The role of reactive N-bromo species and radical intermediates in hypobromous acid-induced protein oxidation

Free Radical Biology & Medicine 39 (2005) 900 – 912 www.elsevier.com/locate/freeradbiomed

Original Contribution

The role of reactive N-bromo species and radical intermediates in hypobromous acid-induced protein oxidation Clare L. Hawkins*, Michael J. Davies The Heart Research Institute, 145 Missenden Road, Camperdown, Sydney, NSW 2050, Australia Received 12 April 2005; accepted 9 May 2005

Abstract Activated eosinophils, and hypobromous acid (HOBr) generated by these cells, have been implicated in the tissue injury in asthma, allergic reactions, and some infections. Proteins are major targets for this oxidant, but limited information is available on the mechanisms of damage and intermediates formed. Reaction of HOBr with proteins is shown to result in the formation of bromamines and bromamides, from side-chain and backbone amines and amides, and 3-bromo- and 3,5-dibromo-Tyr, from Tyr residues; these materials account for ca. 70% of the oxidant consumed. Protein carbonyls, dityrosine, and 3,4-dihydroxyphenylalanine are also formed, though these are minor products (<5% of HOBr added). With BSA, extensive (selective and nonspecific) protein fragmentation and limited aggregation are also observed. The bromamines/bromamides are unstable and induce further oxidation and free radical formation as detected by EPR spin trapping. Evidence was obtained for the generation of nitrogen-centered radicals on side-chain and backbone amide groups of amino acids, peptides, and proteins. These radicals readily undergo rearrangement reactions to give carbon-centered radicals. With proteins, a-carbon (backbone) radicals are detected, which may play a role in protein fragmentation. A novel damage transfer pathway from Gln side-chain amide groups to backbone sites was also observed. D 2005 Elsevier Inc. All rights reserved. Keywords: Hypobromous acid; Bromamine; Bromamide; Protein oxidation; Radicals; EPR; Free radicals

Activation of eosinophils both in vivo and in vitro is known to result in the generation of H2O2 and O2I via a respiratory burst and the release of eosinophil peroxidase [1]. This enzyme catalyzes the reaction of H2O2 with bromide ions (Br ) to form hypobromous acid (HOBr) [1]. At physiological pH, HOBr exists as a mixture with the ionized form OBr (pK a 8.7 [2]); HOBr is employed below to designate this mixture. There is also evidence to support Abbreviations: Br-Tyr, 3-bromo-Tyr; diBr-Tyr, 3,5-dibromo-Tyr; DEPMPO, 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide; DMPO, 5,5V-dimethyl-1-pyrroline-N-oxide; DNPH, 2,4-dinitrophenylhydrazine; DOPA, 3,4-dihydroxyphenylalanine; EPR, electron paramagnetic resonance spectroscopy; HOBr, the physiological mixture of hypobromous acid and its anion; HOCl, the physiological mixture of hypochlorous acid and its anion; MNP, 2-methyl-2-nitrosopropane; MPO, myeloperoxidase; TNB, 5thio-2-nitrobenzoic acid. * Corresponding author. Fax: +61 2 9550 3302. E-mail address: [email protected] (C.L. Hawkins). 0891-5849/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2005.05.011

the generation of HOBr by the enzyme myeloperoxidase (MPO), which is released by activated neutrophils, monocytes, and some macrophages, at plasma levels of halide ions (Cl 100 mM, Br 20 –100 AM, I < 1 AM) [3,4]. However, it has been predicted, on the basis of the specificity constants, that <5% of H2O2 is converted to HOBr by MPO [5]. Thus, it has been suggested that the production of HOBr, via the MPO-dependent pathway, is due to the reaction of hypochlorous acid (HOCl) with Br and the formation of bromine chloride as an intermediate [4]. HOBr plays a major role in the human immune system, killing invading pathogens [1]. Recently, it has been shown that eosinophil activation and HOBr production play a critical role in the tissue damage observed in asthma, allergic reactions, and other malignancies and infections [6 – 8]. However, little information is available on the reactions of HOBr with proteins and other biological molecules.

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There is evidence to support the formation of short-lived N-bromo species including bromamines and dibromamines from amino acids [1,9]. These species have been shown to decompose under various conditions to form aldehydes and nitriles [10,11]. Reaction of HOBr with proteins has been shown to result in bromination of aromatic amino acid residues, including formation of 3-bromo-Tyr (Br-Tyr) and 3,5-dibromo-Tyr (diBr-Tyr) [6,11– 13]. These products have been employed as specific markers of HOBr reactions in vivo, with these species detected at elevated levels on bronchoalveolar lavage proteins [6] and proteins isolated from sputum [7] obtained from patients with asthma. The rates of reaction of HOBr with protein components have been determined recently [14]. The amino acid side-chains were found to be significantly more reactive than the backbone amide groups, with Cys, Met, Trp, His, Tyr, and Lys side-chains and cystine residues the most reactive targets [14]. For most residues, HOBr reacts 30- to 100-fold faster than HOCl, though Cys and Met residues are 10-fold less reactive, and ring halogenation of Tyr is about 5000fold faster [14]. This suggests that Tyr residues may be more important, and Cys and Met residues less important, targets for HOBr compared to HOCl. We have shown recently, in studies employing electron paramagnetic resonance (EPR) spectroscopy, that radicals are generated on decomposition of chloramines formed after reaction of HOCl with a number of substrates, including amino acids, peptides, and proteins [15,16]. The initial radicals observed in these reactions are nitrogen-centered species, formed by the cleavage of the N –Cl bond by thermal and transition metal ion promoted processes. In the case of proteins, the nitrogen-centered radicals are formed predominantly on the side-chain amino group of Lys residues [16]. These species have been shown to undergo further reactions that result in both side-chain modification and protein fragmentation [15,16]. The nitrogen-centered radicals formed on amino acids and proteins can also damage other biological molecules including nucleosides and DNA [17]. As proteins are likely to be major targets for reaction with HOBr in vivo due to their abundance and high reactivity, we have examined the formation and subsequent reactions of bromamine and bromamide intermediates formed on proteins and related materials. Using EPR spectroscopy with spin trapping, we have detected and characterized, for the first time, radical intermediates, on reaction of HOBr with proteins and related materials. The effect of HOBr on protein integrity and the formation of protein oxidation products have also been quantified.

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experiments using oxygen-free nitrogen. pH control was achieved by use of 0.1 M pH 7.4 phosphate buffer treated with Chelex resin (Bio-Rad, Hercules, CA, USA) to remove any contaminating trace transition metal ions. All amino acids, peptides, and derivatives were from Sigma (St Louis, MO, USA), Bachem (Bubendorf, Switzerland), or Roche (Castle Hill, NSW, Australia) and were used as supplied. Isotopically labeled derivatives were from Cambridge Isotope Laboratories (Andover, MA, USA). Solutions of HOCl were prepared daily by dilution of a concentrated stock (BDH, Poole, UK; approx 0.5 M in 0.1 M NaOH) into 0.1 M pH 7.4 phosphate buffer. Solutions of HOBr were prepared by adding HOCl (40 mM) to a small excess of NaBr (45 mM) in water, at 20-C, and incubating for 60 min to ensure complete consumption of HOCl then diluted to the desired concentration immediately before use [18]. The concentrations of HOCl and HOBr were standardized at pH 12 using E292 = 350 M 1 cm 1 [19] and E329 = 332 M 1 cm 1 [20], respectively. EPR spectroscopy

Materials and methods

EPR spectra were recorded at room temperature using a Bruker EMX spectrometer with 100-kHz modulation equipped with a cylindrical (ER4103TM) cavity. Samples were contained in a flattened aqueous sample cell (from Wilmad WG-813-SQ) and recording of the spectra was initiated within 2 min of the start of the reaction. 5,5Dimethyl-1-pyrroline-N-oxide (DMPO; from ICN, Aurora, OH, USA) was purified before use by treatment with activated charcoal. 5-Diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO; from Alexis, San Diego, CA, USA) was purified before use by treatment with K3Fe(CN)6, charcoal, and DEAE Sepharose A25 as described previously [21]. Stock solutions of 2-methyl-2-nitrosopropane (MNP; from Sigma; 0.1 M) were generated by dissolution of the solid dimer in CH3CN overnight at 20-C; these solutions were diluted into the incubations such that the final CH3CN concentration was 12.5% (v/v). Experiments involving the addition of Cu+ ions were carried out in the absence of O2 (by gassing with O2-free nitrogen) to prevent autoxidation of the metal ions and subsequent formation of H2O2. Hyperfine coupling constants were measured directly from the field scan and confirmed by spectral simulation with the program WINSIM [22]. The correlation coefficients between simulated and experimental spectra were >0.90. Typical EPR spectrometer settings were gain 1  106, modulation amplitude 0.05 mT, time constant 0.16 s, scan time 84 s, resolution 1024 points, center field 348 mT, field scan 10 mT, power 25 mW, and frequency 9.76 GHz with four scans averaged.

Chemicals

Quantification of bromamine and bromamide intermediates

Solutions were prepared in high-purity, deionized water; metal ion solutions were degassed before, and during,

The UV –Vis spectra of N-bromo species formed on reaction of HOBr with the substrates were recorded on a

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Perkin –Elmer Lambda 40 spectrophotometer. The concentration and stability of the bromamine/bromamide intermediates were determined by the reaction with 5-thio-2nitrobenzoic acid (TNB) as described previously [16,23]. TNB (typically 35 – 40 AM) was prepared from the disulfide 5,5V-dithio-2-nitrobenzoic acid (1 mM) by exposure to NaOH (50 mM) for 5 min before dilution into 0.1 M pH 7.4 phosphate buffer. The concentration of TNB consumed after reaction with the various N-bromo species for 15 min was determined using E412 = 13,600 M 1 cm 1 [23]. Formation of protein carbonyl groups Carbonyl groups were quantified with 2,4-dinitrophenylhydrazine (DNPH; 10 mM in 2.5 M HCl) as described previously using E370 = 22,000 M 1 cm 1 [16,24]. The protein concentration was determined by measuring the absorbance at 280 nm in identical experiments performed with 2.5 M HCl in the absence of DNPH. The concentration of protein carbonyl groups was corrected for any protein loss assuming that a similar recovery of protein was obtained in the presence and absence of DNPH [24]. HCl hydrolysis and analysis of protein oxidation products Protein was precipitated, after quenching the reaction with freshly prepared sodium borohydride (12.5 mM), by the addition of sodium deoxycholate (0.015% w/v) and trichloroacetic acid (5% w/v) and pelleted by centrifugation (2 min at 4500g), washed twice with ice-cold acetone, and freeze-dried. The samples, contained in 1-ml shell-style vials, were hydrolyzed in Picotag vessels containing 1 ml HCl (6 M) and 50 Al mercaptoacetic acid, gassed thoroughly with N2, evacuated, and heated at 110-C for 16 h. The hydrolysates were freeze-dried, redissolved in Nanopure water, and filtered (0.45-Am pore-size filter, Pall Life Sciences, Ann Arbor, MI, USA) before HPLC analysis. No detectable artifactual protein oxidation was observed after this treatment [25]. The levels of the protein oxidation products and parent p-Tyr were determined in the protein hydrolysates by separation on a Zorbax ODS column (4.6  250 mm, 5-Am particle size; Agilent Technologies, Palo Alto, CA, USA) with a Pelliguard guard column, at 30-C, with a flow rate of 1 ml min 1, using the following gradient: isocratic 2% B for 20 min, then to 50% B over 30 min, washing with 50% B for 5 min, and reequilibration to 2% B for 5 min. Solvent A was 10 mM phosphoric acid and 100 mM sodium perchlorate (pH 2.1) and solvent B was methanol in Nanopure water (80%, v/v). Oxidation products were monitored in series by a UV detector at 280 nm (SPD10A, Shimadzu), a fluorescence detector with kEx 280 nm and kEm 320 or 410 nm (F-1080, Hitachi), and an electrochemical detector set at a potential of 1.2 V (Intro, Antec Leyden). Parent Tyr was quantified by UV absorbance (k 280 nm); DOPA, o-Tyr, m-Tyr, and di-Tyr by fluorescence (kEx 280 nm and kEm 320 or 410 nm for di-Tyr); and

brominated Tyr derivatives by UV absorbance (k 280 nm) and electrochemical oxidation (at a potential of 1.2 V) as described previously [25,26]. All products were quantified with respect to authentic standards. Separation of proteins by SDS – PAGE Proteins were separated by the method of Laemmli [27] using 8% polyacrylamide gels. Protein samples were added to an equal volume of 60 mM Tris/HCl buffer, pH 6.8, containing glycerol (10% v/v), 2-mercaptoethanol (5% v/v), SDS (2% w/v), and bromophenol blue (0.01% w/v). In some cases, samples were reduced by heating at 95-C for 5 min before loading and separation. Bands were visualized using Coomassie blue or silver staining. Gels were scanned using a Bio-Rad GelDoc 2000 system, and the density of the protein bands was determined over a linear range using the Bio-Rad molecular imaging software. Statistical analyses Statistical analyses to compare the effect of HOBr treatment to that of the untreated control were carried out using one-way ANOVA with Dunnett_s or Tukey_s post hoc testing performed with GraphPad Prism 4 (GraphPad Software, San Diego, CA, USA; www.graphpad.com).

Results Reaction of HOBr with proteins Formation and decay of protein bromamines Evidence for the formation of protein bromamine intermediates was obtained using the TNB assay. Treating BSA (100 AM) with a 10-fold molar excess of HOBr (1 mM) resulted in the generation of bromamine species that decomposed rapidly over 60 min (Fig. 1). The rate of bromamine decomposition was found to be temperature-

Fig. 1. Stability of BSA-derived N-bromo species. Graph shows the concentration of N-bromo species observed on reaction of BSA (100 AM) with HOBr (1 mM) and subsequent incubation at ( ) 4-C, ( ) 20-C, and (0) 37-C. These data represent the means T standard deviation of three experiments.

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dependent, with slower rates of decay observed at lower temperatures. The formation of bromamines accounted for ca. 40% of the added HOBr on determination of the concentration of TNB-reactive material 30 s after the addition of the oxidant. This is likely to be an underestimate, given the low stability of these intermediates. Formation of radicals on decomposition of protein bromamines The generation of protein-derived radicals on decomposition of bromamines/bromamides formed on treating BSA with HOBr was investigated using EPR spin trapping techniques. BSA (0.5 mM) was treated with HOBr (5 mM) for 30 s, to ensure complete consumption of the oxidant, before the addition of the spin trap. This sequence of addition prevented confounding direct reaction of HOBr with the spin trap and enabled radical formation from Nbromo species to be examined. Initial experiments carried out with the spin trap DMPO resulted in the detection of weak, poorly resolved signals (data not shown). More intense, broad, signals, consistent with the formation of protein-derived radical adducts, were detected with the spin trap DEPMPO (37.5 mM) (Fig. 2A). These signals were sharpened on addition of the enzyme Pronase (2 units) and incubation of the reaction mixture for 10 min at 37-C (Fig. 2B), due to the release of small, more mobile protein fragments as previously described [16]. No signals were

Fig. 2. EPR spectra observed on reaction of HOBr with BSA using the spin trap DEPMPO. (A) BSA (0.5 mM) treated with HOBr (5 mM) with DEPMPO (37.5 mM); (B) as in (A) but with Pronase (2 units) and 10 min at 37-C; (C) BSA (0.5 mM) and DEPMPO (37.5 mM) only. Signals in (A) are assigned to high-molecular-weight protein radicals. Signals marked with an X in (A) and (B) are attributed to DEPMPO-OH. Signals in (B) are assigned to a carbon-centered radical (?) and a nitrogen-centered radical (unmarked lines).

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observed when excess Met (25 mM) was added to the HOBr-treated BSA, to quench bromamines/bromamides, before the addition of DEPMPO or in control experiments carried out with BSA and DEPMPO alone (Fig. 2C). Experiments with increasing ratios of HOBr:protein (20:1 and 40:1) were unsuccessful due to the detection of high levels of the DEPMPO-OH adduct (data not shown). Intense, broad, signals, from protein-derived radical adducts, were also detected on addition of Cu+ ions [generated on reaction of CuSO4 (625 AM) with TiCl3 (500 AM)] to reaction mixtures containing HOBr-treated BSA (0.5 or 5 mM); this is attributed to metal-ioncatalyzed bromamine/bromamide decay, in accord with previous experiments with the corresponding N-chloro species [15]. The signals observed on Pronase treatment are attributed to the formation of a nitrogen-centered radical, on the basis of the additional 1:1:1 coupling observed, and a carbon-centered radical (for parameters see Table 1). The nature of the protein-derived, carbon-centered radicals was investigated further in experiments with the spin trap MNP. Broad, anisotropic, EPR signals were observed on addition of MNP (12.5 mM) to BSA (0.5 mM) pretreated with HOBr (5 mM) (Fig. 3A). Addition of Cu+ ions [CuSO4 (625 AM) and TiCl3 (500 AM)] to the reaction mixture resulted in an increase in the signal intensity of the radical adducts (Fig. 3B). The distortion in the baseline of the signals observed in the presence of Cu+ ions is attributed to the formation of protein – Cu2+ signals. Again, sharper, more isotropic, EPR signals were observed on Pronase digestion (2 units, 37-C, 10 min) of the proteinderived radical adducts (Fig. 3C). These sharper signals have complex fine structure that was not possible to resolve further, as increasing the incubation time of the protein radical adducts with the protease, to release smaller fragments, resulted in a significant decrease in the signal intensity observed. Thus, the nature of the carbon-centered, protein-derived, radical adducts was investigated further in experiments with smaller (<25 kDa) proteins, which were expected to yield more mobile adducts and hence give sharper EPR signals. Reaction of HOBr with soybean trypsin inhibitor (21.5 kDa), lysozyme (14.3 kDa), ribonuclease A (13.4 kDa), and insulin (5.7 kDa) with addition of MNP and Cu+ ions gave similar broad, anisotropic, EPR signals compared to the species observed with BSA (Fig. 4). However, digestion of the protein adducts with Pronase resulted in the detection of sharper, well-resolved, signals (Fig. 4E). These spin adducts gave rise to a triplet of quartets coupling pattern, consistent with the presence of a carbon-centered radical with couplings, of a similar size, to a hydrogen atom and a nitrogen atom (a N 1.48 mT, a N2 0.27 mT, a H 0.27 mT). Identical signals were observed in experiments with Lysblocked (by reductive methylation) insulin (data not shown), suggesting that Lys side-chains are not the site of radical formation. Thus, this radical is assigned, on the basis of the

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Table 1 Parameters of the radicals observed with DEPMPO, DMPO, and MNP upon reaction of HOBr with amino acids, N-acetyl amino acids, and proteins

a

Hyperfine coupling constants quoted are T0.01 mT.

nitrogen and hydrogen couplings observed, to a backbone a-carbon radical. Formation of protein carbonyls on decomposition of protein bromamines Protein carbonyl groups are known to be generated on decomposition of chloramines [28,29]. The importance of this decomposition pathway with the analogous bromamines was investigated by quantifying carbonyl groups on HOBrtreated BSA using DNPH (see Materials and methods). Reaction of BSA (15 AM) with a 50-fold molar excess of HOBr (750 AM) resulted in the formation of protein carbonyl groups in a time-dependent manner over 15 min (Fig. 5A). Increasing the incubation time of the HOBrtreated BSA further did not lead to additional carbonyl formation. The formation of carbonyl groups was also

dependent on the concentration of HOBr. Thus, reaction of BSA with increasing amounts of HOBr (75 AM –3 mM) for 15 min resulted in the detection of increasing yields of protein carbonyl groups (Fig. 5B). The concentration of carbonyl groups observed accounted for <3% of the initial HOBr added, even with high molar excess of HOBr (>50:1), in which 40% of the HOBr was converted to bromamines. This suggests that protein carbonyl groups are minor products of bromamine decomposition. Formation of Tyr-derived oxidation products The formation of Br-Tyr, diBr-Tyr, and other Tyr-derived oxidation products including DOPA and di-Tyr was quantified by HPLC on reaction of BSA (37.5 AM) with increasing amounts of HOBr (0.19 – 7.5 mM) for 15 min (at 20-C). Increasing concentrations of HOBr resulted in the

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tion of protein Tyr residues to products other than brominated Tyr, DOPA, or di-Tyr; the identity of these other products has not been investigated further. Structural integrity of HOBr-treated proteins Protein fragmentation (and aggregation) was examined by SDS – PAGE under reducing conditions. Reaction of BSA (1.2 AM) with HOBr (0– 240 AM) for 15 min resulted in smearing and loss in staining intensity of the parent protein band with increasing oxidant concentration (Fig. 7). Additional, low-molecular-mass, bands were observed with >10-fold molar excess of oxidant. These bands became darker in staining intensity with increasing concentrations of oxidant. However, at high oxidant concentrations (>50-fold molar excess), these bands became less resolved and smeared, presumably as a result of further reactions. No evidence for high levels of protein aggregation was obtained. The reactions of bromamines/bromamides, and the formation of radicals, were investigated further in experiments with amino acids and peptides to provide more information about the site of radical formation on proteins. Fig. 3. EPR spectra observed on treating BSA with HOBr using the spin trap MNP. (A) BSA (0.5 mM) treated with HOBr (5 mM) and with MNP (12.5 mM); (B) as in (A) but with Cu+ ions [CuSO4 (625 AM) and TiCl3 (500 AM)] added; (C) as in (B) but with Pronase (2 units) and 10 min at 37-C. Signals are assigned to the formation of carbon-centered, proteinderived, radicals. The distortion in the baseline in (B) and (C) is attributed to the formation of Cu2+ – protein complexes.

detection of an increase in the level of brominated Tyr (BrTyr + diBr-Tyr), di-Tyr, and DOPA and a corresponding decrease in the amount of Tyr (Fig. 6). The ratio of Br-Tyr to diBr-Tyr was dependent on the concentration of HOBr added, with a relative increase in the amount of diBr-Tyr detected at the highest concentrations of oxidant employed (Fig. 6B). The brominated Tyr detected accounted for 30% of the initial HOBr added (at 50-fold excess oxidant). Reaction of HOBr with BSA also gave rise to DOPA and di-Tyr under some conditions. Significantly elevated levels of DOPA, compared to control BSA, were detected only with 100-fold molar excess of oxidant (data not shown), whereas significant amounts of di-Tyr were detected with 25-fold molar excess of HOBr (Fig. 6C). The amount of di-Tyr detected decreased at the highest concentration of HOBr employed (200-fold excess), with this attributed to further oxidation of di-Tyr. This hypothesis was confirmed in experiments with isolated di-Tyr (3 AM) and HOBr (3 –30 AM) in which significant loss of di-Tyr was detected by fluorescence (Fig. 6D). Incubation of BSA with HOBr (10- or 50-fold molar excess) for increasing periods (2– 60 min) led to a timedependent decrease in the concentration of protein-bound Tyr detected (Fig. 6E), but no significant increase in the levels of brominated Tyr, DOPA, or di-Tyr (data not shown). These data are consistent with bromamine-mediated oxida-

Fig. 4. EPR spectra observed on treating small proteins with HOBr using the spin trap MNP. (A) Soybean trypsin inhibitor, (B) lysozyme, (C) ribonuclease A, and (D) insulin (all 0.5 mM) treated with HOBr (5 mM) before the addition of MNP (12.5 mM) and Cu+ ions [CuSO4 (625 AM) and TiCl3 (500 AM)]. (E) as in (D) but with Pronase (2 units) and 10 min at 37-C. Signals in (A – D) are assigned to the formation of carbon-centered, protein-derived, radicals. Signals in (E) are attributed to backbone a-carbon radicals.

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absorption from the aromatic side-chains of these amino acids. The conversion of HOBr to bromamine or bromamide was >90% in all cases, as quantified by the TNB assay.

Fig. 5. Formation of protein carbonyl groups on reaction of BSA with HOBr. Graphs represent the concentration of protein carbonyl groups formed on reaction of BSA (15 AM) treated with (A) 50-fold molar excess of HOBr (750 AM) before incubation at 20-C and addition of Met (20 mM) at the required time to quench the reaction and (B) 5- to 200-fold molar excess HOBr (75 AM – 3 mM) for 15 min incubation at 20-C. Statistically significant change compared to the untreated control with *p < 0.05 and **p < 0.01, as determined by one-way ANOVA with Dunnett_s post hoc testing. These data represent the means T standard deviation of at least four experiments.

Reaction of HOBr with amino acids and peptides Formation and stability of bromamine and bromamide species The formation and stability of amino acid and peptide bromamines and bromamides were investigated using UV – visible spectroscopy, as these species and parent HOBr have distinct absorption bands in the 240– 350 nm region (kmax 260, 329, and 280 – 290 nm for HOBr, BrO , and bromamines, respectively) [30]. In all cases, HOBr was reacted with a 100-fold molar excess of substrate to avoid dibromamine formation. The stability of the bromamine/bromamide species formed decreased in the order N-acetyl amino acid amide group (N-acetyl-Gly, N-acetyl-Ala) > side-chain amide group (N-acetyl-Gln, N-acetyl-Asn) > side-chain amine group (N(a)-acetyl-Lys, 6-aminocaproic acid) > cyclic dipeptide amide group (cyclo-(Gly)2, cyclo-(Ala)2) > peptide N-terminal amine group (Gly-Gly-Gly) > a-amino group (Gly, Ala, N(E)-acetyl-Lys) (Table 2). Experiments were not carried out with His and Trp due to the intense UV

Characterization of radicals formed on bromamine/bromamide decomposition The formation of radicals from amino acid and peptide bromamines and bromamides was studied using EPR spin trapping (as above). Typically, spectra were recorded within 2 min of the addition of the spin trap to reaction mixtures containing the HOBr-treated substrate (5 mM HOBr, 25 mM substrate) at pH 7.4. Addition of DMPO (125 mM) to HOBr-treated Lys and His gave intense EPR signals (Figs. 8A and 8B). These have been assigned to nitrogen-centered radical adducts on the basis of their characteristic second nitrogen coupling (for parameters see Table 1). These nitrogen-centered adducts are assigned to radicals centered on the a-amino group of each amino acid, as identical signals were detected with 15NE-Lys and NE-acetyl-Lys, and no signals were observed when this position was blocked or absent (i.e., with Na-acetyl-Lys, imidazole, Na-acetyl-His; data not shown). An additional signal, attributed to a carbon-centered radical adduct, was detected from HOBr-treated His (Fig. 8B, Table 1). A mixture of nitrogen-centered and carbon-centered radical adducts were detected from HOBr-treated N-acetyl-Gln and N-acetyl-Asn (Fig. 8C), though these species were observed only on addition of Cu+ ions [CuSO4 (625 AM) and TiCl3 (500 AM)], to increase the rate of bromamide decomposition. A number of cyclic dipeptides and Nacetyl-amino acids gave similar behavior (Table 1). In experiments in which the substrate was omitted, or DMPO was added to the solution of amino acid or peptide before HOBr, the well-characterized spin trap degradation product DMPOX was detected [31] as a result of direct reaction of HOBr with the spin trap. The nature of the carbon-centered radicals observed was investigated further using the spin trap MNP. Addition of MNP (12.5 mM) to HOBr-treated His gave complex EPR signals with multiple couplings (Figs. 9A and 9B; Table 1). These signals have been assigned to the formation of an imidazole ring-derived, carbon-centered radical, trapped via the ring C2 position, by comparison with previous data [32]. This assignment is supported by the detection of characteristic spectral changes with ring-p-15N-His, consistent with the presence of significant spin density on the ring nitrogen atoms (Figs. 9C and 9D). Reaction of HOBr with the sidechain amide groups of N-acetyl-Gln and N-acetyl-Asn, and the backbone amide groups of the cyclic dipeptides and Nacetyl-amino acids followed by addition of MNP and Cu+ ions also resulted in the detection of carbon-centered radicals. The hyperfine coupling constants of these adducts and proposed assignments are given in Table 1. The formation of carbon-centered radicals is attributed to rearrangement reactions mediated by the initial nitrogencentered species, as described previously [15,16].

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Discussion Bromamines (R-NHBr) and bromamides (R-C(O)-NBrRV) are major products of the reaction of HOBr with proteins, peptides, and amino acids, as evidenced by the conversion of >40% of the initial HOBr to N-bromo species. The stability of these intermediates is structure dependent. Generally, the bromamides are considerably more stable (t 1/2 > 24 h) than the bromamines (t 1/2 < 15 min), with the exception of the cyclic peptide-derived bromamides (t 1/2 12 and 35 min for Gly and Ala, respectively), which are less stable than Lys side-chain bromamines (t 1/2 > 120 min). These data are consistent with previous studies [2,10,11]. The protein-derived N-bromo species decomposed rapidly at temperatures 4-C. It has been reported previously that bromamines are not major products of the reaction of HOBr with proteins [33]. However, it is likely that significant bromamine formation was occurring in this previous study, but not detected, due to the time period between addition of HOBr and assay (30 min) and the incubation temperature used (37-C) [33]. In general, these N-bromo species are less stable than the corresponding N-chloro species, in accord with previous observations [10,11]. Decay of the N-bromo species formed on BSA has been shown to give rise to a number of stable products, including protein carbonyls, brominated Tyr residues, and the Tyr oxidation products DOPA and di-Tyr. The formation of carbonyl groups from amino acid and peptide bromamines has been reported previously [10,11]. This process may arise via a nonradical pathway, in a manner analogous to that of chloramines [16,34], or via a radical pathway (Scheme 1). The yield of carbonyls formed on BSA is, however, low and accounts for <3% of the oxidant added. Furthermore, there was no significant change in carbonyl yield on incubation of the HOBr-treated protein over the time frame 15 –60 min at 20-C, which is surprising given a large proportion of the initial bromamines/bromamides decompose over this period (cf. Fig. 1). This implies that either only some types of bromamine/bromamide decompose to form carbonyls or the carbonyl groups are oxidized further once formed, and Fig. 6. Quantification of Tyr oxidation products produced on reaction of BSA with HOBr after separation by HPLC. Graphs (A – C) show the concentration of (A) Tyr, (B) Br-Tyr (white bars) and diBr-Tyr (hatched bars), and (C) diTyr formed on reaction of BSA (37.5 AM) with 5- to 200-fold molar excess HOBr (187.5 AM – 7.5 mM) for 15 min before quenching the reaction with NaBH4 (12.5 mM) and quantification by HPLC (see Materials and methods). Graph (D) represents the concentration of di-Tyr observed after reaction of diTyr (3 AM) with HOBr (3 – 30 AM). Graph (E) represents the loss of Tyr observed on treating BSA (37.5 AM) with a 50-fold molar excess of HOBr (1.9 mM) at 20-C before addition of Met (20 mM) and NaBH4 (12.5 mM) at the required time to quench the reaction. Statistically significant change with *p < 0.05 and **p < 0.01. In (A) and (C), HOBr-treated samples are compared to the untreated control using one-way ANOVA with Dunnett_s post hoc testing. In (E), all time points are compared using one-way ANOVA with Tukey_s post hoc testing—the asterisks represent a statistically significant change compared to the 2-min time point only. These data represent the means T standard deviation of at least three experiments.

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Fig. 7. SDS – PAGE gel of BSA treated with HOBr. Reaction of BSA (1.2 AM) with increasing amounts of HOBr (12 – 240 AM) for 15 min before the reaction was quenched with Met (20 mM) results in an increasing extent of protein fragmentation as evidenced by the loss in the parent protein band at 66 kDa. Lane 1, BSA only; lanes 2 – 7, BSA with 10-, 25-, 50-, 75-, 100-, 200-fold molar excess HOBr; lane 8, molecular weight markers. Proteins were visualized by silver staining.

hence the yield of these species is underestimated. The yield of carbonyls detected with HOBr-treated BSA is significantly lower than that observed in analogous experiments with HOCl, in which ca. 10% of the initial HOCl gives rise to carbonyls over a period of 24 h [16]; the longer incubation period used in these previous experiments was employed to ensure complete decay of the chloramines. Table 2 Characteristics and lifetimes of the UV absorptions of amino acid and peptide-derived bromamine/bromamide species Substrate

Absorbance kmax

Half-life (t 1/2)a at 20-C, pH 7.4

Glycine Alanine Gly-Gly-Gly Lysine N(E)-Acetyl-Lys N(a)-Acetyl-Lys 6-Aminocaproic acid N-Acetyl-Gln N-Acetyl-Asn N-Acetyl-Gly N-Acetyl-Ala Cyclo-(Gly)2 Cyclo-(Ala)2

289 nm 289 nm 289 nm 289 nm 289 nm 289 nm 289 nm Shoulder at Shoulder at Broad peak Broad peak Broad peak Broad peak

4.2 T 0.5 min 5.2 T 0.2 min 11.2 T 0.2 min 2.4 T 0.3 min 2.7 T 0.3 min >120 min >120 min >24 h >24 h >14 days >14 days 11.9 T 3.0 min 35.5 T 3.7 min

260 nm 260 nm 250 – 300 250 – 300 250 – 300 250 – 300

nm nm nm nm

a t 1/2 = ln 2(s), where s is the lifetime determined by fitting decay data to the first-order exponential curve using Origin 7.0.

Fig. 8. EPR spectra observed on reaction of Lys, His, and N-acetyl-Gln with HOBr using the spin trap DMPO. (A) Reaction of Lys (50 mM) with HOBr (5 mM) followed by the addition of DMPO (125 mM); (B) as in (A) but with His; (C) as in (A) but with N-acetyl-Gln and Cu+ [CuSO4 (625 AM) and TiCl3 (500 AM)] added. Signals in (A) are assigned to the a-amino, nitrogen-centered radical and DMPO-OH, marked with an X. Signals in (B) are assigned to the a-amino, nitrogen-centered radical, a carbon-centered radical, marked ( ), and DMPO-OH, marked (X). The signals in (C) are assigned to the formation of the side-chain, amide nitrogen-centered radical.

&

Similar data have been reported for low-density lipoproteins [33]. Brominated Tyr residues on BSA account for ca. 30% of the initial oxidant with 50-fold molar excess of HOBr. These products (Br-Tyr and diBr-Tyr) are believed to arise predominantly via direct reaction of HOBr with Tyr residues and not bromine transfer from bromamines/bromamides, as no time-dependent increase was observed on incubation of the HOBr-treated protein over 60 min. This was unexpected, as previous studies have reported that the primary bromamines formed on taurine and Na-acetyl-Lys can brominate Tyr residues [26]. There was, however, a small, but significant ( p < 0.05, one-way ANOVA, Tukey’s post hoc test), decrease in parent Tyr residues over this time period, consistent with oxidation of Tyr residues by bromamines/ bromamides to further products. Thus, it may be that bromamines induce oxidation of brominated Tyr residues to more heavily brominated species. This suggestion is consistent with previous studies that have indicated that secondary bromination of Br-Tyr to give diBr-Tyr occurs more rapidly than monobromination [14]; this is also likely to be true for more extensive bromination. This observation

C.L. Hawkins, M.J. Davies / Free Radical Biology & Medicine 39 (2005) 900 – 912

Fig. 9. EPR spectra observed on reaction of His and 15N-ring-k-His with HOBr using MNP. (A) Reaction of His (50 mM) with HOBr (5 mM) and MNP (12.5 mM); (C) as in (A) but with ring k-15N-His. (B) and (D) are computer simulations of the experimental spectra shown in (A) and (C), respectively, using the parameters listed in Table 1, with correlation coefficients >0.94. Signals in (A) and (C) are assigned to the formation of the His side-chain imidazole C2 ring-derived radical, an additional carboncentered radical, marked ( ), and di-tert-butylnitroxide, marked (X).

&

has implications for the use of Br- and diBr-Tyr as markers of HOBr-induced reactions, as further oxidation of these products may result in the underestimation of HOBrinduced damage in vivo. The extent of halogenation of BSA observed with HOBr is significantly higher ( p < 0.001, two-way ANOVA, Bonferroni post hoc test) than that observed with HOCl, where the formation of Cl-Tyr and diCl-Tyr accounted for <5% of the added HOCl (Hawkins and Davies, unpublished

909

data). These data are consistent with the much higher rate constant for reaction of HOBr compared to HOCl with Tyr residues (k = 2.3  105 and 44 M 1 s 1, respectively [14,35]). Evidence was also obtained for the formation of di-Tyr (and DOPA at high excess of HOBr). Di-Tyr is known to be formed via the dimerization of Tyr phenoxyl radicals [36], supporting the hypothesis that radicals are formed during the decomposition of bromamines and bromamides. The yield of this product is low, which is unsurprising given that the formation of this product requires a radical –radical reaction, is presumably affected significantly by steric interactions, and is likely to be underestimated given the observed susceptibility of di-Tyr to further oxidation by brominated oxidants. Reaction of BSA with HOBr results in protein fragmentation as evidenced by a loss in staining intensity, and smearing, of the parent protein band with increasing oxidant concentration. The band smearing is attributed to multiple heterogeneous side-chain fragmentation and modification reactions that result in minor changes to the mass and charge density of the protein, and hence a complex mixture of poorly resolved products, as previously observed with HOCl [16,37]. Specific protein fragments were also detected, in contrast to the situation with HOCl-treated BSA [16], suggesting that reaction of HOBr with proteins gives rise to both site-specific and nonspecific fragmentation. Fragmentation was observed with lower molar excess of HOBr than HOCl [16], in accord with the predicted greater extent of reaction of HOBr at backbone sites [14]. It is unlikely that fragmentation results solely from direct reaction of HOBr with backbone amides, as the rate constants for reaction of HOBr with side-chains are significantly higher [14]. A proportion of the fragmentation observed with low concentrations of HOBr may therefore arise from secondary reactions mediated by side-chain bromamines and bromamides (e.g., those formed on Lys, His, Gln, and Asn) and radicals derived therefrom. Direct EPR evidence has been obtained for radical formation from bromamines and bromamides when these decompose either spontaneously or on reaction with metal ions. The source of the observed radicals has been

Scheme 1. Potential mechanisms of bromamine-mediated protein carbonyl group formation.

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Scheme 2. Formation of a-carbon backbone radicals on rearrangement of Gln side-chain nitrogen-centered radicals.

confirmed by both scavenger experiments with excess Met, which removes bromamines/bromamides, and the sequence of addition of reactants in the EPR studies, in which radical formation is detected even when the spin trap is added into the reaction mixture 30 s after the HOBr. At this time point all of the HOBr has been consumed based on computer modeling [14]. In addition, the rate of increase in EPR signal intensity correlated well with the rate of bromamine/ bromamide decomposition, with both processes enhanced by the addition of Cu+ ions. Experiments using the spin trap DMPO, and HOBrtreated amino acids and peptides, typically gave nitrogencentered radicals as the major adduct species detected, with

these signals assigned to radicals formed on the a-amino group of free amino acids (e.g., Lys and His), the side-chain amide group of Gln or Asn residues, and the backbone amide group of cyclic dipeptides (see Table 1). Evidence for the formation of similar radicals was also obtained with proteins, though the signals were weaker and detected only with the spin trap DEPMPO. These species were observed only with moderate doses of HOBr. With higher concentrations of HOBr, oxidation of the spin trap predominated, with this attributed to bromamine/bromamide-mediated processes. The nature of these protein-derived radicals is not certain. The data obtained suggest that, unlike with HOCl [16], these species are not formed on Lys side-chains,

Scheme 3. Summary of proposed reactions of HOBr with proteins such as BSA.

C.L. Hawkins, M.J. Davies / Free Radical Biology & Medicine 39 (2005) 900 – 912

or the N-terminal a-amino group, as the EPR signals were not affected by reductive methylation (in the case of insulin) and analogous signals were not detected with free Lys or Lys-containing model peptides. These species may therefore arise from the decomposition of bromamides formed on backbone or side-chain amide groups. Carbon-centered radicals, believed to arise via subsequent rearrangement reactions of the initial nitrogencentered species (as observed with HOCl [15,16]), were detected in some experiments using the spin trap MNP. With His, complex signals from carbon-centered, ring-derived radicals were observed, with these assigned to a ringderived radical trapped via C2 (Table 1) [32]. With peptides containing side-chain (Asn and Gln) or backbone (cyclo(Gly)2 and N-acetyl-Gly) amide groups, a-carbon radicals were detected as evidenced by the presence of additional hydrogen and nitrogen couplings of similar magnitude. With cyclo(Gly)2 and cyclo(Ala)2, these acarbon radicals are believed to be generated via 1,2hydrogen shift reactions of an initial nitrogen-centered radical formed from a bromamide generated at the backbone amide site. In contrast, with N-acetyl-Gly and N-acetyl-Asn, decarboxylation seems to be the favored reaction of the initial nitrogen-centered radicals, as these compounds, unlike the cyclic peptides, have a free carboxyl group. The parameters of the resulting a-carbon radical are significantly different from those observed with the cyclic peptides and are consistent with those observed in HOClmediated reactions [15]. In contrast to the behavior of Nacetyl-Asn, HOBr-treated N-acetyl-Gln gave rise to an adduct with an additional nitrogen coupling, but no hydrogen coupling. This signal has been assigned to an a-carbon radical generated via a 1,5-hydrogen shift rearrangement reaction of an initial side-chain amide, nitrogen-centered, radical via a 6-membered ring transition state (Scheme 2). This species was not observed previously with HOCl [15]. This difference between the behavior of N-acetyl-Asn and N-acetyl-Gln with HOBr is believed to be due to the shorter side-chain in the former species, preventing formation of a 6-membered ring transition state. The behavior of these peptides sheds some potential light on the protein-derived species. Digestion of the protein radical adducts with Pronase resulted in the release of small fragments from the proteins with well-resolved couplings. These spectra have been interpreted in terms of the coupling of the unpaired electron with additional hydrogen and nitrogen atoms; this pattern is similar to some of the acarbon radicals detected with the peptides. Such a species might be generated via a 1,2-hydrogen shift reaction of a nitrogen-centered radical formed on Gly residues, or possibly decarboxylation at the C-terminus, but in the absence of further information it is impossible to provide an exact assignment. If these species are indeed a-carbon radicals, they may be of considerable biological significance, as such radicals are implicated in backbone fragmentation via rapid reaction with O2 (k = 1.2  108 T

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0.1 M 1 s 1 and 1.0  108 T 0.2 M 1 s 1 for cyclo(Gly)2 and cyclo(Ala)2, respectively [38]) to give the peroxyl, and potentially alkoxyl, radicals that mediate fragmentation (reviewed in [36,39]. The generation of specific fragments in the PAGE experiments would be consistent with the selective formation of these backbone radicals at particular residues in the protein sequence. In summary, it has been shown that reaction of HOBr with proteins results in the formation of high yields (>40% of initial oxidant) of unstable bromamines and bromamides from amine and amide residues and that subsequent catalyzed or spontaneous decomposition of these materials results in the induction of further damage including protein fragmentation. Br-Tyr and diBr-Tyr are also major products, whereas protein carbonyls, di-Tyr, and DOPA are formed to only a minor extent. A summary of the proposed processes is given in Scheme 3. Significant differences therefore exist between the reactions of HOCl and HOBr with proteins and the extent and nature of protein modifications thus formed.

Acknowledgments The authors thank the National Health and Medical Research Council (Australia) and the Australian Research Council for funding and Dr. David Pattison and Dr. Martin Rees for helpful discussions.

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