B). A MS/MS analysis of the modified peptide with m/z 1832.5 (D) revealed an addition of 16 atomic mass units to the Trp7 residue (y°10 + 16) but not to the Trp14 residue in the same peptide. Note that the masses before (b°13 + 16, indicated by arrow in D) and after (b°14 + 16, indicated by arrow in D) the Nterminus cleavage of Trp14 from the peptide both contain oxidized Trp7, whereas the Trp14 mass remained unchanged as in the native peptide. MS/MS analysis of the modified peptide m/z 1847.5 (F) revealed an addition of 16 atomic mass units to both Trp7 and Trp14 residues (y10 + 32 and b14 + 32, respectively; refer to arrows in F). b⁎ and y⁎ ions refer to ions that lost ammonia (NH3), b° and y° ions refer to ions that lost a water molecule (H2O). Data are representative of four experiments using independent preparations of hhMb.

differences in reactivity between Met131 and Trp7/Trp14 or between Trp7 and Trp14 requires further study. In a previous study, a correlation between the time-dependent oxidation of aromatic residues and the extent of chloramine decay was suggested [43]. However, the oxidation of Met and Trp residues detected here occurred in parallel with measurable chloramine formation but was independent of chloramine decay. His and Lys residues are the most likely sites of chloramine formation, though low concentrations may also be formed at the N-terminus [22]. Reaction of HOCl at His residues to give chloramines is known to occur at a significantly faster rate than at Lys residues [33] and thus, it might be expected that these would be the major species formed. However, His chloramines are short-lived (lifetimes of minutes at most) and undergo fast chlorine transfer reactions that result in regeneration of the parent amino acid and secondary damage to other targets [46]. In contrast, chloramines generated on Lys residues are significantly more stable than the His species [46], and it is likely that the chloramines detected

in this study are predominantly Lys derived. It is possible that shortlived His-derived chloramines play a role in the observed oxidation of Met and Trp residues, whereas chlorination of tyrosine was not evident. The amino acid sequence of hhMb indicates 11 His residues, with at least 2 within 10 Å of the Met and Trp residues. The rate constant for reaction of His chloramines with Met is 2 orders of magnitude lower than the reaction with HOCl (but still a fast process), whereas with Trp the rate constants are similar [46]. Irrespective of the involvement or not of chloramines, our findings are in agreement with previous studies showing that Cys and Met residues are almost exclusively oxidized at low doses of HOCl (either directly or via the intermediacy of chloramines), followed by amine groups and disulfides, with Tyr or Trp residues less-favored targets [43]. HOCl-induced modifications to aromatic side chains have been reported to include oxidation or chlorination of Tyr (phenolic) side chains [50] and oxidation of Trp (indolic) residues [51]. Fu et al. have shown that in MMP-7 oxidized by a 50-fold molar excess of HOCl [51]

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Fig. 10. Stability of protein chloramines formed after reaction of reagent HOCl with hhMb. Samples of hhMb (50 μM) were treated with 0.5 (square), 1 (triangle), 2.5 (inverted triangle), or 5 (diamond) mol excess of HOCl and protein chloramine formation was determined from 5 to 180 min and again after 24 h of incubation as indicated under Experimental Procedures. ⁎p b 0.05 or ⁎⁎p b 0.01, significantly different compared to data at 5 min as determined by two-way ANOVA with Bonferroni post hoc test. Where error bars are not shown, the symbol is bigger than the error. Data are the means ± SD of two separate experiments, each performed in triplicate. Fig. 8. Relative quantification of Trp7/Trp14 peptides in hhMb exposed to reagent HOCl. Samples of hhMb (50 μM) were exposed to 0- to 5-fold molar excess of HOCl and the protein was then digested with trypsin. The resultant peptides were then separated by LC/MS. (A) The Trp7/Trp14-containing peptide (m/z 1816.5) and (B) the corresponding [M + 32 + H]+ oxidized species (m/z 1847.5) were quantified and expressed relative to the corresponding peak areas, either in control samples (for the native Trp7/Trp14 peptide) or in the fully oxidized samples (for the [M + 32 + H]+ peptide). ⁎p b 0.05 or ⁎⁎p b 0.01, significantly different from the control as determined by one-way ANOVA with Tukey's multiple comparison test. Data are the means ± SEM of four independent experiments.

an oxidative cross-link can be generated between a Trp residue and an adjacent Gly residue. The mechanism for this process was investigated using a range of synthetic peptides and the available data suggest that HOCl initially chlorinates the indole ring of Trp, which subsequently reacts with the main chain nitrogen of an adjacent Gly residue. The resulting species contains a Trp-Gly cross-link comprising a stable aromatic six-membered ring. In contrast, in the present studies with hhMb only the addition of oxygen atoms to Trp has been detected, with no evidence for other forms of modification, despite the presence of Gly15 adjacent to the Trp14 in hhMb. Consistent with this

Fig. 9. Relative quantification of Met55 and Met131 oxidized peptides in hhMb exposed to freshly isolated human neutrophils. Samples of hhMb (50 μM) were exposed to isolated human PMN (final density 4 × 106 or 20 × 106 cells/ml) for 2 h at 20°C in Hanks' balanced salt solution. Where indicated, 1.25 μg/ml PMA was added. At the end of the incubation, cells were centrifuged, and the supernatant was collected and treated with trypsin and then subjected to analyses by LC/MS/MS. The extent of oxidation of the (A) Met55 (peptide m/z 1101.3) and (B) Met131 (peptide m/z 1518.4) peptides was quantified by normalizing the product peak areas to those measured after exposure of hhMb to 10 × 106 neutrophils/ml. No modifications to the Trp7/Trp14-containing peptide were detected under the experimental conditions employed. ⁎p b 0.05 or ⁎⁎p b 0.01, significantly different from the control (samples incubated in the presence of PMN and the absence of PMA) as determined by one-way ANOVA with Tukey's multiple comparison test. Data are the means ± SD of three independent experiments.

observation, both Trp residues incorporate only a single oxygen atom, probably yielding an oxindole derivative (reviewed in [22,52]). Other studies have shown that the carbonyl content of hhMb increased after exposure to HOCl at HOCl/hhMb ratios similar to those employed in this study [30]. These carbonyls may arise from decay of Lys chloramines (a known reaction [50]) over longer incubation times. An alternative explanation for some of these differences arises from the source and HOCl/protein ratio in these studies. Formation of the Trp-Gly cross-link occurs in the presence of HOCl at an oxidant:protein ratio of 50:1 mol/mol [53] or with synthetic peptides. Thus, it is possible that Gly plays a role in Trp oxidation at high molar ratios, whereas at the lower ratios (up to 5 mol/mol) used here, direct oxygenation of Trp occurs. The disparity in products may also arise from differences in the protein conformation and/or the use of short peptides. The precise HOCl concentration and the rate of oxidant production in inflamed biological tissues are not clear; therefore it remains to be determined whether Trp modifications of the type described here, and previously, are pathologically relevant. The biological consequences of protein damage by HOCl have been studied extensively [13,17,43,54]. The inflammatory response and cytokine elaboration are integral components of the host response to tissue injury and play a particularly active role after myocardial infarction [55]. Inflammation of the heart is typically distinguished by a significant increase in the accumulation of leukocytes, neutrophils, and macrophages in the affected myocardium [6]. Activated neutrophils are a major source of reactive oxidants and are likely to be contributors to the oxidative damage associated with a variety of diseases in which inflammatory cells participate [8,56]. Here it has been shown that activated neutrophils yield hhMbox with Met (particularly Met55), but not Trp, residues being susceptible to oxidation. Interestingly, the extent of protein oxidation was significantly lower than that determined from a bolus addition of reagent HOCl; this may be a consequence of cell-derived HOCl reacting with the neutrophils (or material released from these) as alternative targets. Thus, the amount of oxidant to which the Mb protein is exposed may be considerably lower than with the reagent system. This may (partially) explain the lower extent of oxidation seen with the cells. The pathophysiological relevance of the present study depends on the likelihood of intracellular Mb (within cardiac myocytes) being exposed to HOCl generated in the extracellular compartment, the degree of activation of neutrophils in the damaged myocardium (as

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the latter will dictate the rate and extent of production of HOCl), and competition between cytosolic Mb and membrane-bound proteins as a target for oxidizing HOCl. Notably, within cardiac myocytes cytosolic Mb is present in relatively high concentrations, and in its role as an oxygen transport protein involves facilitated transport from the sarcolemma (on the cell surface) to intracellular mitochondria for oxidative phosphorylation [39,40]. Whether the modifications we have identified in this study can have an impact on intracellular oxygen transport and maintenance of mitochondrial function is not clear. During myocardial ischemia an influx of Ca2+ promotes mitochondrial membrane depolarization [57] and disruption of the KATP channel [1]. If the posttranslational modifications identified here were to decrease cardiac Mb’s ability to bind and transport molecular oxygen, then this may represent a form of specific protein damage that exacerbates mitochondrial dysfunction and disruption of the KATP channel in myocytes, effectively decreasing their capacity to produce ATP-further studies are necessary to validate this idea. The utility of Mb as a diagnostic marker for AMI has been examined in several studies [58–60]. However, because Mb lacks cardiac specificity, its elevation was not necessarily associated with cardiac damage. Several biochemical markers for the early detection of myocardial damage have been proposed, of which troponin T [61], myoglobin [62], and CK-MB [63] are most commonly employed. However, recent evidence suggests that Mb levels may be a strong independent predictor for cardiac mortality in patients admitted for exclusion of myocardial infarction [64]. Irrespective of the biomarker, Mb serum levels change rapidly in the early stages after the onset of AMI; therefore, sensitivity and specificity of any particular marker will necessarily change rapidly over time (on the scale of several hours). The current study demonstrates that HOCl can mediate the production of oxidized Mb, with specific targeting of Met and Trp residues. It is proposed that these selective modifications to Mb induced by reagent HOCl or neutrophil-derived oxidants may generate a suitable marker for myocardial inflammation. Horse heart Mb differs in its composition from human Mb, by the presence of three Mets and a single Cys residue in the latter. Despite these differences, preliminary results with wildtype and a Cys110Ala variant of human Mb show similar oxidation patterns in the presence of HOCl, where the susceptibility of protein side chains to oxidation is Cys N Met N Trp (Sapir, Pattison, Davies, and Witting, unpublished data). Future studies will focus on the characterization of oxidatively modified human Mb and establishing whether these modifications have an impact on the binding of molecular oxygen. Acknowledgments This work was performed with support from the ARC Discovery Projects (M.J.D. and P.K.W.), the ARC Fellowship (P.K.W.) and Centres of Excellence programs (M.J.D.), and an NHMRC (fellowship to C.L.H.). References [1] Jovanovic, A.; Jovanovic, S.; Lorenz, E.; Terzic, A. Recombinant cardiac ATPsensitive K+ channel subunits confer resistance to chemical hypoxia-reoxygenation injury. Circulation 98:1548–1555; 1998. [2] Richard, V.; Murry, C. E.; Reimer, K. A. Healing of myocardial infarcts in dogs: effects of late reperfusion. Circulation 92:1891–1901; 1995. [3] Reimer, K. A.; Vander Heide, R. S.; Richard, V. J. Reperfusion in acute myocardial infarction: effect of timing and modulating factors in experimental models. Am. J. Cardiol. 72:13G–21G; 1993. [4] Solomon, A.; Gersh, B. The open-artery hypothesis. Annu. Rev. Med. 49:63–76; 1998. [5] Winterbourn, C. C.; Vissers, M. C.; Kettle, A. J. Myeloperoxidase. Curr. Opin. Hematol. 7:53–58; 2000. [6] Frangogiannis, N. G.; Smith, C. W.; Entman, M. L. The inflammatory response in myocardial infarction. Cardiovasc. Res. 53:31–47; 2002. [7] Kyne, L.; Hausdorff, J. M.; Knight, E.; Dukas, L.; Azhar, G.; Wei, J. Y. Neutrophilia and congestive heart failure after acute myocardial infarction. Am. Heart J. 139:94–100; 2000. [8] Klebanoff, S. J. Myeloperoxidase: friend and foe. J. Leukocyte Biol. 77:598–625; 2005.

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