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The understanding of the mechanism, oxidant(s) involved, and how/what protein radicals are produced during the reaction of wild type Cu, Zn-superoxide dismutase (SOD1) with H2O2 and their fate is incomplete, but a better understanding of the role of this reaction is needed. We used immuno-spin trapping and mass spectrometry analysis to study the protein oxidations driven by human (h) and bovine (b) SOD1 when reacting with H2O2 using human serum albumin (HSA) and mouse brain homogenate (mBH) as target models. In order to gain mechanistic information about this reaction, we considered both copper- and 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 (to suppress copper chemistry), CO3 •− radical produced distinct radical sites in both SOD1 and HSA, which caused protein aggregation without causing protein fragmentation. The CO3 •− produced by reaction of hSOD1 with H2O2 also produced distinctive DMPO nitrone adduct positive protein bands in the mBH. Finally, we propose a biochemical mechanism to explain CO3 •− production from carbon dioxide, enhanced protein radical formation, and protection by (bi)carbonate against H2O2-induced fragmentation of the SOD1 active site. Our 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 analyzing the critical targets of SOD1-driven oxidation during such pathological processes as neuroinflammation.
Much of the mechanistic biochemical evidence regarding Cu,Zn-superoxide dismutase (SOD1)1-driven pathogenic oxidative mechanisms has been obtained by studying the reaction between bovine or human SOD1 and H2O2 [1–4]. 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 . 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 familial amyotrophic lateral sclerosis (fALS).
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)1+ ↔ (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 , and SOD1 fragmentation at its active site [10, 12].
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, 13–16]. Recently, 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 •− ; however, the chemical mechanism of this reaction remains controversial [7, 17–19]. Much of our current knowledge of SOD1-driven oxidation has been gained by studying the oxidation of chemical compounds (e.g., NADPH , dichlorofluoresceine , peroxidase substrates [12, 21], ethanol, spin trap compounds [21, 22], formate and azide , etc.), the loss of superoxide dismutase activity, or the activity or the structure of protein or nucleic acid targets . Because of its diffusibility and powerful oxidative nature, CO3 •− may form distinct radicals in SOD1 itself or tissue proteins, which may be trapped with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) 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 electron spin resonance (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 [25–27] and DNA radicals [24, 28] called immuno-spin trapping [26, 27] that helps to identify protein radicals induced by SOD1-driven oxidation in cells and tissues. Immuno-spin trapping  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  by using heterogeneous immunoassays and also by mass spectrometry . 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, 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 . Here we have used immuno-spin trapping and mass spectrometry to understand the mechanism of protein radical formation induced by the bovine and human SOD1/HSA or mouse brain homogenate (mBH)/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. Further, we have analyzed 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 this 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).
Bovine Cu,Zn-superoxide dismutase (bSOD1, from bovine erythrocytes) and beef liver catalase were purchased from Roche Applied Science (Indianapolis, IN). Sodium bicarbonate (99.7–100.3%) was purchased from Alfa Aesar (Ward Hill, MA). The spin trap DMPO was purchased from Alexis Biochemicals (San Diego, CA), purified twice by vacuum sublimation at room temperature, 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 7,800 M−1 cm−1. Reagent grade 30% H2O2 was obtained from Fisher Scientific Co. (Fair Lawn, NJ). The H2O2 concentration was verified using UV-vis absorption at 240 nm (ε240nm = 43.6 M−1 cm−1). Recombinant human SOD1 (expressed in E. coli) was from BioVision (Cat#4802, Mountain View, CA). Erythrocyte human SOD1 (hSOD1, Cat#S-9636) and all other reagents were purchased from Sigma Chemical Co. (St. Louis, MO). Buffers were treated with Chelex® 100 ion-exchange resin (Bio-Rad Laboratories, Hercules, CA) 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 gas mixture of 5% CO2 and 95% N2.
Our bSOD1 and hSOD1 (from erythrocytes and recombinant) solutions were prepared and determined to be free of detectable unbound copper as previously described . SOD1 concentration is expressed as micromolar (µM), determined by measuring the absorbance at 258 nm (ε258 nm = 10.3 mM−1 cm−1) at pH 7.4 . HSA solutions were prepared as recently described [31, 33]. Our HSA preparations were free of detectable copper contamination, and the free sulfhydryl /albumin ratio was similar to that reported [31, 33]. The HSA concentration was determined by using the BCA assay (Pierce) or assuming its UV-visible extinction coefficient of ε280 nm= 35,700 M−1 cm−1. Male C576J/BL mice (25–30 g body weight) were euthanized according to institutionally approved protocols, and brains were collected in ice-cold 10 mM sodium phosphate buffer, pH 7.4, washed in the same buffer and homogenized (1g/ml). The homogenate was centrifuged at 11,700×g and the supernatant was dialyzed (3 kDa cut-off) against 10 mM sodium phosphate buffer, pH 7.4. The protein concentration in the dialyzed mouse brain homogenate (mBH) was determined using the BCA assay.
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 chelexed sodium (bi)carbonate buffer, pH 7.4 (BB), or 100 mM sodium phosphate buffer, pH 7.4 (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 stopped by adding 10 IU catalase to eliminate excess H2O2. The pH of the reaction mixtures after the reactions was completed was between 7.3–7.6.
The H2O2-induced Cu2+ release from bSOD1 or hSOD1 and its reduction to Cu1+ was monitored using the specific Cu1+ chelator BCDS . Cu1+ binds to BCDS and produces a complex (Cu1+(BCDS)2)−3 that exhibits a characteristic absorbance maximum at 480 nm, with ε480 = 12,540 M−1 cm−1
Bovine and human SOD1 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) .
A rabbit antiserum against the nitrone form of DMPO was obtained in our laboratory  and used to develop immuno-spin trapping [26, 27, 29]; this antiserum has been successfully used to detect protein [27, 29] and DNA radicals [24, 28]. The anti-DMPO serum is commercially available from Alexis Biochemicals, Cayman Chemicals, AbCam, Chemicon International, and Oxford Biomedical Research.
DMPO-protein radical-derived nitrone adducts were determined using a standard enzyme-linked immunosorbent assay (ELISA) and Western blot as previously described [12, 29]. Briefly, the reaction mixture was separated by reducing SDS-PAGE (1.2 µg proteins/ 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 blot. Briefly, immuno-complexes were detected by exposing the membrane to NBT/BCIP One Step reagent from Pierce (Rockford, IL.) for 15 min or, when indicated, by enhanced chemiluminescence (ECL) using a CDP-Star II (Roche Molecular Biochemicals) / Nitro Block II (Tropix, Bradford, MA) system. Where indicated, a MagicMark™ XP Western Protein Standard (Invitrogen) was used as a molecular weight marker that glows after Western blot development .
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 chelexed 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. An extinction coefficient of 26.6 mM−1 cm−1 was used to quantify tetraguaiacol formation.
The protein bands were manually excised from the gel, cut into small pieces, and transferred into a 96-well microtiter plate. Gel pieces were subjected to automatic tryptic digestion using an Investigator™ Progest protein digestion station (Genomic Solutions, Ann Arbor, MI). 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 hrs. The enzyme solution used was sequencing grade modified trypsin (Promega Corporation, Madison, WI) at a concentration of 0.01 mg/ml in 25 mM ammonium bicarbonate buffer (pH 7). Resulting tryptic peptides were extracted from the gel, lyophilized, and stored at −80 °C. Prior to mass spectrometric analysis, the peptides were reconstituted in 40 µl of a 97:3 solution of water:acetonitrile (0.1% formic acid).
For the nanoLC/ESI/MS/MS analyses, we used an Agilent XCT Ultra ion trap (Agilent Technologies, Inc., Santa Clara, CA) equipped with an HPLC-Chip Cube MS interface and an Agilent 1100 nanoLC system. Injections of 30 µl of the peptide digests were made onto 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 (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 prior to scan out), 100,000 or 200 milliseconds 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 10,000 counts) from each spectrum selected for MS/MS analysis.
Following the analyses, the MS/MS data were extracted and analyzed using Spectrum Mill MS Proteomics software (Agilent Technologies, Inc). To generate peak lists, the raw data files were processed using the Data Extractor function with the following parameters: deconvoluted ions of 300–6,000 Da and a retention time of 10 to 60 min. MS scans with the same precursor m/z were merged based on a +/− 1.4 m/z window and a +/− 15 sec retention time window. Using the extracted data, searches were performed against the NCBI nonredundant 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 2 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 manually validated.
We observed H2O2-induced bovine and human erythrocyte SOD1 inactivation in (bi)carbonate buffer (Fig. 1A). In agreement with our previous report  and 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 phosphate buffer with DTPA.
When (bi)carbonate was added to a bSOD1/H2O2 or hSOD1/H2O2 system in fresh argon-bubbled (to purge carbon dioxide) phosphate buffer (PB) containing the copper chelator DTPA, the amount of H2O2-induced bSOD1-centered radicals observed as monomers and dimers increased, as seen by Western blot (Fig. 1B). The hSOD1 enzyme produced similar results (data not shown). Although bSOD1-centered 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 chelexed phosphate buffer, pH 7.4, containing 0.1 mM DTPA in a closed system, but we could detect them by Western blot when phosphate buffer was equilibrated with air at room temperature for 15 min, presumably due to the absorption of CO2 (data not shown). In phosphate buffer with 0.1 mM DTPA, the SOD1 nitrone adduct (SOD1-self oxidized) formation was totally dependent on (bi)carbonate. It increased with incubation time (Fig. 1C) and was completely prevented by cyanide (data not shown), suggesting that (bi)carbonate and copper redox cycling at the enzyme active site is essential for producing SOD1 nitrone adducts.
In Figs. 1A–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 quickly with DTPA (rate constant ~ 1.7 × 107 M−1 s−1 ), 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 (Fig. 1D), suggesting that high concentrations of DTPA scavenge CO3 •−. Accordingly, in subsequent experiments shown in this report, we used 0.1 mM DTPA, which is over three-fold higher than the copper content of the SOD1 solutions used in our experiments.
In order to investigate the potential for the induction of SOD1-driven oxidation in surrounding proteins, we used human serum albumin (HSA) as a model target. We observed that HSA blocked H2O2-induced SOD1 self-inactivation in (bi)carbonate buffer, but in phosphate buffer it had no effect (Fig. 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 in SDS-PAGE and stained the gels with Coomassie blue, we observed that both proteins were extensively fragmented when the reaction was carried out in phosphate buffer (Fig. 2B, left 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] (Fig. 2B, left panel). However, when we performed the reaction in (bi)carbonate buffer with DTPA, the active site of SOD1 was protected against site-specific fragmentation (Fig. 2B, right panel, ), but extensive fragmentation at other sites of both SOD1 and HSA still occur 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 erythrocyte bSOD1 was replaced by hSOD1 (data not shown).
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 (Fig. 2C, left panel). At the same H2O2 concentration, the number of radical sites was higher when the reaction was carried out at pH 7.4 in 100 mM (bi)carbonate buffer (BB) rather than in 100 mM phosphate buffer (PB) (Fig. 2C, central panel). But in (bi)carbonate buffer with 0.1 mM DTPA, we observed distinct protein nitrone adducts of monomers and aggregates from both proteins, but not of fragments (Fig. 2C, B). 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 Cu-binding sites other than the active site of SOD1.
We further examined SOD1-driven, copper-triggered radical chemistry in proteins by reacting SOD1 and HSA with H2O2 in phosphate buffer without DTPA (Fig. 3). Under these conditions, HSA and SOD1 itself were targets of copper-mediated, site-specific fragmentation (Fig. 3A, left panel). H2O2-induced fragmentation of SOD1 at its active site was evidenced 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 (Fig. 3A, right 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 rebound 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 Cu1+ as its red complex (Fig. 3B). Depending on the concentration of H2O2, fifteen minutes of reaction was enough to release a significant amount of copper, an amount that would be sufficient to produce protein radicals, as we previously found by immuno-spin trapping in the HSA/Cu2+/H2O2 system . After two hours, 5 mM H2O2 released approximately one-half of the available copper at the active site.
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 (Fig. 4A). We did not observe guaiacol oxidation when SOD1 was reacted with H2O2 in argon-purged (to eliminate CO2) phosphate buffer with or without DTPA. The strongest oxidation of guaiacol was observed when the reaction was carried out in 100 mM (bi)carbonate buffer; this oxidation was inhibited by almost 30% with 0.1 mM DTPA and by over 50% with 100 mM phosphate. 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 (data not shown). Other peroxidase substrates such as ABTS and NADPH were used with similar results (data 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 (Fig. 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.
With or without DTPA, (bi)carbonate was observed to enhance the production of H2O2-induced SOD1 and HSA nitrone adducts (Fig. 4C). However, 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 Fig. 4C, left vs right panel).
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 •−  (see also scheme 1), 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 sodium phosphate buffer with 0.1 mM DTPA in order to specifically study CO3 •− -triggered free radicals in SOD1 itself and HSA (Fig. 5A). Under these conditions, SOD1 and HSA radicals were observed as their monomer and dimer and required the addition of all components (Fig. 5A, right panel). This result confirms the proposal of Bonini at al. that the CO3 •− formed at the active site of SOD1 can diffuse out the channel to react with bovine serum albumin forming diverse radicals . CO3 •− -triggered protein radicals increased with incubation time (data not shown). The oxidation profile of HSA (Fig. 5A) was independent of whether bSOD1 or hSOD1 was used, establishing that the tryptophan radical formed from hSOD1 has little role in HSA oxidation (see Fig. 6). In addition, SOD1-driven, CO3 •− -triggered oxidation of HSA, but not of SOD1 itself, was partially prevented by adding guaiacol to the reaction mixture (Fig. 5B) with concomitant oxidation to tetraguaiacol (data not shown). These results suggest that by scavenging CO3 •−, guaiacol protected HSA against SOD1-driven oxidation.
Gaining further insights into the species oxidized by the (bi)carbonate-dependent species formed at the active site of SOD1, we observed that carbonic anhydrase/dehydratase (CA) enhanced H2O2-induced SOD1 and HSA nitrone adducts and also was a target for its own catalyzed reactions, producing CA nitrone adducts (Fig. 5C). Figure 6A shows a protein staining and a Western blot of hSOD1, isolated from human erythrocytes, reacted with H2O2 and DMPO in the absence of copper chemistry. Fig. 6A central panel, also shows the (bi)carbonate-dependent formation of a protein radical in the enzyme. The right panel in Fig. 6A shows that the formation of hSOD1 and HSA-centered 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 Fig. 6B. Human SOD1 isolated from transformed E. coli produced results similar to those of the human and bovine erythrocyte enzymes (data 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.
We have devised experimental conditions to study protein oxidation driven by the bovine and human SOD1/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 hydroxyl radical-like-induced oxidations. As a target protein mixture for CO3 •−, we used a mouse brain homogenate (mBH) (Fig. 7). We partially characterized the corresponding Coomassie blue stained gel bands using LC/ESI/MS/MS analyses. The Coomassie stained protein bands (corresponding to the anti-DMPO bands observed by Western blot) were excised from the gel, digested, and then subjected to LC/MS/MS analyses for protein identification.
Following LC/MS/MS analyses, the raw data were extracted and searched against the NCBI nonredundant 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 6 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 mass spectrometry to detect potential targets for oxidation by CO3 •− in a complex system such as mouse brain homogenate.
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 this report we investigated the H2O2-induced, wild type bovine and human SOD1-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 (Scheme 1).
In some studies of the SOD1 and H2O2 reaction, the role of copper- and CO3 •− -triggered oxidations is difficult to distinguish (Scheme I). In the absence of copper chelators , Bonini et. al. were the first to detect bovine serum albumin 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 bovine serum albumin. In the absence of DTPA, the SOD1/HSA/H2O2 reaction in (bi)carbonate buffer produced greater oxidative damage, mainly to side chain residues (Table 1), than that in phosphate buffer, as demonstrated by increases in DMPO-trappable SOD1 radicals and SOD1 inactivation (Figs. 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 previously found that (bi)carbonate protected against site-specific fragmentation, but enhanced side-chain radical formation in HSA .
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 . In addition, the oxidants formed at the SOD1 active site (reaction 1 and 2) are generated in systems whether or not they contain DTPA due 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 (a hydroxyl radical-like species) at the SOD1 active site may well inactivate SOD1 at a different site than CO3 •− does. 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 . (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 Trp. Compare Fig. 1B and Fig. 6A.
Previously, we have shown that carbonic anhydrase enhances the (bi)carbonate-enhanced, H2O2-induced, copper-catalyzed oxidation of a peroxidase substrate, suggesting the involvement of CO2 in the formation of CO3 •− . Indeed, in the present study (Fig. 5C) we show that when carbonic anhydrase 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 nonphysiological concentrations above 25 mM, (bi)carbonate increases the rate of inactivation of SOD1 (Fig. 1A) . The peroxidase substrate guaiacol competes with HSA for CO3 •−; thus, guaiacol acts as an alternative target to HSA with a consequent decrease in HSA-centered radical formation (Fig. 5B), but does not completely prevent SOD1 inactivation (data not shown). This could be related to the proximity of SOD1 to CO3 •− as it is formed .
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 carbonic anhydrase on protein radical formation (Fig. 5C). Indeed, peroxymonocarbonate , which has been proposed to be an important physiological oxidant , oxidizes biological targets by a two-electron mechanism, and its oxidizing power is enhanced when reduced to CO3 •− by catalysis with metal centers [4, 31, 37] (Scheme 1). For the formation of CO3 •−, we propose the formation of a free peroxymonocarbonate anion intermediate (HOOCO2 −, see Scheme I); CO2 and the hydrogen peroxide (−OOH/H2O2) are in equilibrium with HOOCO2 − with a Keq = 0.33 M−1 (reaction 3) [19, 38–40]. The addition of the deprotonized water (−OH) to carbon dioxide is catalyzed by carbonic anhydrase. Analogously, we make the novel proposal that addition of deprotonized hydrogen peroxide (−OOH) to CO2 is likewise catalyzed by carbonic anhydrase (reaction 3)
Accordingly, the equilibrium shown in reaction 3 most likely proceeds via the intermediacy of CO2, where HOOCO2 − is essentially a CO2 adduct of the −OOH . The reduction of HOOCO2 − by H2O2-induced Cu1+-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 recently been suggested that the equilibrium constant for Reaction 3 is increased in the presence of biological targets such as proteins, lipids, and carbonic anhydrase mimetics, which may alter the equilibrium of gaseous CO2/dissolved CO2/HCO3 − . Our proposal of a direct role for carbonic anhydrase in peroxymonocarbonate anion formation (reaction 3) is supported by the fact that the established mechanism of carbonic anhydrase for the formation of (bi)carbonate (reaction 4)  is very similar for peroxymonocarbonate  except the base of hydrogen peroxide (−OOH) nucleophilically attacks CO2 instead of the base of water (−OH) where the Zn2+ of carbonic anhydrase catalyzes the desprotonation of HOOH and H2O, respectively.
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 favored 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  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 . Our results suggest that CO3 •− can produce protein radicals or oxidize proteins at side chains , 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 Cu1+ reduces peroxymonocarbonate to form CO3 •−, oxidizes a set of well distinguished bands of proteins in mouse brain homogenate (Fig. 7). Immuno-spin trapping in combination with mass spectrometry technology [30, 44] has allowed the identification of potential targets of CO3 •− -mediated oxidations in mouse brain homogenate. The identification of the protein radicals and their subcellular location using murine models of fALS may be important to understanding 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.
This research was supported by the Intramural Research Program of the NIEHS/NIH. DCR acknowledges the National Institute of Environmental Health Sciences support (Award# R00ES015415) and the start-up funds from the Presbyterian Health Foundation. We thank Ms. Mary Mason and Dr. Ann Motten for helping in the editing of this manuscript.
1The abbreviations used are: ABTS, 2,2′-azino-bis-[3-ethylbenzothiazoline]-6-sulfonic acid; CA, carbonic anhydrase; CO3 • −, carbonate radical anion; DMPO, 5,5-dimethyl-1-pyrroline N-oxide; DTPA, diethylenetriamine-pentaacetic acid; ELISA, enzyme-linked immuno-sorbent assay; ESR, electron spin resonance; fALS, familial amyotrophic lateral sclerosis; HSA, human serum albumin; H2O2, hydrogen peroxide; SOD1, Cu,Zn-superoxide dismutase.