Protective mechanisms against peptide and protein peroxides generated by singlet oxygen

Free Radical Biology & Medicine, Vol. 36, No. 4, pp. 484 – 496, 2004 Copyright D 2003 Elsevier Inc. Printed in the USA. All rights reserved 0891-5849/$-see front matter

doi:10.1016/j.freeradbiomed.2003.11.021

Original Contribution PROTECTIVE MECHANISMS AGAINST PEPTIDE AND PROTEIN PEROXIDES GENERATED BY SINGLET OXYGEN PHILIP E. MORGAN, ROGER T. DEAN, 1 and MICHAEL J. DAVIES Free Radical Group, The Heart Research Institute, Camperdown, Sydney, NSW, Australia (Received 3 July 2003; Revised 16 October 2003; Accepted 14 November 2003)

Abstract—Reaction of certain amino acids, peptides, and proteins with singlet oxygen yields substrate-derived peroxides. Recent studies have shown that these species are formed within intact cells and can inactivate key cellular enzymes. This study examines potential mechanisms by which cells might remove or detoxify such peroxides. It is shown that catalase, horseradish peroxidase, and Cu/Zn superoxide dismutase do not react rapidly with these peroxides. Oxymyoglobin and oxyhemoglobin, but not the met (Fe3+) forms of these proteins, react with peptide but not protein, peroxides with oxidation of the heme iron. Glutathione peroxidase, in the presence of reduced glutathione (GSH) rapidly removes peptide, but not protein, peroxides, consistent with substrate size being a key factor. Protein thiols, GSH, other low-molecular-weight thiols, and the seleno-compound ebselen react, in a nonstoichiometric manner, with both peptide and protein peroxides. Cell lysate studies show that thiol consumption and peroxide removal occur in parallel; the stoichiometry of these reactions suggests that thiol groups are the major direct, or indirect, reductants for these species. Ascorbic acid and some derivatives can remove both the parent peroxides and radicals derived from them, whereas methionine and the synthetic phenolic antioxidants Probucol and BHT show little activity. These studies show that cells do not have efficient enzymatic defenses against protein peroxides, with only thiols and ascorbic acid able to remove these materials; the slow removal of these species is consistent with protein peroxides playing a role in cellular dysfunction resulting from oxidative stress. D 2003 Elsevier Inc. All rights reserved. Keywords—Protein oxidation, Protein peroxides, Protein radicals, Singlet oxygen, Photo-oxidation, Antioxidants, Glutathione peroxidase, Free radicals

play a key role in the development of a number of human pathologies, including cataract [7 – 10], sunburn, some skin cancers [11], and aging (reviewed in [12 – 14]). 1 O2 reacts with a range of biological molecules including DNA [15], cholesterol [16], lipids [17], and amino acids and proteins [18 –20]. Proteins are major biological targets for 1O2, owing to their abundance and high rate constants for reaction [20,21]. Reaction with proteins occurs primarily at Trp, Met, Cys, His, and Tyr side-chains (reviewed [19,20]), with this resulting in the formation of short-lived endo- or hydroperoxides on Trp, His, and Tyr residues; the structure of these materials has been reviewed [19,20]. Similar species have been detected on intact proteins [22,23] and on proteins within intact cells [23]. Peroxides are also generated on exposure of proteins, peptides, and amino acids, in the presence of O2, to high energy radiation (e.g., g-sources, X-rays), metal ion/peroxide systems, thermal sources of peroxyl radicals, per-

INTRODUCTION

Singlet oxygen (molecular oxygen in its 1Dg state; 1O2) has been reported to be generated in myeloperoxidase- and eosinophil peroxidase-catalyzed reactions [1– 3] and by certain activated cells including neutrophils [4], eosinophils [3,5], and macrophages [6]. Direct exposure of a number of compounds, including proteins, to UV light has been shown to generate 1O2, and it has been demonstrated that a large number of dye molecules and endogenous cellular chromophores also generate 1O2 on exposure to visible light. As a result of the widespread exposure of humans to UV and visible light, 1O2 has been suggested to

Address correspondence to: Dr. Michael Davies, The Heart Research Institute, 145 Missenden Road, Camperdown, Sydney, NSW 2050, Australia; Fax: +61 2 9550 3302; E-mail: [email protected]. 1 Present address: University of Canberra, ACT 2601, Australia. 484

Detoxification of protein peroxides

oxynitrite, and activated white cells among others [24,25]. These peroxides are long-lived in vitro in the absence of light, heat, or redox-active transition metal ions [24 – 27]. In the presence of these agents rapid decomposition occurs with the formation of reactive radicals [22,26 –28]. The formation of such protein radicals can propagate oxidative damage and chain reactions, as well as the transfer of oxidative damage to other biomolecules including lipids, antioxidants, and DNA [25,29 – 31]. Cellular defenses against such protein peroxides are poorly understood. Previous studies have shown that peroxides generated by g-radiation can be removed by ascorbate, and thiols and, for amino acid and peptide peroxides, by reaction with glutathione peroxidase and some heme enzymes [24,25,29,32,33]. However the nature of these radiation generated species is very different from those generated by 1O2, as the former are primarily present on aliphatic residues (Val, Leu, Ile, Glu, Pro, Lys) [24,34], whereas 1O2-generated peroxides are predominantly present on Tyr, His, and Trp residues, which are typically buried within protein structures [20,27,35,36] and hence would be expected to have a very different accessibility and hence reactivity. In particular these peroxides may be poorly removed by enzymes, and other detoxification systems, due to steric and electronic effects, and this may explain the long-lived nature of these peroxides when generated within cells [23]. In this study the removal of these 1O2-generated peroxides by both enzymatic and low-molecular-weight systems is examined. MATERIALS AND METHODS

Materials Proteins, enzymes, and cofactors were obtained from Sigma– Aldrich (St. Louis, MO; BSA, fraction V, >96% or >98%, free of fatty acids; lysozyme, chicken egg white, approx. 95%; catalase, from bovine liver; superoxide dismutase, from bovine erythrocytes; horseradish peroxidase; glutathione peroxidase, from bovine erythrocytes; metmyoglobin from horse heart, minimum 90%; methemoglobin, from bovine blood), Bachem (Bubendorf, Switzerland; H – Gly – Tyr – Gly – OH), or Boehringer Mannheim (Roche, Basel, Switzerland; pronase, from Streptomyces griseus). All other chemicals were commercial samples of high purity and used as supplied. Water used in all experiments was passed through a four-stage Milli Q system equipped with a 0.2 Am-pore-size final filter. Solutions of Fe2+-EDTA (1:1 complex) were prepared using de-oxygenated water and maintained under an atmosphere of oxygen-free N2 during use. When ethanol (for ebselen, Probucol and BHT) or acetone (for ascorbic acid-6-palmitate) was used as a co-solvent, final concentrations were 9.5% v/v, and 33% v/v, respectively. All

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concentrations are final concentrations in the reaction mixture. Peroxide generation and quantification Peroxides were generated on BSA and lysozyme (50 mg ml 1) and N-Ac – Trp– OMe and Gly– Tyr –Gly (2.5 mM) by photolysis with visible light (250 W, Kodak S-AV 2050 slide projector) through a 345 nm cutoff filter in the presence of 10 AM rose bengal [22,27]. Solutions were kept on ice during photolysis (30 or 60 min for BSA, 60 min for N-Ac – Trp– OMe, lysozyme, Gly –Tyr– Gly) and were continually aerated. Except where indicated, H2O2 formed during the photolysis period was removed by the addition of catalase (65 U ml 1) and incubation at room temperature for 15 min. Aliquots were subsequently frozen at 80jC until used. Peroxide concentrations were determined by use of a modified FOX assay as described in [37]. This assay involves the spectrophotometric quantification of the oxidation of an acidic Fe(II) – xylenol orange complex to the corresponding Fe(III) species, by the peroxide, in the absence of sorbitol [37]. Diluted samples of commercial H2O2 of known concentration were used to construct standard curves. This assay gives values similar to those of iodometric analysis (A. Wright, C.L. Hawkins, and M.J. Davies, unpublished data). Protein concentrations were determined by the BCA assay (Pierce, Rockford, IL) using BSA standards unless otherwise stated. BSA peptide peroxides were prepared by photooxidation of BSA (10 mg ml 1) for 60 min as described above, with subsequent treatment with catalase (as above) and pronase (1 mg ml 1) for 15 min at 37jC. Samples were subsequently cooled to 4jC, and the pronase was removed by centrifugation (ca. 1500  g, 4jC) through a 10 000 molecular weight cut-off filter (Amicon Ultrafree 15; Millipore, North Ryde, NSW, Australia). The eluent was subsequently frozen at 80jC until used. Cell culture J774A.1 cells (a murine macrophage-like cell line) were cultured under sterile conditions in a humidified 5% CO2 atmosphere. Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Sigma, St. Louis, MO), with added 10% (v/v) heat-inactivated fetal calf serum (Gibco BRL, Life Technologies, Melbourne, Australia), 2 mM L-glutamine (Trace Scientific, Melbourne, Australia), 100 U ml 1 penicillin, and 0.1 mg ml 1 streptomycin (Sigma, St. Louis, MO). Prior to lysis, cells were washed twice with PBS, followed by scraping from the flasks in PBS, and pelleting by centrifugation at 1500 rpm for 5 min. The cells were resuspended and lysed in water at either 2  106 or 10  106 cells mL 1 and were divided

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into 0.5 ml aliquots prior to storage at 80jC. Cell protein concentrations were determined using the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL) with BSA standards. Reaction of peroxides with proteins and enzymes The effects of enzymes on peroxide levels were examined by the addition of the enzyme (catalase, 65 U ml 1; superoxide dismutase (SOD), 20 U ml 1, horseradish peroxidase (HRP), 25 U ml 1) to otherwise untreated samples and incubation at room temperature with residual peroxide concentrations determined at 0, 5, 10, 15, and 30 min. The effects of glutathione peroxidase were determined using catalase pretreated samples. The enzyme was activated before use by incubation with 2 mM GSH for 15 min at 37jC, followed by PD-10 column chromatography (Amersham Biosciences, Uppsala, Sweden) to remove low-molecular-weight materials, and storage at 4jC until use. Preactivated enzyme (ca. 2.5 U ml 1) was then incubated with peroxide-containing samples (ca. 80 nmol peroxide per unit GPX activity), or controls, at room temperature, in the presence, or absence, of a 10-fold excess of GSH (at pH 7.4) over peroxide concentration, with residual peroxide concentrations measured at 0, 5, 10, 15, and 30 min. Reaction of protein and peptide peroxides with myoglobin and hemoglobin were carried out by incubation with the met and oxy forms (25 – 50 AM with respect to heme) at pH 7.0 F 0.5 and 25jC in a cuvette within a UV-visible spectrophotometer. Reactions were followed by monitoring changes in absorbance between 450 and 700 nm, with scans performed before and immediately after addition of peroxides, and every 5 min thereafter for 60 min. Heme concentrations were determined by use of Drabkin’s reagent [38] with 1 ml added to 100 Al of 0.2 –0.3% (w/v) solutions of the heme proteins. Samples were incubated in the dark for 15 min at room temperature, followed by measurement of the absorbance at 540 nm using q540 = 11 000 M 1 cm 1. The oxy forms of the proteins were prepared on the day of use by treatment with sodium dithionite (20 mg ml 1) followed by immediate gassing with O2 for ca. 3 min. Excess sodium dithionite was then removed by elution through PD-10 columns, and the protein fractions were kept at 4jC under an O2 atmosphere; such solutions were stable for several hours under these conditions. Reaction of peroxides with low-molecular-weight agents The effect of added reagents on BSA peroxide concentrations was examined by incubation of the samples with a 10-fold excess (5-fold for Probucol and BHT) of reagent over peroxide concentration for 30 min at 37jC. Controls were incubated under identical conditions, with

water (plus co-solvent when used) in place of the reagent. Immediately postincubation, samples were loaded onto a PD-10 column and eluted with water; the protein fractions were collected and residual peroxide was determined using the modified FOX assay after correction for sample dilution (assessed by protein absorbance at 280 nm). None of the added reagents interfered significantly with these procedures. The pH of representative incubations was checked, and in all cases was 7.0 F 0.5. Reaction of peptide and protein peroxides with cell lysates Peroxide concentrations were determined upon incubation of photolyzed BSA, pronase-digested BSA, N-Ac – Trp– OMe, or Gly– Tyr –Gly (all ca. 20 AM peroxides initially), for up to 120 min at 37jC, in the presence and absence of J774A.1 cell lysates (0.4 mg ml 1 protein). Aliquots (100 Al) were removed at the indicated time points and added to 900 Al of water, and residual peroxides were determined using the modified FOX assay. To determine whether there was a correlation between peroxide loss and thiol loss, free thiol concentrations were determined after J774A.1 cell lysates (0.4 mg ml 1 protein) were incubated with nonphotolyzed or photolyzed (ca. 20 AM peroxides) BSA, pronase-digested BSA, N-Ac – Trp– OMe, or Gly – Tyr– Gly for up to 120 min at 37jC. Aliquots (100 Al) were removed at the stated time intervals and added to 1 ml of 500 AM 5,5V-dithionitrobenzoic acid (in 100 mM phosphate buffer, pH 7.4). The released 5-thionitrobenzoic acid anion was quantified by its absorbance at 412 nm after 30 min at room temperature using q412 = 13 600 M 1 cm 1. EPR spectroscopy EPR samples were prepared by addition of the peroxide (80 –200 AM, either in the presence of the putative antioxidant or after pretreatment and PD-10 elution as described above) to solutions containing the spin trap PBN (9.4 mM in 50 mM phosphate buffer, pH 7.4) at 20jC. Fe2+ – EDTA (100 AM, 1:1 complex) was added where stated. Samples were subsequently incubated for 5 min at 20jC before examination by EPR using a standard, flattened, aqueous solution cell (WG813-SQ; Wilmad, Buena, NJ). Relative radical adduct concentrations were determined by measurement of peak-to-peak line heights on spectra recorded under identical conditions. EPR spectra were recorded at 20jC using a Bruker EMX X-band spectrometer equipped with 100 kHz modulation and a cylindrical ER4103TM cavity. Typical spectrometer settings were: gain, 1.0  106; modulation amplitude, 0.1 mT, time constant, 163.8 ms; sweep time, 81.9 s; center field,

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348.0 mT; field sweep width, 8.0 mT; microwave power, 25.0 mW, frequency, 9.7 GHz; with four acquisitions averaged. Statistical analysis Statistical analyses comparing the efficacy of enzymes in removing peroxides compared to the corresponding controls were performed at the final time point using a one-way ANOVA with Tukey’s post-hoc test. Comparisons of multiple peroxide concentrations/EPR signal intensities to corresponding controls (at the final time point where multiple samples were analyzed) were performed using a one-way ANOVA with Dunnett’s post-hoc test. Where only one condition was compared to its corresponding control a Student’s t test was used. Where percentages were used in analysis the data were first transformed using the formula p V= arcsine Mp. In all cases significance was assumed if P < 0.05. RESULTS

Reaction of peptide and protein peroxides with protective enzymes Photolysis of solutions of N-Ac – Trp– OMe, Gly – Tyr –Gly, lysozyme, or BSA in the presence of rose bengal with visible light (E > 345 nm) in the presence of O2 resulted in the generation of peroxides as detected by a modified FOX assay. The identification of the FOX assay-positive material as substrate-derived peroxides, and not H2O2, was confirmed by the addition of catalase to remove the latter prior to analysis. Omission of the rose bengal, photolysis in the absence of O2, or incubation of complete samples in the absence of light, resulted in substrate peroxide concentrations of < 5 AM. This is in accord with previous studies [22,27,39]. Treatment of (non-catalase-treated) N-Ac –Trp –OMeand BSA-derived peroxides with superoxide dismutase (ca. 20 U ml 1) for up to 30 min at room temperature did not result in any statistically significant loss of peroxides when compared to controls (Fig. 1). Incubation with catalase (ca. 65 U ml 1) resulted in a rapid initial decrease in peroxide concentration due to the consumption of H2O2, followed by a further, much slower, decay over the remaining period which occurred at a similar rate in the untreated samples (Fig. 1). The identical rates of loss of peroxide between the control and catalase-treated sample, after the removal of H2O2, is consistent with a lack of activity of the enzyme toward the substrate-derived peroxides. These data are consistent with previous work with a range of photolyzed peptides and proteins and higher catalase levels [39].

Fig. 1. Effects of Cu/Zn superoxide dismutase, catalase, and horseradish peroxidase (HRP) on BSA and N-Ac – Trp – OMe peroxides generated by rose bengal in the presence of visible light and O2. (A) BSA (50 mg ml 1) and (B) N-Ac – Trp – OMe (2.5 mM) were photolyzed in the presence of rose bengal (10 AM) with visible light for 30 min (BSA) or 60 min (N-Ac – Trp – OMe), with continual gassing with air at 4jC. Immediately following photolysis, superoxide dismutase (ca. 20 U ml 1), catalase (ca. 65 U ml 1), or HRP (ca. 25 U ml 1) was added individually to aliquots, and peroxide levels were determined at the indicated times using a modified FOX assay. (w) No additions; () superoxide dismutase added; ( ) catalase added; (E) HRP added. Initial peroxide concentrations were in the range of 160 – 260 AM for BSA and 630 – 1250 AM for N-Ac – Trp – OMe. Data are means F SD of three or more independent experiments. As the initial peroxide concentrations varied between experiments, each graph is a composite of data converted to percentages of initial peroxide concentrations; individual peroxide concentrations after enzymatic treatment were compared to their corresponding control, which was an aliquot of the same preparation without added enzyme.

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Horseradish peroxidase (ca. 25 U ml 1) decreased the peroxide concentration in photolyzed, non-catalase-treated, BSA solutions by a small, but statistically significant, amount when compared to controls (one-sided paired t test at 30 min, P < 0.005; Fig. 1A). Analogous behavior was observed with photolyzed N-Ac – Trp – OMe (Fig. 1B). Approximately 80% of the initial peroxides

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remained after 30 min of incubation at room temperature in both cases, indicating that this peroxidase does not rapidly remove these materials. Preincubation with catalase (5 Ag ml 1; ca. 65 U ml 1) for a 15 min period was employed in all subsequent experiments to ensure complete removal of H2O2 from all photolyzed solutions. Reaction of peptide and protein peroxides with hemoglobin and myoglobin As previous studies have indicated that some radiationgenerated amino acid peroxides can react with hemoglobin [32], similar studies were carried out with 1O2-mediated peroxides. Oxidation of the heme group in iron (II) (oxy-) and iron (III) (met-) hemoglobin and myoglobin was monitored spectrophotometrically (E 450 – 700 nm) at 25jC; consumption of peroxides could not be readily examined due to the intense optical absorption bands of the heme proteins. Incubation of N-Ac – Trp– OMe peroxides and BSA peroxides with methemoglobin and metmyoglobin did not result in any significant changes (data not shown). In contrast, incubation with oxyhemoglobin (Fig. 2A) or oxymyoglobin (Fig. 2B) resulted in rapid oxidation of the heme iron in some cases, with NAc – Trp – OMe peroxides (4:1 molar ratio relative to heme) having the greatest effect (Fig. 2). These reactions yielded good isobestic points (data not shown) consistent with a clean conversion of the Fe2+ – O2 form to the Fe3+ state. The peroxide dependence of these reactions was confirmed by use of N-Ac –Trp –OMe peroxide solutions that had been allowed to decay (incubation at 37jC for 7 days) before use (Fig. 2). Lysozyme peroxides (39 AM peroxides, 25 AM heme) slowly oxidized oxyhemoglobin, but not oxymyoglobin, but BSA peroxides (100 AM peroxides, 25 AM heme) did not oxidize the heme group of either protein (Fig. 2). In contrast, peptide peroxides (20 AM peroxides, 25 AM heme) obtained from enzymatic digestion of BSA peroxides (see Materials and Methods) gave rise to significant oxidation (Fig. 2). Gly –Tyr– Gly peroxides (100 AM peroxides, 25 Am heme) had similar behavior. These data are consistent with the size of the peroxide-containing molecule being a key determinant of the rate of oxidation of the heme groups in oxyhemoglobin and oxymyoglobin. Nonphotolyzed controls, which contained rose bengal, did not generate any significant changes in the observed spectra (data not shown). Reaction of peptide and protein peroxides with glutathione peroxidase Incubation of BSA or lysozyme peroxides with glutathione peroxidase in the presence of GSH at room temperature did not result in any enhancement in the

Fig. 2. Effects of peptide and protein peroxides on the absorbance spectra of oxyhemoglobin and oxymyoglobin at selected wavelengths. Oxyhemoglobin and oxymyoglobin were prepared as detailed under Materials and Methods. Incubations with peroxides were performed at 25jC for 60 min, with absorbance measured before, and immediately after, mixing and every 5 min thereafter. Changes in oxyhemoglobin spectra (A) were measured at 576 nm; changes in oxymyoglobin spectra (B) at 581 nm. (n) BSA (100 AM peroxides, 25 AM heme); (+) lysozyme (39 AM peroxides, 25 AM heme); (x) N-Ac – Trp – OMe (200 AM peroxides, 50 AM heme); () Gly – Tyr – Gly (100 AM peroxides, 25 AM heme); (E) BSA-derived peptide peroxides, obtained by digestion of photolyzed BSA with pronase (20 AM peroxides, 25 AM heme); (o) N-Ac – Trp – OMe peroxides which had been preincubated at 37jC for 7 days to allow the peroxides to decay.

rate of peroxide loss above that generated by GSH alone (Fig. 3). In contrast, addition of glutathione peroxidase (ca. 2.5 U ml 1) to N-Ac – Trp– OMe and Gly – Tyr – Gly peroxides in the presence of GSH resulted in a rapid loss of these peroxides (Fig. 3). BSA-derived peptide peroxides, generated by pronase digestion, were also removed rapidly (Fig. 3). Treatment with glutathione peroxidase alone did not result in significant loss of any of these peroxides when compared to controls without enzyme (Fig. 3). GSH alone

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Fig. 3. Effects of glutathione peroxidase (GPX) on peptide and protein peroxides generated by rose bengal in the presence of visible light and O2. Peptide and protein peroxides were generated as described in Fig. 1 and the text, with BSA photolyzed for 60 min. Immediately following photolysis, catalase (ca. 65 U ml 1) was added and incubated at room temperature for 15 min to remove H2O2. BSA peptide peroxides were prepared by incubation of the oxidized BSA at 37jC in the presence of 1 mg ml 1 pronase. Peroxide levels were determined during further incubation with GPX (ca. 2.5 U ml 1), in either the presence or the absence of a 10-fold excess of GSH over the peroxide concentration, at the indicated times using a modified FOX assay. (x) Control; (5) GSH added; (D) GPX added; () GSH and GPX added. In all cases the ratio of peroxides to GPX was ca. 80 nmol of peroxides per unit of GPX. Data are means F SEM of three independent experiments. Statistical analysis (at 30 min) was by a one-way ANOVA with Tukey’s post-hoc test. Lines with different letters indicate statistically significant differences at the P < 0.05 level. (A) BSA peroxides; (B) lysozyme peroxides; (C) NAc – Trp – OMe peroxides; (D) Gly – Tyr – Gly peroxides; (E) BSA peptide peroxides generated by pronase digestion.

gave a statistically significant loss of peroxides, in most cases, when compared to controls (Fig. 3), although these reactions were slower than those with the complete system. Analogous experiments with GSH alone at 37jC gave a more rapid consumption of peroxide, with ca. 70% of BSA peroxides removed after 30 min (Table 1).

Reaction of peptide and protein peroxides with low-molecular-weight compounds The efficacy of other low-molecular-weight compounds, in addition to GSH, in removing BSA peroxides was examined by preincubating BSA peroxides (ca. 100 AM peroxide) with a 10-fold excess (5-fold for Probucol

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Table 1. Depletion of BSA Peroxides by Putative Antioxidants as Assessed by Residual Peroxide Levels and EPR Signal Intensities of Peroxide-derived Radicals Compound

Protein peroxides remaining (%)

EPR signal intensity retained (%)

Control (no additions) Ebselen Reduced glutathione (GSH) Dithiothreitol (DTT) Reduced lipoic acid N-(2-Mercaptopropionyl)glycine (2MPG) N-Acetyl-L-cysteine Meso-2,3-dimercaptosuccinic acid 2-Mercaptoethanesulfonic acid Captopril Oxidized lipoic acid L-(+)-Ergothioneine 3,3V-Thiodipropionic acid Methionine Ascorbic acid-6-palmitate Dehydroascorbic acid Ascorbic acid Ascorbic acid-2-phosphate Trolox C BHT Probucol

100 8 29 31 26 36

F5 F 2** F 7** F 1** F 10a F 11**

100 16 19 12

38 52 51 52 40 68 66 91 8 33 29 96 81 97 93

F 1** F 4** F 15** F 4** F 6** F 24* F 8* F2 F 7** F 1** F 8** F6 F1 F6 F 23

23 23 24 29 42 59 65 91

F F F F F F F F

8** 3** 2** 12** 5** 13** 6* 13

24 32 90 48 90 103

F F F F F F

3** 16** 8 17** 1 17

F F F F

12 1** 8** 5**

bate itself and ascorbate-6-palmitate were highly effective. The concentration-dependence and time course of peroxide removal by GSH, N-(2-mercaptopropionyl)glycine (2MPG), Trolox C, and ascorbic acid are given in Fig. 4. Scavenging of peroxide-derived radicals by low-molecular-weight compounds Decomposition of BSA peroxides by Fe2+ – EDTA (100 AM) in the presence of the spin trap PBN (9.4 mM)

20 F 1**

b

Note. BSA peroxide samples (66 – 133 AM initial peroxide content) were incubated with a 10-fold (5-fold for Probucol and BHT) excess of antioxidant and analyzed as described in the legend to Fig. 4. EPR samples were prepared as described in the legend to Fig. 5. The intensity of the radical adduct signal derived from the BSA peroxides was determined by measurement of the peak to peak line height of the central absorption line and compared to controls. Data are the means F range of triplicate determinations from two independent experiments. Statistical analysis was by one-way ANOVA with Dunnett’s post-hoc test. a Triplicate determinations from a single experiment. b Broad ascorbyl radical signal detected (see Fig. 5 (E)) which precluded accurate measurement of the intensity of the BSA peroxidederived radical signal. * P < 0.05 compared to photolyzed BSA without added antioxidant. ** P < 0.01 compared to photolyzed BSA without added antioxidant.

and BHT) of the added compound for 30 min, before separation of residual BSA peroxides from excess reagent by chromatography on PD-10 columns, and quantification of residual peroxide by the FOX assay. This separation was necessitated by the interference induced by some of these low-molecular-weight materials in the FOX assay. As low-molecular-weight peroxides (e.g., N-Ac – Trp – OMe-peroxides) could not be separated from the added reagent using this method, studies could not be carried out with these species. The data obtained with a range of compounds are summarized in Table 1. The organoselenium compound ebselen removed nearly all of the BSA peroxides, and a number of thiols had similar behavior, though larger species were less effective. The thioethers (3,3V-thiodipropionic acid and methionine), the phenols (Trolox C, BHT, and Probucol), and ascorbic acid-2-phosphate were poorly effective, whereas ascor-

Fig. 4. Loss of BSA-derived protein peroxides on incubation with putative antioxidant compounds at 37jC. (A) BSA peroxides (120 AM peroxide) were incubated with various excesses of low-molecularweight compounds for 30 min in H2O at 37jC, followed by measurement of residual peroxide levels. (B) Time course of loss of BSA peroxides (120 AM) incubated in H2O at 37jC with a twofold excess of the indicated compound over 30 min. In both series of experiments reactions were halted by the loading and subsequent elution of the reaction mixtures through PD-10 columns. Peroxide levels were assessed by a modified FOX assay after correction for dilution caused by the chromatography. Data are the means of triplicate determinations F SD from two independent experiments; where no error bar is visible it is obscured by the symbol. (o) No additions; (x) ascorbic acid; (E) Trolox C; (5) N-(2-mercaptopropionyl)glycine; () glutathione.

Detoxification of protein peroxides

gave intense EPR signals from large, slowly tumbling, protein-derived radical adducts, as described previously for other photooxidized proteins (Fig. 5A) [22,23,40]. The intensity of the EPR signals detected in these control experiments was then compared with those obtained in experiments where the BSA peroxides were pretreated with low-molecular-weight compounds (as described above) before reaction with the Fe2+ –EDTA (Figs. 5B and 5C). The percentage of the EPR signal remaining after such pretreatment follows a trend similar to that of the values for the residual protein peroxide concentrations determined by the FOX assay (Table 1), though the EPR values were generally lower. This was particularly noticeable with Trolox C and is ascribed to the occurrence of both peroxide reduction and radical-scavenging activities of the tested compounds. In the case of ascorbic acid-6palmitate an additional set of EPR features was detected which have been assigned to the ascorbyl radical formed on oxidation of this substrate, confirming its radical scavenging activity (Fig. 5E). Competition experiments in which Trolox C was added to the BSA peroxide/PBN mixture at the same time as Fe2+ –EDTA (i.e., without separation of the treated protein sample on a size-exclu-

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sion column) confirmed that this compound readily scavenges protein peroxide-derived radicals, with EPR signals from the Trolox C phenoxyl radical detected (Fig. 5F) [41]. This is consistent with Trolox C being a potent scavenger of radicals derived from BSA peroxides, though a poor direct detoxification agent for these species. Reaction of peptide and protein peroxides with cell lysates The above data suggest that reaction of peptide and protein peroxides with thiols is a major route to the removal of these compounds. Previous studies have demonstrated that protein thiols also react readily with peptide peroxides, and to a lesser extent with protein peroxides [39]. These reactions can result in the inactivation of thiol-dependent enzymes (e.g., glyceraldehyde3-phosphate dehydrogenase, glutathione reductase, caspases [39,42]). To test this hypothesis, the kinetics of loss of peroxides and thiol groups was examined in lysates from J774A.1 cells (a murine macrophage-like cell line). To confirm that these lysates retained enzymatic activity, aliquots (0.4 mg ml 1 protein) were incubated with 20

Fig. 5. EPR spectra of PBN radical adducts detected on reaction of Fe2+ – EDTA with untreated BSA peroxides (A) or antioxidant-treated BSA peroxides (B) – (F). Samples employed in experiments (A) – (E) were prepared by pre-incubating peroxidized BSA (66 – 123 AM peroxide) with a 10-fold excess of added agent (or water for controls) for 30 min at 37jC, before elution through a PD-10 column to stop further reaction. The resulting protein fractions were subsequently diluted to identical protein concentrations and PBN (9.4 mM, 50 mM phosphate buffer, pH 7.4) and 100 AM Fe2+ – EDTA were added. Samples were incubated for 5 min at 20jC before EPR spectra were recorded. (A) No additions; (B) after pretreatment with methionine; (C) after pretreatment with DTT; (D) after pretreatment with 33% v/ v acetone; (E) after pretreatment with ascorbic acid-6-palmitate in 33% v/v acetone. (F) EPR spectrum observed on incubation of BSA peroxides (80 AM) with Trolox C (800 AM), PBN (9.4 mM, 50 mM phosphate buffer, pH 7.4), and Fe2+ – EDTA (100 AM); the Trolox C was added simultaneously with the PBN. The broad signals present in spectra (A) – (D) are assigned to BSA-derived radical adducts. The sharp signals in (E) are assigned to the ascorbyl radical from ascorbic acid-6-palmitate. The sharp signals in (F) are assigned to the Trolox C phenoxyl radical. All spectra were recorded with identical spectrometer settings except for (F) where the gain was 5.

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AM H2O2 at 37jC. This resulted in a rapid loss of peroxide, as assessed by the FOX assay, with 50% depletion after 60 s and complete loss in under 5 min (data not shown). In the absence of

lysate, no loss of peroxide was detected under identical conditions (data not shown, c.f. data in [39]). In subsequent experiments, lysate samples (0.4 mg ml 1 protein)

Fig. 6. Loss of peroxide groups (A, C, and E) and consumption of thiol groups (B, D, and F) during incubation of J774 cell lysates with peptide and protein peroxides at 37jC. Peroxides were generated on BSA (A, B), BSA-derived peptides (by pronase digestion of BSA peroxides) (C, D), and N-Ac – Trp – OMe (E, F) as detailed in the legend to Fig. 1 and the text, with photolysis for 60 min. Subsequent incubations for 2 h at 37jC employed ca. 20 AM peroxide in the presence or absence of J774A.1 cell lysates (0.4 mg ml 1 protein). Peroxide concentrations were determined by a modified FOX assay, and thiol concentrations were measured by reaction with DTNB at the indicated times. Initial free thiol concentrations in (B) and (D) were derived from both BSA and J774A.1 lysates as follows: (B) 66 AM derived from J774A.1 lysates, remainder BSA free thiols; (D) 59 AM derived from J774A.1 lysates, remainder free thiols on BSA-derived peptides. (n) Protein or peptide peroxides in absence of added lysate; (o) protein or peptide peroxides with added J774A.1 lysates; (D) thiol concentrations in J774 A.1 lysates incubated with nonphotolyzed protein or peptide; (x) thiol concentrations in J774A.1 lysates incubated with protein or peptide peroxides. Data are the means F range of three independent experiments; where no error bar is visible it is obscured by the symbol. Statistical analysis was by one-tailed t test. In the statistical calculations in (B) and (D), percentage losses of the initial thiol concentrations (data not shown) were used, as the initial thiol concentrations were not equal; * P < 0.001.

Detoxification of protein peroxides

were incubated at 37jC with ca. 20 AM peroxides present on N-Ac – Trp – OMe, Gly – Tyr – Gly, BSA, and BSAderived peptides. In each case a time-dependent loss of peroxides was observed (Figs. 6A, 6C, and 6E; Gly – Tyr –Gly results not shown). With N-Ac – Trp– OMe and Gly – Tyr – Gly little peroxide loss was observed over similar time periods in the absence of added lysate, whereas in the case of BSA and BSA-derived peptide peroxides a significant time-dependent loss of peroxide was also observed in the absence of added lysate (Fig. 6). This is ascribed to either thermal decomposition of the peroxide or reduction of these species by the single free thiol group (Cys-34) present on BSA [43]. Measurement of lysate thiol groups (both low-molecular-weight and protein-bound) in identical incubations demonstrated that thiol groups were lost with kinetics similar to those of the loss of peroxides (Figs. 6B, 6D, and 6F). Previous studies have shown that, in the absence of added peroxide, J774A.1 lysate thiols are stable over 2 h at 37jC [44], thus the thiol losses cannot be ascribed to thermal decomposition. The bulk of the thiols lost were derived from cell lysate components, and not the single free thiol group (Cys-34) present on the added BSA. The total thiols lost in solutions containing BSA peroxides and J774A.1 lysates (initially 88 AM free thiols) was 26 AM over 2 h, of which 3 AM was due to thiol loss from the BSA (Fig. 6B) and the total thiol loss in the solution containing pronase-digested BSA peroxides and J774A.1 lysates (initially 65 AM free thiols) was 35 AM over 2 h, of which 1 AM was BSA thiol loss (Fig. 6D). In the case of N-Ac – Trp– OMe peroxides 3 mol of thiol were consumed per mole peroxide lost (12 Amol peroxides, 36 Amol free thiols, Fig. 6F), whereas for Gly– Tyr –Gly the ratio was approximately 1:1 (15 Amol peroxide, 14 Amol free thiols, data not shown). As additional decay mechanisms occur with the BSA- and pronase-digested BSA – peroxides in the absence of J774A.1 lysates, the ratio of thiols consumed per mole of peroxides lost could not be determined accurately; in both cases, however, greater than one mole of thiol was consumed per mole peroxide lost.

DISCUSSION

Reaction of amino acids, peptides, and proteins with O2 has been previously shown to generate peroxides which are primarily located on Tyr, Trp, and His sidechains ([22,27,35,36,45], reviewed [20]). The structures of some of these peroxides have been determined [27,36,45,46]. Similar species can be formed on isolated proteins [22,23] and proteins within intact cells [23], as well as by exposure to peroxyl radicals [47] or g radiation [48]. The residues on which these peroxides are formed within proteins have not been determined. 1

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Previous studies have shown that 1O2-generated peptide and protein peroxides are not removed by the protective enzyme catalase [22,39]; this has been confirmed here. Other heme-containing proteins (myoglobin, hemoglobin, and horseradish peroxidase) also react slowly, if at all, with these species. No reaction was seen with Cu/Zn SOD, and the selenium-containing enzyme glutathione peroxidase only removed the low-molecular-weight peroxides. Steric bulk therefore appears to be a major factor in determining which species are detoxified, though the buried position of most Tyr, His, and Trp residues (and hence the likely site of peroxides) within proteins and hindered access to the active sites in these proteins (e.g. [49 –51]) probably also play a role. In accord with this explanation, the smallest peroxides (e.g., those formed on N-Ac –Trp –OMe) were the only species that reacted at any significant rate. Similar behavior has been observed with radiation-generated peroxides exposed to lactoperoxidase, glutathione peroxidase, and phospholipid glutathione peroxidase [25,29,33]. The differences observed within the 1O2-generated peroxides are unlikely to be due to different peroxide populations, as peptide peroxides generated from BSA by enzymatic digestion reacted more rapidly than those on the parent protein, which will presumably have the same product distribution. The observation that only the oxy (Fe2+) forms of myoglobin and hemoglobin react was unexpected, as the met (Fe3+) forms of these proteins react rapidly with other alkyl peroxides (e.g., tert-BuOOH) and H2O2 to give higher oxidation states [50 – 52]. The reason for this difference is unknown, as the oxidation potentials of these peroxides are likely to be similar. The clean stoichiometric removal of 1O2-generated peroxides by the oxy Fe2+ species may be a protective process in cells that contain met –heme (ferriheme) reductase enzymes (e.g., myocytes and erythrocytes [53,54]). In contrast to the above data, BSA peroxides were removed, albeit slowly, by some low-molecular-weight thiols, the organo-selenium compound ebselen, and ascorbic acid and some derivatives. This removal was species-, time-, and concentration-dependent. The efficiency of removal of peroxides by equimolar concentrations of ascorbic acid, 2MPG, and GSH were 0.6, 0.35, and 0.15, respectively (Fig. 4A). The kinetics of removal followed a similar trend (ascorbate > 2MPG > GSH). The efficiency of GSH remained at ca. 0.15 per mole GSH added over a range of excesses, although the overall extent of peroxide removal increased; thus a 5-fold excess of GSH, removed ca. 70% of the starting peroxide after 30 min (Fig. 4A). Peroxide removal by 2MPG, GSH, and ebselen is likely to occur via two-electron molecular reactions with formation of disulfides (from the thiols) and the selenoxide from ebselen. This has not been confirmed here, but

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is in accord with previous studies [55,56]. Peroxide removal by ascorbate (and derivatives) may be via oneelectron, radical-mediated processes catalyzed by trace transition metal ions; this interpretation is supported by the detection of the ascorbyl radical (and derivatives) by EPR. In contrast to the above compounds, the phenols Trolox, BHT, and Probucol were inefficient at removing the peroxides (Fig. 4 and Table 1). Trolox was however an efficient scavenger of BSA peroxide radicals generated by Fe2+ – EDTA. The detection of the Trolox phenoxyl radical by EPR confirmed this action (Fig. 5), and this is in accord with the rate constants for reaction of these compounds with other radicals [57,58]. Methionine can remove both lipid peroxides [59] and radiationgenerated amino acid-peroxides and protein peroxides [33], but was inefficient at removing both the 1O2generated peroxides and radicals derived from these. This may be due to its zwitterionic nature, as the related compound 3,3V-thiodipropionic acid was more efficient (Table 1). Removal by thiols and ascorbate may be the major detoxification pathways for 1O2-mediated protein peroxides within cells. The former may be more important due to their higher concentration within most cells [60]. In support of this conclusion, we have shown previously that ascorbate-deficient and ascorbate-replete cells contain identical protein peroxides levels after illumination, which is inconsistent with ascorbate being a major detoxification agent [23]. Furthermore, the parallel kinetics of peroxide consumption and thiol loss in cell lysates is consistent with thiol oxidation (directly, or indirectly via unknown enzyme reactions) being the major pathway. The stoichiometry of thiol lost to peroxide consumed, which was nearly always between 2:1 and 1:1, is consistent with molecular oxidation of the thiol to a sulfenic acid (RSOH), and subsequent rapid reaction of this species with either a second thiol molecule (to give the disulfide and a 2:1 stoichiometry) or other nucleophiles (with resultant formation of higher oxyacids, e.g., RSO3H, [61]). While these reactions might be considered as protective, thiol depletion can inhibit a range of cellular processes as they are key enzyme cofactors, and oxidation of protein thiols can result in rapid inactivation of key thiol-dependent enzymes. This has been shown to occur with a range of enzymes (e.g., glyceraldehyde-3-phosphate dehydrogenase, glutathione reductase, and caspases) [39,42]. Even with large reagent excesses, or with added cell lysates, complete removal of the peroxides was difficult to achieve even after 120 min at 37jC (Figs. 4 and 6, Table 1). This suggests the presence of particularly stable protein peroxide populations. These may be species buried within protein structures which are inaccessible to aqueous phase reagents. In this regard, it is interesting to note that the

most efficient low-molecular-weight species examined (ebselen, ascorbic acid-6-palmitate) are hydrophobic, and hence most likely to penetrate into protein structures. This slow removal may underlie the ready detection of protein peroxides in intact cells exposed to visible light in the presence of a range of photosensitizers ([23], Policarpio, Hawkins, and Davies, unpublished data). The above conclusions are in broad agreement with data obtained for radiation-generated peroxides [24,29, 33], even though the peroxides are formed on different residues. Two major differences are in the behavior of ebselen, which was efficacious in the current study but not with radiation-generated peroxides [33], and methionine, which showed the inverse; this may reflect the peroxide locations within proteins and their accessibility. Such variations may make the design of broad spectrum protective agents against protein and peptide peroxides more complex.

Acknowledgments—This work was supported in part by the Australian Research Council, the Juvenile Diabetes Foundation International, and the National Health and Medical Research Council.

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ABBREVIATIONS

BSA—bovine serum albumin DTT—dithiothreitol EDTA—ethylenediaminetetraacetic acid disodium salt GSH—reduced glutathione 2MPG—N-(2-mercaptopropionyl)glycine N-Ac-Trp-OMe—N-acetyl tryptophan methyl ester 1 O2—molecular oxygen in its first excited singlet (1Dg) state PBN—N-tert-butyl-a-phenylnitrone SOD—superoxide dismutase HRP—horseradish peroxidase GPX—glutathione peroxidase