Biochemical Journal

Research article

Cu,Zn-superoxide dismutase-driven free radical modifications: copper- and carbonate radical anion-initiated protein radical chemistry

Dario C. Ramirez, Sandra E. Gomez-Mejiba, Jean T. Corbett, Leesa J. Deterding, Kenneth B. Tomer, Ronald P. Mason

Abstract

The understanding of the mechanism, oxidant(s) involved and how and what protein radicals are produced during the reaction of wild-type SOD1 (Cu,Zn-superoxide dismutase) with H2O2 and their fate is incomplete, but a better understanding of the role of this reaction is needed. We have used immuno-spin trapping and MS analysis to study the protein oxidations driven by human (h) and bovine (b) SOD1 when reacting with H2O2 using HSA (human serum albumin) and mBH (mouse brain homogenate) as target models. In order to gain mechanistic information about this reaction, we considered both copper- and CO3•− (carbonate radical anion)-initiated protein oxidation. We chose experimental conditions that clearly separated SOD1-driven oxidation via CO3•− from that initiated by copper released from the SOD1 active site. In the absence of (bi)carbonate, site-specific radical-mediated fragmentation is produced by SOD1 active-site copper. In the presence of (bi)carbonate and DTPA (diethylenetriaminepenta-acetic acid) (to suppress copper chemistry), CO3•− produced distinct radical sites in both SOD1 and HSA, which caused protein aggregation without causing protein fragmentation. The CO3•− produced by the reaction of hSOD1 with H2O2 also produced distinctive DMPO (5,5-dimethylpyrroline-N-oxide) nitrone adduct-positive protein bands in the mBH. Finally, we propose a biochemical mechanism to explain CO3•− production from CO2, enhanced protein radical formation and protection by (bi)carbonate against H2O2-induced fragmentation of the SOD1 active site. Our present study is important for establishing experimental conditions for studying the molecular mechanism and targets of oxidation during the reverse reaction of SOD1 with H2O2; these results are the first step in analysing the critical targets of SOD1-driven oxidation during pathological processes such as neuroinflammation.

  • carbonate radical anion
  • immuno-spin trapping
  • nitrone adduct
  • peroxymonocarbonate
  • protein radical
  • superoxide dismutase

INTRODUCTION

Much of the mechanistic biochemical evidence regarding SOD1 (Cu,Zn-superoxide dismutase)-driven pathogenic oxidative mechanisms has been obtained by studying the reaction between bovine or human SOD1 and H2O2 [14]. The reaction of SOD1 and H2O2 may be particularly important in sites of inflammation, particularly in peroxisomes, which are known to have high local concentrations of H2O2 and SOD1, and where the degradation of damaged proteins occurs [5]. The study of radical pathways induced by H2O2/SOD1-driven peroxidation may be important to the understanding of the molecular mechanisms of tissue damage during neuroinflammation, as occurs, for example, in fALS (familial amyotrophic lateral sclerosis).

Mechanistically, the reaction of SOD1 with H2O2 proceeds in several sequential steps (Reactions 1 and 2) that end with the formation of an enzyme-bound oxidant [i.e. (CuO)+↔(Cu-OH•)2+↔Cu3+] at the enzyme active site [6,7]. This species promotes oxidation of one or more histidine residues at the SOD1 active site [8,9], partial copper release [10,11], enzyme inactivation [6] and SOD1 fragmentation at its active site [10,12].

Embedded Image (Reaction 1)Embedded Image (Reaction 2)

The copper-bound oxidant at the enzyme active site (Reactions 1 and 2) is generally proposed to oxidize (bi)carbonate to the diffusible carbonate radical anion (CO3•−) [7,1316]. CO2 has been postulated to be the chemical entity in (bi)carbonate buffer that reacts with the strong oxidant at the SOD1 active site to give CO3•− [14]; however, the chemical mechanism of this reaction remains controversial [7,1719]. Much of our current knowledge of SOD1-driven oxidation has been gained by studying the oxidation of chemical compounds (e.g. NADPH [20], dichlorofluorescein [11], peroxidase substrates [12,21], ethanol, spin-trap compounds [21,22], formate and azide [13]), the loss of superoxide dismutase activity, or the activity or the structure of protein or nucleic acid targets [23]. Because of its diffusibility and powerful oxidative nature, CO3•− may form distinct radicals in SOD1 itself or tissue proteins, which may be trapped with DMPO (5,5-dimethylpyrroline-N-oxide) to form stable nitrone adducts for further characterization [12,24]. However, in cells and tissues, it is difficult to study the formation of protein radicals using ESR with or without spin trapping because of their rapid decay and the severe overlap of their spectra.

We have developed a new biochemical tool with which to study protein [2527] and DNA [24,28] radicals called immuno-spin trapping [26,27] that helps to identify protein radicals induced by SOD1-driven oxidation in cells and tissues. Immuno-spin trapping [29] involves the trapping of protein radicals with the spin trap DMPO in situ and in real time and the further detection of the protein radical–DMPO nitrone adducts with an anti-DMPO serum [25] by using heterogeneous immunoassays and also by MS [30]. In principle, this technology allows the simultaneous detection of more than one protein radical at the same time and in the same reaction system, as they are formed during tissue oxidative damage [24,29].

We have previously shown that (bi)carbonate, but not DTPA (diethylenetriaminepenta-acetic acid), protects SOD1 against H2O2-induced fragmentation at its active site and that, in the presence of DTPA, (bi)carbonate is required in order to observe SOD1 nitrone adducts with immuno-spin trapping [12]. In the present study, we have used immuno-spin trapping and MS to understand the mechanism of protein radical formation induced by the bovine and human SOD1/HSA (human serum albumin) or mBH (mouse brain homogenate)/H2O2 system. To accomplish this goal, we chose experimental conditions that would separate the two major radical pathways of protein modification by H2O2-induced SOD1-driven oxidation: copper (both active site and released)- and CO3•−-triggered radical reactions. Furthermore, we have analysed the way in which these two different initiators of radical reactions contribute to oxidizing target proteins and SOD1 itself using HSA and mBH as models. In the present study, we also propose a novel mechanism to explain how (bi)carbonate blocks the fragmentation of the SOD1 active site by the enzyme-bound oxidant (Reaction 2).

EXPERIMENTAL

Materials

bSOD1 (bovine SOD1) (from bovine erythrocytes) and beef liver catalase were purchased from Roche Applied Science. Sodium bicarbonate (99.7–100.3% purity) was purchased from Alfa Aesar. The spin-trap DMPO was purchased from Alexis Biochemicals, purified twice by vacuum sublimation at room temperature (15–25 °C), and stored under an argon atmosphere at −80 °C until use. The DMPO concentration was measured at 228 nm, assuming a molar absorption coefficient of 7800 M−1·cm−1. Reagent-grade 30% H2O2 was obtained from Fisher Scientific. The H2O2 concentration was verified using UV–visible absorption at 240 nm (ε240=43.6 M−1·cm−1). Recombinant hSOD1 (human SOD1) (expressed in Escherichia coli) was from BioVision. Erythrocytic hSOD1 and all other reagents were purchased from Sigma Chemical Co. Buffers were treated with Chelex® 100 ion-exchange resin (Bio-Rad Laboratories) to remove transition metals usually found as contaminants. The pH of the buffer solutions containing bicarbonate was adjusted to 7.4 by bubbling with a 19:1 N2/CO2 gas mixture.

SOD1, HSA and mBH preparation

Our bSOD1 and hSOD1 (from erythrocytes and recombinant) solutions were prepared and determined to be free from detectable unbound copper as described previously [31]. SOD1 concentration was determined by measuring the absorbance at 258 nm (ε258=10.3 mM−1·cm−1) at pH 7.4 [32]. HSA solutions were prepared as described previously [31,33]. Our HSA preparations were free from detectable copper contamination, and the free sulfhydryl/albumin ratio was similar to that reported in [31,33]. The HSA concentration was determined by using the BCA (bicinchoninic acid) assay (Pierce) or assuming its UV–visible molar absorption coefficient of ε280=35700 M−1·cm−1. Male C576J/BL mice (25–30 g of body weight) were killed following institutionally approved protocols, and brains were collected in ice-cold 10 mM PB [sodium phosphate buffer (pH 7.4)], washed in the same buffer and homogenized (1 g/ml). The homogenate was centrifuged at 11700 g and the supernatant was dialysed (3 kDa cut-off) against 10 mM PB. The protein concentration in the dialysed mBH was determined using the BCA assay.

Chemical reactions

Typically, the reactions of 15 μM SOD1, 7.5 μM (0.5 mg/ml) HSA or 0.5 mg/ml mBH and 0.1 mM H2O2 were carried out in the presence of 100 mM DMPO in 100 mM Chelex®-bound BB [sodium (bi)carbonate buffer (pH 7.4)], or 100 mM Chelex®-bound PB, or in PB containing a physiological concentration of (bi)carbonate (i.e. ∼25 mM) and with or without 0.1 mM DTPA. Solutions were incubated at 37 °C for 1 h, and the reaction was stopped by adding 10 IU of catalase to eliminate excess H2O2. The pH of the reaction mixtures after the reactions was completed was between 7.3 and 7.6.

Measurement of H2O2-induced bathocuproine-assisted reduction of released Cu2+

The H2O2-induced Cu2+ release from bSOD1 or hSOD1 and its reduction to Cu+ was monitored using the specific Cu+ chelator BCDS (bathocuproine disulfonic acid) [34]. Cu+ binds to BCDS and produces a complex [Cu+(BCDS)2]3− that exhibits a characteristic absorbance maximum at 480 nm, with ε480=12540 M−1·cm−1.

Measurement of SOD1 activity

bSOD1 and hSOD1 activity was measured using the ferricytochrome c reduction assay [11,32]. For controls, the ratio of ferricytochrome c reduction was measured in samples containing 15 μM active or heat-inactivated SOD1 (incubation for 40 min at 75 °C) [32].

Anti-DMPO serum

A rabbit antiserum against the nitrone form of DMPO was obtained in our laboratory [25] and was used to develop immuno-spin trapping [26,27,29]; this antiserum has been used successfully to detect protein [27,29] and DNA [24,28] radicals. The anti-DMPO serum is commercially available from Alexis Biochemicals, Cayman Chemicals, AbCam, Chemicon International and Oxford Biomedical Research.

Immuno-spin trapping assays

DMPO–protein radical-derived nitrone adducts were determined using a standard ELISA and Western blot as described previously [12,29]. Briefly, the reaction mixture was separated by reducing SDS/PAGE [4–12% NuPAGE Novex Bis-Tris gels (Invitrogen); 1.2 μg of protein/lane]. After the separation of protein, gels were stained using Coomassie Blue, or the proteins were blotted to a nitrocellulose membrane, and the nitrone adducts were detected by Western blotting. Briefly, immunocomplexes were detected by exposing the membrane to NBT (Nitro Blue Tetrazolium)/BCIP (5-bromo-4-chloroindol-3-yl phosphate) One Step reagent from Pierce for 15 min or, when indicated, by enhanced chemiluminescence using a CDP-Star II (Roche Molecular Biochemicals)/Nitro Block II (Tropix) system. Where indicated, a MagicMark™ XP Western Protein Standard (Invitrogen) was used as a molecular-mass marker that glows after Western blot development [24].

Measurement of H2O2-induced SOD1-driven oxidation of guaiacol

The peroxidative activity of bSOD1 or hSOD1/H2O2 with or without (bi)carbonate or DTPA was measured using guaiacol as a substrate. The reaction was initiated by adding 5 μM SOD1 to 100 mM Chelex®-bound buffers (pH 7.4), containing 0.5 mM guaiacol and 1 mM H2O2 (final volume 1 ml). Oxidation of guaiacol to tetraguaiacol was measured spectrophotometrically in a Beckman DU® 640 spectrometer at 470 nm and at room temperature for 15 min. A molar absorption coefficient of 26.6 mM−1·cm−1 was used to quantify tetraguaiacol formation.

Mass spectrometric identifications from gel bands

In-gel tryptic digestion

The protein bands were manually excised from the gel, cut into small pieces and transferred into a 96-well microtitre plate. Gel pieces were subjected to automatic tryptic digestion using an Investigator™ Progest protein digestion station (Genomic Solutions). Briefly, gel bands were sequentially washed twice with 25 mM ammonium bicarbonate buffer (pH 7) and acetonitrile, dehydrated, rehydrated with 25 μl of the enzyme solution and digested at 37 °C for 8 h. The enzyme solution used was sequencing-grade modified trypsin (Promega Corporation) at a concentration of 0.01 mg/ml in 25 mM ammonium bicarbonate buffer (pH 7). Resulting tryptic peptides were extracted from the gel, freeze-dried and stored at −80 °C. Before mass spectrometric analysis, the peptides were reconstituted in 40 μl of a 97:3 solution of water/acetonitrile (containing 0.1% formic acid).

ESI (eletrospray ionization) MS

For the nano-LC (liquid chromatography)–ESI–MS/MS (tandem MS) analyses, we used an Agilent XCT Ultra ion trap (Agilent Technologies) equipped with an HPLC-Chip Cube MS interface and an Agilent 1100 nano-LC system. Injections of 30 μl of the peptide digests were made on to a 40 nl enrichment column followed by a 43 mm×75 μm analytical column packed with Zorbax 300SB C18 particles. Peptides were separated and eluted using a linear gradient of 3–50% acetonitrile (containing 0.1% formic acid) over 40 min, followed by a linear gradient of 50–95% acetonitrile over 7 min at a flow rate of 500 nl/min. The ion-trap mass spectrometer was operated in the positive-ion mode, standard enhanced mode using the following settings: capillary voltage, −2150 V; mass range, 300–1500; ICC smart target (number of ions in the trap before scan out), 100000 or 200 ms of accumulation; and MS/MS fragmentation amplitude, 1.0 V. During the LC–MS/MS analyses, automated data-dependent acquisition software was employed with the six most abundant ions (threshold requirement of 10000 counts) from each spectrum selected for MS/MS analysis.

Following the analyses, the MS/MS data were extracted and analysed using Spectrum Mill MS Proteomics software (Agilent Technologies). To generate peak lists, the raw data files were processed using the Data Extractor function with the following parameters: deconvoluted ions of 300–6000 Da and a retention time of 10–60 min. MS scans with the same precursor m/z were merged based on a ±1.4 m/z window and a ±15 s retention time window. Using the extracted data, searches were performed against the NCBI non-redundant protein database using the MS/MS search function. Parameters used for the searches included: precursor mass tolerance, ±1.5 Da; product mass tolerance, ±1 Da; enzyme specificity, trypsin, with maximum two missed cleavage sites; variable modifications, oxidized methionine and N-terminal pyroglutamic acid; and at least two unique peptides matched peak intensity, 80%; species, mouse. Proteins with a summed MS/MS search score of 30 or greater were considered for validation. At this scoring threshold, the false-positive rate was essentially 0% as determined by searching against a reversed sequence database. All MS/MS sequence assignments used for protein identifications were validated manually.

RESULTS

Parallel SOD1-centred radical and enzyme inactivation induced by H2O2

We observed H2O2-induced bSOD1 and erythrocytic hSOD1 inactivation in BB (Figure 1A). In agreement with our previous work [12] and that of other authors [11,18], we did not find any effect on H2O2-induced SOD1 inactivation when the (bi)carbonate concentration was 25 mM or less in PB with DTPA.

Figure 1 Parallel SOD1 inactivation and SOD1-centred radical formed by CO3•− and the effect of DTPA

(A) SOD1 activity determined after reacting 15 μM erythrocytic hSOD1 or erythrocytic bSOD1 with 0.1 mM H2O2 for 1 h at 37 °C in 100 mM PB containing various concentrations of (bi)carbonate (pH 7.4) and 0.1 mM DTPA. *P<0.05 with respect to reactions with no (bi)carbonate added. (B) Western blot of nitrone adducts produced when 15 μM bSOD1, 100 mM DMPO and 0.1 mM H2O2 were allowed to react for 1 h at 37 °C in 100 mM fresh argon-bubbled (to purge CO2) PB containing various concentrations of (bi)carbonate (pH 7.4), with 0.1 mM DTPA. Reactions were stopped by removing the excess H2O2 with 10 IU of catalase. M, molecular-mass markers (masses are indicated in kDa); (m), monomer; (d), dimer. (C) ELISA of nitrone adducts produced from reactions of 15 μM bSOD1 or hSOD1 with 0.1 mM H2O2 and carried out in 100 mM PB containing 25 mM (bi)carbonate (pH 7.4). Reactions were stopped at various times with 10 IU of catalase. RLU, relative light units. (D) Tetraguaiacol formed from reactions that contained the same reagent concentrations, except that the H2O2 concentration was 1 mM and the buffer was as in (B), but the reaction buffer contained various concentrations of DTPA. The oxidation of guaiacol (0.5 mM) to tetraguaiacol was determined at 15 min as indicated in the Experimental section. *P<0.05 compared with the sample without DTPA. Results are either from a representative experiment or are means±S.E.M. for three experiments, each in triplicate, as appropriate.

When (bi)carbonate was added to a bSOD1/H2O2 or hSOD1/H2O2 system in fresh argon-bubbled (to purge carbon dioxide) PB containing the copper chelator DTPA, the amount of H2O2-induced bSOD1-centred radicals observed as monomers and dimers increased, as seen by Western blotting (Figure 1B). The hSOD1 enzyme produced similar results (results not shown). Although bSOD1-centred radicals were detected at the physiological (bi)carbonate concentration of 25 mM, higher (bi)carbonate concentrations led to more nitrone adduct formation, demonstrating that the (bi)carbonate-dependent mechanism was not saturated under physiological conditions. We could not find any H2O2-induced SOD1 nitrone adducts in argon-purged freshly prepared Chelex®-bound PB containing 0.1 mM DTPA in a closed system, but we could detect them by Western blotting when PB was equilibrated with air at room temperature for 15 min, presumably due to the absorption of CO2 (results not shown). In PB with 0.1 mM DTPA, the SOD1 nitrone adduct (SOD1-self-oxidized) formation was totally dependent on (bi)carbonate. It increased with incubation time (Figure 1C) and was completely prevented by cyanide (results not shown), suggesting that (bi)carbonate and copper redox cycling at the enzyme active site is essential for producing SOD1 nitrone adducts.

In Figures 1(A)–1(C), the copper chelator DTPA was added to eliminate the contribution of H2O2-induced free copper-mediated SOD1 oxidation; however, it is known that CO3•− reacts relatively rapidly with DTPA (rate constant ∼1.7×107 M−1·s−1 [35]), and thus the concentration of DTPA was carefully adjusted to avoid underestimating CO3•−-triggered oxidations. Indeed, we observed that concentrations of DTPA higher than 0.5 mM inhibited CO3•−-mediated guaiacol oxidation (Figure 1D), suggesting that high concentrations of DTPA scavenge CO3•−. Accordingly, in subsequent experiments, we used 0.1 mM DTPA, which is over 3-fold higher than the copper content of the SOD1 solutions used in our experiments.

Albumin protects bSOD1 and hSOD1 activity by acting as an alternative target for radical modification

In order to investigate the potential for the induction of SOD1-driven oxidation in surrounding proteins, we used HSA as a model target. We observed that HSA blocked H2O2-induced SOD1 self-inactivation in BB, but in PB it had no effect (Figure 2A). This suggested that HSA protects SOD1 activity by scavenging the diffusible CO3•− that enhances H2O2-induced SOD1 inactivation. When we separated the reaction mixture using SDS/PAGE and stained the gels with Coomassie Blue, we observed that both proteins were extensively fragmented when the reaction was carried out in PB (Figure 2B, left-hand panel). DTPA protected HSA against fragmentation, but did not affect SOD1 fragmentation at its active site, as assessed by the ∼5 and ∼10 kDa SOD1 fragments [10,12] (Figure 2B, left-hand panel). However, when we performed the reaction in BB with DTPA, the active site of SOD1 was protected against site-specific fragmentation (Figure 2B, right panel; [12]), but extensive fragmentation at other sites of both SOD1 and HSA still occurs until DTPA is added. Although active-site fragmentation is not occurring, copper must still be released, presumably as a result of histidine oxidation (Table 1). This released copper must be causing protein fragmentation through Fenton-type chemistry. Similar results were obtained when erythrocytic bSOD1 was replaced by hSOD1 (results not shown).

Figure 2 Structural and functional modification of SOD1 by H2O2 and the effect of HAS

(A) SOD1 activity determined in a reaction mixture that contained 15 μM bSOD1, 0.1 mM H2O2 and various concentrations of HSA. Reactions were carried out at pH 7.4 in 100 mM BB or 100 mM PB, with 0.1 mM DTPA for 1 h. (B) Coomassie Blue-stained gels of reaction mixtures containing 15 μM bSOD1 and 7.5 μM HSA incubated as in (A), but carried out in PB (left-hand panel) or BB (right-hand panel) with or without 0.1 mM DTPA. The reactions were started by adding 0.1 mM H2O2 and carried out at 37 °C then stopped with 10 IU of catalase. (C) Western blots produced when 15 μM SOD1, 7.5 μM HSA and 100 mM DMPO were mixed in 100 mM PB, 100 mM BB or BB containing 0.1 mM DTPA (BB+DTPA) (pH 7.4) and allowed to react with various concentrations of H2O2. Reaction mixtures were incubated for 1 h at 37 °C and stopped by adding 10 IU of catalase. M, molecular-mass markers (masses are indicated in kDa); (f), (m) and (d) indicate fragment, monomer and dimer respectively. SOD1 activity assay and SDS/PAGE with Coomassie Blue staining were performed as described in the Experimental section. Results either are from a representative experiment or are means±S.E.M. for three experiments, each in triplicate, as appropriate.

View this table:
Table 1 Approximate rate constants (M−1·s−1) for the reactions of OH• and CO3•− with some amino acids at pH∼7.4

Chemical kinetic rate constants are from http://www.rcdc.nd.edu/Solnkin2/.

As modulated by the copper chelator DTPA and (bi)carbonate, two structural consequences of free radical chemistry can be delineated in the formation of H2O2-induced SOD1-driven protein radicals: fragmentation and aggregation. Fragmentation other than at the active site appears to be mediated by copper released from the active site of SOD1. In the absence of DTPA and (bi)carbonate, we saw evidence of both pathways, as protein nitrone adducts (both fragments and intact proteins) increased with the concentration of H2O2 (Figure 2C, left-hand panel). At the same H2O2 concentration, the number of radical sites was higher when the reaction was carried out in 100 mM BB rather than in 100 mM PB (Figure 2C, middle panel). But in BB with 0.1 mM DTPA, we observed distinct protein nitrone adducts of monomers and aggregates from both proteins, but not of fragments (Figure 2C, right-hand panel). These results suggest that DTPA inhibits the Fenton-like chemistry triggered by copper released from the SOD1, which results in protein fragmentation. Once the released copper rebinds to specific residues on SOD1 and HSA, in the presence of excess H2O2, these residues can also act as additional sites for CO3•− generation (see below). It is noteworthy that this amplifying copper-dependent radical chemistry occurs at protein copper-binding sites other than the active site of SOD1.

SOD1-driven copper-dependent radical damage

We further examined SOD1-driven copper-triggered radical chemistry in proteins by reacting SOD1 and HSA with H2O2 in PB without DTPA (Figure 3). Under these conditions, HSA and SOD1 itself were targets of copper-mediated site-specific fragmentation (Figure 3A, left-hand panel). H2O2-induced fragmentation of SOD1 at its active site was evident by its ∼5 and ∼10 kDa fragments [10,12] and was not prevented by DTPA, although DTPA did strongly inhibit nitrone adduct formation of the protein (Figure 3A, right-hand panel). This result suggests that the formation of H2O2-induced copper-bound oxidant at the SOD1 active site precedes release of copper. Released copper would then be re-bound to HSA and other sites in SOD1 itself to react with excess H2O2 and induce free-radical-mediated site-specific fragmentation. To test this hypothesis, we measured H2O2-induced copper release from the bSOD1 active site by BCDS-assisted reduction of Cu2+ to Cu+ as the formation of its red [Cu+(BCDS)2]3− complex (Figure 3B). Depending on the concentration of H2O2, 15 min of reaction was enough to release a significant amount of copper, an amount that would be sufficient to produce protein radicals, as we found previously by immuno-spin trapping in the HSA/Cu2+/H2O2 system [31]. After 2 h, 5 mM H2O2 released approx. one-half of the available copper at the active site.

Figure 3 Copper-mediated oxidative modification to SOD1 itself and HSA

(A) Left-hand panel: gel stained with Coomassie Blue; 15 μM bSOD1, 7.5 μM HSA and various concentrations of H2O2 were allowed to react in 100 mM PB for 1 h at 37 °C. M, molecular-mass markers (masses are indicated in kDa). Right-hand panel: anti-DMPO Western blot analysis of a reaction mixture containing bSOD1 and HSA and incubated with 0.1 mM H2O2 and 100 mM DMPO in the same buffer as in the left-hand panel with or without DTPA. (B) Copper released and measured as [Cu+(BCDS)2]3− from bSOD1 when 15 μM bSOD1 was treated with various concentrations of H2O2 in 100 mM PB. Results are representative of at least three independent experiments, each performed in triplicate.

To study the effect of phosphate, (bi)carbonate and DTPA on oxidation of substrates too bulky to access the SOD1 active site, we examined the oxidation of guaiacol to tetraguaiacol (Figure 4A). We did not observe guaiacol oxidation when SOD1 was allowed to react with H2O2 in argon-purged (to eliminate CO2) PB, with or without DTPA. The strongest oxidation of guaiacol was observed when the reaction was carried out in 100 mM BB; this oxidation was inhibited by almost 30% with 0.1 mM DTPA and by over 50% with 100 mM PB. Phosphate may compete with (bi)carbonate for copper binding and also for access to the positively charged channel to the SOD1 active site. We obtained similar results when bSOD1 was replaced with hSOD1 (results not shown). Other peroxidase substrates such as ABTS [2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)] and NADPH were used with similar results (results not shown). We also observed that H2O2-induced loss of copper from the SOD1 active site was enhanced by (bi)carbonate and inhibited by phosphate (Figure 4B). Presumably, the oxidation of histidine residues at the SOD1 active site by CO3•− is responsible for this copper release, which occurs without SOD1 active-site fragmentation.

Figure 4 Effect of (bi)carbonate on SOD1-driven copper- and CO3•−-mediated oxidations

(A) Guaiacol (0.5 mM) oxidation when 15 μM SOD1 was allowed to react with 1 mM H2O2 in 100 mM PB, 100 mM BB or 100 mM PB containing 100 mM BB (BB/PB), with or without 0.1 mM DTPA. Tetraguaiacol production was determined after 15 min of incubation at 37 °C. (B) Copper released from the bSOD1 active site when 15 μM bSOD1 was added to (bi)carbonate diluted in either distilled water (BB) or 100 mM phosphate buffer (BB/PB), with 1 mM BCDS. The reaction was started with 0.1 mM H2O2, and the formation of [Cu+(BCDS)2]3− was determined after 1 h as described in the Experimental section. (C) Anti-DMPO Western blots produced when 15 μM bSOD1, 7.5 μM HSA, 100 mM DMPO and 0.1 mM H2O2 were incubated for 1 h at 37 °C in 100 mM PB containing various amounts of BB. Solutions were without (left-hand panel) or with (right-hand panel) the copper chelator DTPA. M, molecular-mass markers (masses are indicated in kDa); (f), (m) and (d) indicate fragments, monomer and dimer respectively. Results either are from a representative experiment or are means±S.E.M. for three experiments, each in triplicate, as appropriate.

With or without DTPA, (bi)carbonate was observed to enhance the production of H2O2-induced SOD1 and HSA nitrone adducts (Figure 4C). However, levels of protein radicals were higher in the absence of DTPA than in its presence. Products of copper-mediated site-specific modification of SOD1 and HSA were observed when the reaction was carried out without DTPA, but they disappeared when DTPA was included (compare smears in Figure 4C left- and right-hand panels).

Radical chemistry induced by SOD1-driven CO3•− oxidations

Thus (bi)carbonate enhances H2O2-induced loss of copper from the enzyme active site, presumably by oxidizing active-site histidine residues, and this copper then re-binds to other SOD1 sites or to target proteins such as HSA. The re-bound copper acts as additional sites for peroxymonocarbonate reduction to CO3•− [31] (see also Figure 5), thus preventing site-specific fragmentation. Accordingly, in the following experiments, we used a physiological concentration of (bi)carbonate (i.e. ∼25 mM) in 100 mM PB with 0.1 mM DTPA in order to specifically study CO3•−-triggered free radicals in SOD1 itself and HSA (Figure 6A). Under these conditions, SOD1 and HSA radicals were observed as their monomer and dimer and required the addition of all components (Figure 6A, right-hand panel). This result confirms the proposal of Bonini at al. [4] that the CO3•− formed at the active site of SOD1 can diffuse out the channel to react with BSA, forming diverse radicals. CO3•−-triggered protein radicals increased with incubation time (results not shown). The oxidation profile of HSA (Figure 6A) was independent of whether bSOD1 or hSOD1 was used, establishing that the tryptophan radical formed from hSOD1 has little role in HSA oxidation (see Figure 7). In addition, SOD1-driven CO3•−-triggered oxidation of HSA, but not of SOD1 itself, was partially prevented by adding guaiacol to the reaction mixture (Figure 6B), with concomitant oxidation to tetraguaiacol (results not shown). These results suggest that, by scavenging CO3•−, guaiacol protected HSA against SOD1-driven oxidation.

Figure 5 Mechanisms of H2O2-induced, SOD1-driven, copper- and CO3•−-initiated protein radicals

The reaction between H2O2 and SOD1 involves the formation of a strongly bound oxidant (indicated as Cu2+/OH) at the enzyme active site (see Reactions 1 and 2). H2O2-induced SOD1-driven oxidations are as follows. (i) Copper-initiated: this oxidizing species oxidizes key histidine residues at the enzyme active site and releases Cu2+. Released Cu2+ is reduced to Cu+ by excess H2O2. Cu+ re-binds to proteins and redox cycles with H2O2, producing site-specific fragmentation (indicated by the symbol ∼). DTPA chelates Cu2+ and prevents its redox cycling and binding to proteins, thus preventing copper-catalysed site-specific fragmentation of proteins. (ii) CO3•−-initiated: H2O2 reacts with CO2 to produce another oxidizing species, the peroxymonocarbonate anion (HOOCO2). Peroxymonocarbonate anion is an adduct between the deprotonated form of H2O2, OOH, and CO2 [37,38]. Peroxymonocarbonate can be reduced to CO3•− by metal centres [12,19,31,37,43]. Peroxymonocarbonate is a small anionic species that we propose can diffuse through the anionic channel to the enzyme active site and be reduced to CO3•− by the product of Reaction 1 (Cu+-SOD1). As a result, Reaction 2 does not occur and the bound oxidant formation is prevented. CO3•− oxidizes amino acid side chains (marked with bold white dots), but does not appear to fragment proteins [31]. Although CO3•− is highly reactive, it is, of course, more diffusible than the copper-bound oxidant. CO3•− oxidizes peroxidase substrates such as guaiacol or proteins such as HSA. Thus SOD1 drives CO3•−-mediated oxidation that promotes its own and other proteins' oxidation. CA enhances CO3•−-mediated protein-centred radicals, including that of CA, that can be trapped by the nitrone spin trap DMPO. DMPO traps radical sites produced by copper- and CO3•−-mediated oxidations (marked with *). Moreover, CO3•− oxidizes key residues for the activity of the enzyme and promotes an enhanced release of copper presumably by oxidizing the Cu2+-co-ordinating histidine residues.

Figure 6 SOD1-driven CO3•−-mediated oxidation of proteins

(A) Coomassie Blue-stained gel (left-hand panel) and Western blots (right-hand panel) produced when reaction mixtures containing the components indicated in each lane and their final concentrations were the same as in Figure 4(C), but reactions were performed in 25 mM (bi)carbonate added to 100 mM PB containing 0.1 mM DTPA. Reactions were stopped after 1 h with 10 IU of catalase. (B) Western blot produced from reactions containing the same components as in (A) (last lane), but various concentrations of guaiacol. The reaction mixture was incubated for 1 h and stopped by adding 10 IU of catalase. (C) Western blot produced from reaction mixtures containing the same components as in (A) (last lane) at various concentrations of CA. Incubations were stopped after 1 h with 10 IU of catalase. The analyses of the reaction mixtures by Coomassie Blue staining and Western blotting for the detection of protein nitrone adducts were carried out as described in the Experimental section. Blots are representative of three independent experiments, each performed in duplicate. M, molecular-mass markers (masses are indicated in kDa); (m), (d) and (t) indicate monomer, dimer and trimer respectively.

Figure 7 hSOD1 (isolated from erythrocytes)-driven and bicarbonate-dependent protein radical formation

(A) Left-hand panel: a protein staining of the reaction mixture containing 10 μM hSOD1, 100 mM DMPO and 100 μM H2O2 in 100 mM Chelex®-bound PB containing 100 μM DTPA, and with or without 10 or 25 mM BB. After incubation for 1 h at 37 °C, the reaction was stopped by adding 10 IU of catalase. The final pH of the reaction mixture was between 7.3 and 7.6. Middle panel: as in the left-hand panel, but the nitrone adducts where detected by Western blot with the anti-DMPO antibody. Right-hand panel: Western blot of the reaction mixtures containing the same reagents and concentrations as in the left-hand panel, but 7.5 μM HSA was included in the reaction mixture and the reactions were performed in PB or in PB containing 25 mM BB (25 BB/PB), and various concentrations of H2O2 were added. Mr, molecular-mass markers (Sea Blue®, Invitrogen) (masses are indicated in kDa). (B) Western blot with the DMPO antiserum of the reaction mixtures containing similar concentrations of reagents and performed as in (A), except that the effect of various concentrations of BB was evaluated. Blots are representative of three independent experiments. (m) and (d) indicate monomer and dimer respectively

Gaining further insights into the species oxidized by the (bi)carbonate-dependent species formed at the active site of SOD1, we observed that CA (carbonic anhydrase/dehydratase) enhanced H2O2-induced SOD1 and HSA nitrone adducts and was also a target for its own catalysed reactions, producing CA nitrone adducts (Figure 6C). Figure 7(A) shows a protein staining and a Western blot of hSOD1, isolated from human erythrocytes and allowed to react with H2O2 and DMPO in the absence of copper chemistry. Figure 7(A), middle panel, also shows the (bi)carbonate-dependent formation of a protein radical in the enzyme. The right-hand panel in Figure 7(A) shows that the formation of hSOD1 and HSA-centred radicals are dependent on (bi)carbonate and H2O2. Control experiments and dose-dependent effect of (bi)carbonate on hSOD1-driven protein radical production are shown in Figure 7(B). Human SOD1 isolated from transformed E. coli produced results similar to those of the human and bovine erythrocytic enzymes (not shown). Once again, our results show the importance of a (bi)carbonate-dependent and diffusible species capable of generating DMPO-trappable radical sites in SOD1 itself and in any other protein in the reaction microenvironment when SOD1 reacts with H2O2 in the presence of DTPA.

Protein radical formed by the hSOD1-driven CO3•−-mediated oxidations in mBH

We have devised experimental conditions to study protein oxidation driven by the bSOD1 or hSOD1/H2O2 systems that are important to understanding the mechanism of possible protein modifications in vivo. Here we used the SOD1/H2O2/DTPA/(bi)carbonate system to generate CO3•− without copper-derived OH-like-induced oxidations. As a target protein mixture for CO3•−, we used mBH (Figure 8). We partially characterized the corresponding Coomassie Blue-stained gel bands using LC–ESI–MS/MS analyses. The Coomassie Blue-stained protein bands (corresponding to the anti-DMPO bands observed by Western blotting) were excised from the gel, digested and then subjected to LC–MS/MS analyses for protein identification.

Figure 8 hSOD1-driven CO3•−-triggered protein oxidations in mBH

Coomassie Blue stain and Western blot showing the pattern of protein oxidations induced by the hSOD1/H2O2/DTPA (20 μM)/(bi)carbonate system in mBH. Left-hand panel: Coomassie Blue stain of a mixture of 20 μM hSOD1 with 500 μg/ml mBH in 100 mM PB. The numbers on the left indicate LC/MS/MS assignments (see Table 2) based on positive bands detected by the anti-DMPO Western blot. The middle panel shows an anti-DMPO Western blot of a reaction mixture containing 2 μM hSOD1, 500 μg/ml mBH and 50 μM DTPA, in 25 mM BB, 100 mM PB or PB equilibrated with CO2 (gas). Right-hand panel: Western blot of a reaction mixture between 10 μM bSOD1, 500 μg/ml mBH and 50 μM DTPA in argon-purged (to eliminate dissolved CO2) PB or in 100 mM BB. For Western blot analysis, 50 mM DMPO was added. In all cases, the reaction was performed at pH 7.4, started by adding 100 μM H2O2 and stopped by extensive dialysis against 50 mM ammonium bicarbonate (pH 8.0) to eliminate excess reagents for MS analysis [30]. M, Sea Blue® Plus2 pre-stained molecular-mass marker; M*, MagicMark™ Western Standard. The stains shown are representative of three separate experiments.

Following LC–MS/MS analyses, the raw data were extracted and searched against the NCBI non-redundant database (species: mouse only) (Table 2). Only those proteins with a summed MS/MS score greater than 30 and at least two distinct tryptic peptides are listed. The immuno-spin trapping analysis detected six distinct protein bands. As shown in Table 2, many proteins were identified in each band of the Western blot. One or more of the identified proteins in each band may be a target of CO3•−-triggered modification, leading to formation of protein radicals that are trapped by DMPO. These protein radicals can be assigned to mitochondrial proteins, heat-shock proteins and cell cytoskeleton proteins (Table 2). The above results exhibit the utility of the combined approaches of immuno-spin trapping and MS to detect potential targets for oxidation by CO3•− in a complex system such as mBH.

View this table:
Table 2 LC–MS/MS protein identifications from Coomasie Blue-stained gel bands (Figure 8) which correspond to anti-DMPO-positive bands in mBH exposed to hSOD1-driven CO3•−-initiated oxidations

Identifications are based on a unique score of at least 30 and on at least two unique peptides. The band number is from Coomassie Blue-stained gel shown in Figure 7. The number of unique tryptic peptides was observed by LC–MS/MS. Protein score was based on Spectrum Mill scoring algorithm. The accession number is the gi number from a search of the NCBI non-redundant database limited to mouse species only. The entry name listed is the first name listed within the family of proteins.

DISCUSSION

The study of the mechanism of protein radical formation by the wild-type SOD1/H2O2 system is important to the understanding of tissue damage in neuroinflammatory pathologies such as fALS. In the present study, we investigated the H2O2-induced wild-type bSOD1- and hSOD1-driven production of protein radicals in SOD1 itself and the model protein targets HSA and mBH under experimental conditions that clearly separated copper- from CO3•−-initiated radical chemistry (Figure 5).

In some studies of the SOD1 and H2O2 reaction, the role of copper- and CO3•−-triggered oxidations is difficult to distinguish (Figure 5). In the absence of copper chelators, Bonini et al. [4] were the first to detect BSA radicals formed by CO3•−-triggered radical chemistry following the H2O2-induced peroxidative activity of SOD1 in the presence of (bi)carbonate; however, SOD1 radicals were not detected [4,19]. According to our results, this system would involve the reduction of peroxymonocarbonate to CO3•− by copper-bound oxidants located at the SOD1 active site and at many other sites in both SOD1 and BSA. In the absence of DTPA, the SOD1/HSA/H2O2 reaction in BB produced greater oxidative damage, mainly to side-chain residues (Table 1), than that in PB, as demonstrated by increases in DMPO-trappable SOD1 radicals and SOD1 inactivation (Figures 1A and 1B). In the absence of DTPA, reduction of peroxymonocarbonate and formation of CO3•− will occur at many sites outside the enzyme active site where copper is re-bound. In agreement with these observations, we have found previously that (bi)carbonate protected against site-specific fragmentation, but enhanced side-chain radical formation in HSA [31].

DTPA prevents the re-binding of copper to SOD1 or HSA, but, as previously reported, does not prevent oxidative damage to SOD1 at the enzyme active site [12]. In addition, the oxidants formed at the SOD1 active site (Reactions 1 and 2) are generated in systems whether or not they contain DTPA, owing to the fact that this chelating agent does not have access to the SOD1 active site [8,13]. This can explain the failure of HSA, guaiacol or ABTS, even at high concentrations, to protect against SOD1's active-site fragmentation (∼10 and ∼5 kDa fragments) and to completely block SOD1 inactivation.

The copper-bound oxidant (an OH-like species) at the SOD1 active site may well inactivate SOD1 at a different site from that inactivated by CO3•−. CO3•− is the species responsible for the oxidation of peroxidase substrates [11,13,21] and for oxidation of a key tryptophan residue and formation of a Trp–Trp cross-linkage in human SOD1 [36]. (Bi)carbonate was required for DMPO-trappable protein radical formation and caused protein aggregation of SOD1, possibly due to the formation of side-chain radicals (Table 1). In any case, in our results, we have found very little difference between hSOD1 (recombinant or erythrocytic) and bSOD1, although bSOD1 does not contain any tryptophan (compare Figures 1B and 7A).

Previously, we have shown that CA enhances the (bi)carbonate-enhanced, H2O2-induced, copper-catalysed oxidation of a peroxidase substrate, suggesting the involvement of CO2 in the formation of CO3•− [31]. Indeed, in the present study (Figure 6C), we have shown that when CA is included in the SOD1/H2O2/HSA/DTPA system with physiological concentrations of (bi)carbonate, it produces a dramatic concentration-dependent increase in (bi)carbonate-enhanced, H2O2-induced, SOD1-driven protein radicals. Our results indicate that, under our experimental conditions, peroxidase substrates, such as guaiacol, ABTS, NADPH, and proteins protect SOD1 against inactivation by 0.1 mM H2O2 in 100 mM (bi)carbonate with 0.1 mM DTPA because they scavenge CO3•−. Our results also suggest that the diffusible CO3•− can oxidize critical residues for SOD1 activity or for the transport of the superoxide radical anion through the enzyme cationic channel. In fact, at non-physiological concentrations above 25 mM, (bi)carbonate increases the rate of inactivation of SOD1 (Figure 1A) [17]. The peroxidase substrate guaiacol competes with HSA for CO3•−, thus guaiacol acts as an alternative target to HSA with a consequent decrease in HSA-centred radical formation (Figure 6B), but does not completely prevent SOD1 inactivation (results not shown). This could be related to the proximity of SOD1 to CO3•− as it is formed [15].

In order to explain the pathways of CO3•−-initiated radical chemistry and the apparent discrepancy between protein radicals and dismutase activity, we propose a novel mechanism involving CO2 based on the enhancing effect of CA on protein radical formation (Figure 6C). Indeed, peroxymonocarbonate, which has been proposed to be an important physiological oxidant [4], oxidizes biological targets by a two-electron mechanism, and its oxidizing power is enhanced when reduced to CO3•− by catalysis with metal centres [4,31,37] (Figure 5). For the formation of CO3•−, we propose the formation of a free peroxymonocarbonate anion intermediate (HOOCO2, see Figure 5); CO2 and hydrogen peroxide (OOH/H2O2) are in equilibrium with HOOCO2 with a Keq=0.33 M−1 (Reaction 3) [19,3840]. The addition of the deprotonized water (OH) to CO2 is catalysed by CA. Analogously, we make the novel proposal that addition of deprotonized hydrogen peroxide (OOH) to CO2 is likewise catalysed by CA: Embedded Image (Reaction 3)

Accordingly, the equilibrium shown in Reaction 3 probably proceeds via the intermediacy of CO2, where HOOCO2 is essentially a CO2 adduct of the OOH [38]. The reduction of HOOCO2 by H2O2-induced Cu+-SOD1 (Reaction 1) forms CO3•− directly, without any need for the formation of the high-energy bound oxidant at the enzyme's active site (Reaction 2). It has been suggested recently that the equilibrium constant for Reaction 3 is increased in the presence of biological targets such as proteins, lipids and CA mimetics, which may alter the equilibrium of gaseous CO2/dissolved CO2/HCO3 [19]. Our proposal of a direct role for CA in peroxymonocarbonate anion formation (Reaction 3) is supported by the fact that the established mechanism of CA for the formation of (bi)carbonate (Reaction 4) [41] is very similar for peroxymonocarbonate [38], except that the base of hydrogen peroxide (OOH) nucleophilically attacks CO2 instead of the base of water (OH) where the Zn2+ of CA catalyses the deprotonation of HOOH and H2O respectively: Embedded Image (Reaction 4)

The ease of diffusion of peroxymonocarbonate anion through the anion channel of SOD1 and the substantially lower oxidation potential of CO3•− (E° ∼1.78 V) relative to that of the strong oxidant at the active site (E° ∼2.31 V) should be reflected in a much more kinetically favoured formation of protein radicals and would explain the protection afforded by (bi)carbonate against the fragmentation at the SOD1 active site induced by H2O2, which probably is initiated by the abstraction of the hydrogen of the amide bond [42] linking two amino acids near the active-site copper of SOD1. In addition, CO3•− diffuses and produces additional DMPO-trappable radical sites in the side chains of SOD1 itself and HSA (Table 1), but it does not produce protein fragmentation [31]. Our results suggest that CO3•− can produce protein radicals or oxidize proteins at side chains [43], but it cannot induce backbone cleavages and further protein fragmentation as the copper-bound oxidant does [12,31]. The enhanced inhibition of the enzyme by (bi)carbonate may be the consequence of the oxidation of residues by the diffusible CO3•−, which may be important for driving superoxide radical anion through the SOD1 anion channel to its active site.

Our model of hSOD1-driven CO3•−-initiated protein oxidation is supported by our finding that the SOD1/H2O2/DTPA/(bi)carbonate system, in which Cu+ reduces peroxymonocarbonate to form CO3•−, oxidizes a set of well-distinguished bands of proteins in mBH (Figure 8). Immuno-spin trapping in combination with MS technology [30,44] has allowed the identification of potential targets of CO3•−-mediated oxidations in mBH. The identification of the protein radicals and their subcellular location using murine models of fALS [45] may be important to our understanding of the molecular mechanisms of fALS.

We have established experimental conditions that separate copper- from CO3•−-initiated protein oxidations in the SOD1/H2O2 system, which may help us to develop an experimental model to characterize biological targets in fALS. The identification of these proteins may be a starting point for investigations aimed at identifying critical proteins in neurons overexpressing the hSOD1 mutant proteins that are targets for oxidation and thus may have a critical role in this neuroinflammatory disease.

FUNDING

This research was supported by the Intramural Research Program of the National Institute of Environmental Health Sciences of the National Institutes of Health. D.C.R. acknowledges support from the National Institute of Environmental Health Sciences [grant number R00ES015415] and the start-up funds from the Presbyterian Health Foundation.

Acknowledgments

We thank Ms Mary Mason and Dr Ann Motten for helping in the pre-submission editing of this manuscript.

Abbreviations: ABTS, 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid); BB, sodium (bi)carbonate buffer (pH 7.4); BCA, bicinchoninic acid; BCDS, bathocuproine disulfonic acid; CA, carbonic anhydrase; DMPO, 5,5-dimethylpyrroline-N-oxide; DTPA, diethylenetriaminepenta-acetic acid; ESI, electrospray ionization; fALS, familial amyotrophic lateral sclerosis; HSA, human serum albumin; LC, liquid chromatography; mBH, mouse brain homogenate; MS/MS, tandem MS; PB, sodium phosphate buffer (pH 7.4); SOD1, Cu,Zn-superoxide dismutase; bSOD1, bovine SOD1; hSOD1, human SOD1

References

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