Cysteine Oxidation of Tau and Microtubule-associated Protein-2 by Peroxynitrite
MODULATION OF MICROTUBULE ASSEMBLY KINETICS BY THE THIOREDOXIN REDUCTASE SYSTEM*
- ↵‡ To whom correspondence should be addressed: Dept. of Chemistry, The College of William and Mary, P.O. Box 8795, Williamsburg, VA 23187-8795. Tel.: 757-221-2554; Fax: 757-221-2175; E-mail: lmland{at}wm.edu.
Abstract
Alterations in the redox status of proteins have been implicated in the pathology of several neurodegenerative conditions including Alzheimer and Parkinson diseases. We report that peroxynitrite- and hydrogen peroxide-induced disulfides in the neuron-specific microtubule-associated proteins tau and microtubule-associated protein-2 are substrates for the ubiquitous thioredoxin reductase system composed of thioredoxin reductase, human or Escherichia coli thioredoxin, and NADPH. Tau and microtubule-associated protein-2 cysteine oxidation and reduction were quantitated by monitoring the incorporation of 5-iodoacetamidofluorescein, a thiol-specific labeling reagent. Cysteine oxidation of tau and microtubule-associated protein-2 to disulfides altered the ability of the proteins to promote the assembly of microtubules from purified porcine tubulin. Treatment of tau and microtubule-associated protein-2 with either the thioredoxin reductase system or small molecule reductants fully restores the ability of the MAPs to promote microtubule assembly. Thus changes in the redox state of microtubule-associated proteins may regulate microtubule polymerization in vivo.
Strong evidence implicates oxidative damage to proteins as well as cytoskeletal abnormalities in the pathogenesis of several neurodegenerative diseases including Alzheimer disease, Parkinson disease, and amyotrophic lateral sclerosis (1, 2). Microtubules formed by reversible polymerization and depolymerization of tubulin, a heterodimer composed of similar 50 kDa α and β subunits, are key components of the neuronal cytoskeleton and are required for proper neuron function (3, 4). In addition to tubulin, the neuron-specific microtubule-associated proteins (MAPs),1 MAP2 and tau, play a vital role in promoting and maintaining the neuronal cytoskeleton (5).
Given the abundance of tubulin, tau, and MAP2 and the critical function they play in neurons, we are interested in studying the interaction they have with reactive oxygen species including peroxynitrite anion (ONOO-) and hydrogen peroxide (H2O2). ONOO-, formed from the reaction of nitric oxide and superoxide, is a strong oxidant that can damage several amino acids in proteins (6, 7).
Previously we showed that tubulin, with 20 free sulfhydryl groups, is readily oxidized by ONOO-, and the extent of tubulin cysteine oxidation rather than other types of ONOO--induced damage, correlates well with inhibition of microtubule polymerization (8). Addition of disulfide reducing agents restores a significant portion of the polymerization activity that is lost following ONOO- addition. Recently we reported (9, 10) that intra- and inter-subunit tubulin disulfides are substrates for two endogenous protein reductase systems including the thioredoxin reductase system (TRS) and the glutaredoxin reductase system.
The TRS, composed of thioredoxin reductase (TrxR), thioredoxin (Trx), and NADPH, reduces a diverse group of both small-molecule
and protein disulfides including 5,5′-dithiobis(nitrobenzoic acid), α-lipoic acid, insulin, and protein disulfide isomerases
(11). TrxR catalyzes the NADPH-dependent reduction of an active site disulfide in oxidized Trx, Trx-S2, to the dithiol, Trx-(SH)2. Reduced Trx then undergoes thiol/disulfide exchange with oxidized protein substrates, protein-S2 (11, 12). 
Tau is the major protein component of the paired helical filaments found in neurofibrillary tangles of Alzheimer disease brain, and tau is known to be abnormally phosphorylated and glycated in these aberrant structures (13–15). Tau isolated from Alzheimer disease brains no longer binds to microtubules; thus, microtubule stability in neurons is compromised. Sulfhydryl oxidation of tau has been suggested as a possible early step in the formation of paired helical filaments (16, 17). For example, cysteine 322 in the microtubule-binding domain of human tau has been identified as a residue capable of forming disulfide cross-links (16). In a model suggested by Schweers et al. (16), tau-tau disulfide-linked dimers form first, and then the dimers serve as a template for noncovalent paired helical filament formation.
Herein we report that the neuron-specific MAPs, MAP2 and tau, are oxidized in vitro by ONOO- and H2O2 to form disulfide-linked species. Cysteine oxidation of these MAPs alters the ability of these proteins to promote the assembly of microtubules from purified porcine tubulin. We observed that ONOO--damaged tau and MAP2 are substrates for the TRS. Treatment with either the TRS or small molecule reductants fully restores the ability of the MAPs to promote microtubule assembly.
EXPERIMENTAL PROCEDURES
Materials
Porcine brains were obtained from the Smithfield Packing Company in Smithfield, VA. Human thioredoxin and rat thioredoxin reductase were obtained from American Diagnostica, Inc. Escherichia coli thioredoxin, bovine tau protein, a mouse monoclonal antibody against bovine tau (Tau-1), and a horseradish peroxidase-conjugated goat anti-mouse secondary antibody were obtained from Sigma. X-ray film, SuperSignal West Pico chemiluminescent substrate, bicinchoninic acid protein assay reagent, and 5-iodoacetamidofluorescein (IAF) were obtained from Pierce. All other reagents were obtained from either Sigma or Fisher.
Methods
Purification of Brain Tubulin and Heat-stable MAPs—Tubulin and heat-stable MAPs were purified from porcine brains by cycles of temperature-dependent polymerization and depolymerization and phosphocellulose chromatography as described (18). Tubulin (typically 3–4 μg/μl) in PME buffer (0.1 m PIPES, pH 6.9, 1 mm MgSO4, 2 mm EGTA) was aliquoted and stored at -80 °C. Total MAPs were eluted from the phosphocellulose column in PME buffer containing 500 mm NaCl. Heatstable MAPs were obtained as described by Vallee (19), desalted, and stored at -80 °C. Tubulin and MAP concentrations were determined by the bicinchoninic acid protein assay (Pierce).
Synthesis of ONOO-—ONOO- was synthesized from acidified H2O2 and sodium nitrite as described (20). The concentration of ONOO- was determined by measuring the absorbance at 302 nm (ϵ302 = 1670 m-1 cm-1) in 0.4 m NaOH.
Labeling of Cysteines with IAF—Heat-stable MAPs (1.2 μg/μl, 20 μl) were treated with NaOH (control), ONOO- for 2 min, or H2O2 for 15–20 min at 25 °C. ONOO- stock solutions were diluted with 0.4 m NaOH immediately prior to use, and the volume of ONOO- solution added to achieve the indicated concentrations was normalized (1.5 μl) to avoid variations in pH. Likewise, H2O2 samples were also treated with an equivalent volume of NaOH. Varying concentrations of TCEP, Trx, TrxR, and NADPH in a final volume of 5–15 μl were then added to the samples and incubated at 25 °C for 15 min. IAF in N,N-dimethylformamide was added to final concentrations of 1.5 mm, and samples were incubated at 37 °C for 30 min. Proteins were resolved by SDS-PAGE on a 7.5% gel under reducing conditions, and gel images were captured using a Kodak DC290 system and a UV transilluminator. The intensity of the fluorescein-labeled protein bands was measured using Kodak 1D image analysis software.
Microtubule Polymerization Assays—Aliquots of heat-stable MAPs (1.2 μg/μl, 20 μl) in PME buffer were treated with ONOO- or NaOH (control) at 25 °C for 5 min or H2O2/NaOH for 20 min. Following treatment with the oxidants, the components of the TRS including E. coli or human Trx, TrxR, and NADPH or the disulfide reducing agent TCEP were added to the MAP samples in a total volume of 5–15 μl. All reagents added after ONOO- were dissolved in PME buffer. Following a 15-min incubation at 25 °C, the MAP samples were combined with purified porcine tubulin (80 μl, 2.0 μg/μl) on ice. GTP was added to a final concentration of 1.25 mm. The entire sample (105–115 μl) was transferred to a 96-well plate, and microtubule assembly was monitored at 340 nm for 18 min using a thermostated (37 °C) Bio-Tek ELx808-UV plate reader. Following assembly, samples were transferred to microcentrifuge tubes, and microtubules were collected by centrifugation at 16,000 × g for 30 min. Supernatant protein was removed following centrifugation, and protein concentrations were determined by the bicinchoninic acid assay. Supernatant and protein pellet samples were also analyzed by SDS-PAGE under reducing and nonreducing conditions.
Western Blots of Oxidized Tau—Bovine tau (0.5 μg/μl, 20 μl) was treated with 0.4 m NaOH (1.5 μl) or with ONOO- diluted with 0.4 m NaOH to final concentrations of 100, 250, or 500 μm. Following a 5-min incubation at 25 °C, half of each sample was removed and treated with gel loading buffer containing SDS and βME. The remaining portion of each sample was treated with gel loading buffer minus βME. Protein samples were separated by SDS-PAGE on 7.5% gels, transferred to nitrocellulose membranes, and tau proteins (1:2000 for 2 h) were detected using a mouse anti-tau antibody. Antibody complexes were visualized by chemiluminescence using horseradish peroxidase-conjugated secondary antibodies.
RESULTS AND DISCUSSION
To assess the susceptibility of a mixture of heat-stable MAPs, composed primarily of the neuron-specific proteins tau and MAP2, to cysteine oxidation, we treated the proteins with micromolar concentrations of ONOO-. Approximate concentrations of tau and MAP2 used in these experiments are 12 and 2 μm, respectively. However, the molar concentration of the cysteine target is greater because mammalian tau and MAP2 contain 1–2 and 7 cysteines, respectively (21). Also of note, a significant fraction of the ONOO- added to any buffered solution at neutral pH isomerizes to nitrate, and all ONOO- reacts within 2 min (7).
Following treatment of tau and MAP2 with ONOO-, IAF, a sulfhydryl-specific fluorescein labeling reagent, was added. Oxidized cysteines, whether composed of disulfides or higher oxidation states of sulfur, cannot be labeled; thus, as the concentration of ONOO- increases, the incorporation of fluorescein into tau and MAP2 decreases. Fig. 1A shows a dose-dependent decrease in IAF labeling of both tau and MAP2 as the concentration of ONOO- increases. Multiple tau isoforms derived from a single gene can be seen in the region marked tau in Fig. 1A (22). Likewise, the band marked MAP2 refers to two high molecular weight isoforms, MAP2A and MAP2B (21). Cysteine oxidation of both tau and MAP2 was detected with as little as 50 μm ONOO-. Band intensities for tau and MAP2 treated with increasing concentrations of ONOO- are presented in Fig. 1B.
Sulfhydryl-specific fluorescein labeling of tau and MAP2. A, heat-stable porcine MAPs (1.2 μg/μl, 20 μl) were treated with NaOH (Control), ONOO- for 5 min, or H2O2 for 20 min at room temperature followed by 1.5 mm IAF for 30 min at 37 °C. Lane 1, Control MAPs. Lanes 2–5, MAPs treated with 50, 100, 250, and 500 μm, respectively. IAF labeling was terminated by the addition of SDS gel loading buffer containing βME. B, bar graphs of tau and MAP2 band intensities are shown. C, proteins were resolved by SDS-PAGE on 7.5% gels under reducing conditions, gel images were digitized, and band intensities were measured using Kodak 1D image analysis software. Lane 1, control MAPs. Lane 2, MAPs treated with 0.5 mm ONOO-. Lane 3, MAPs treated with 1.0 mm H2O2.
In addition to ONOO-, we also assessed cysteine oxidation of tau and MAP2 by H2O2. Although roughly 1000-fold less reactive toward cysteines than ONOO-, substantial H2O2 oxidation of cysteines was observed when the MAPs were treated with 1 mm H2O2 for 20 min (23). Fig. 1C compares levels of cysteine labeling for control MAPs and for MAPs treated with 0.50 mm ONOO- and 1 mm H2O2. Labeling of MAP2 was reduced to 40% of control with 0.50 mm ONOO- but only reduced to 60% of control with 1 mm H2O2. Tau labeling was reduced to 30% of control with both ONOO- and H2O2.
To determine whether cysteine oxidation of tau and MAP2 yielded disulfides exclusively, rather than higher oxidation states of sulfur, we treated the MAPs first with ONOO-, then with TCEP, a phosphine-based disulfide reducing agent, and finally with IAF. Because TCEP is not a sulfhydryl-containing compound like βME or dithiothreitol, the presence of TCEP does not affect the IAF labeling step. Fig. 2A shows that treatment of ONOO--damaged MAPs with TCEP fully restores IAF labeling to control levels; tau and MAP2 band intensities in lane 1 (control MAPs) are equal to those in lane 3 (ONOO-/TCEP treatment). This observation is consistent with disulfide bonds as the only type of cysteine oxidation of tau and MAP2 induced by ONOO-.
Restoration of sulfhydryl-specific fluorescein labeling of tau and MAP2. A, heat-stable MAPs (1.2 μg/μl, 20 μl) were treated with NaOH (Control) or ONOO- for 5 min at 25 °C. After a 5–15 min treatment with either TCEP or varying components of the TRS at 25 °C, IAF was added (1.5 mm), and samples were incubated for an additional 30 min at 37 °C. Lane 1, control MAPs. Lane 2, MAPs treated with 0.5 mm ONOO-. Lane 3, oxidized tubulin treated with 1.5 mm TCEP. B, MAPs were resolved by SDS-PAGE on a 7.5% gel under reducing conditions, gel images were digitized, and band intensities were measured using Kodak 1D image analysis software. Lane 1, control MAPs. Lane 2, MAPs treated with 0.5 mm ONOO-. Lane 3, oxidized tubulin treated with 6 μm human Trx (hTrx) and 750 μm NADPH. Lane 4, oxidized tubulin treated with 6 μm human Trx (hTrx), 100 nm TrxR, and 750 μm NADPH. Lane 5, oxidized tubulin treated with 6 μm E. coli Trx, (bTrx), 100 nm TrxR, and 750 μm NADPH.
The observation that disulfides were formed by ONOO- treatment of MAPs, coupled with our recent work on ONOO--damaged tubulin, led us to ask whether oxidized tau and MAP2 could be repaired by the TRS. Fig. 2B shows the results of the TRS repair assay after SDS-PAGE separation of the fluorescein-labeled MAP samples. Fluorescein labeling of both tau and MAP2 is reduced following the addition of 0.5 mm ONOO- relative to control MAPs (Fig. 2B, lanes 1 and 2). Treatment of ONOO--damaged MAPs with Trx and NADPH restored a modest number of sulfhydryl groups that were then labeled by IAF (lane 3). The addition of reduced Trx (Trx-(SH)2) to the oxidized MAPs even in the absence of TrxR has the potential to show some repair because reduced Trx undergoes thiol-disulfide exchange with oxidized tau and MAP2 according to Reaction 2 (11).
Treatment of ONOO--damaged MAPs with the complete TRS (Fig. 2B, lanes 4 and 5) restored fluorescein labeling of tau and MAP2 to 92 and 94%, respectively, of control, whereas TCEP treatment restored labeling of both to 94–97% of control. Equimolar concentrations of human and E. coli Trx were used in the repair assays with mammalian TrxR, and identical results were obtained.
Once we had established that tau and MAP2 were sensitive targets for ONOO- damage and that oxidized tau and MAP2 were substrates for the TRS, we sought to assess the effect of MAP cysteine oxidation on microtubule polymerization. First, we treated heat-stable MAPs with ONOO- or H2O2 in a separate reaction, combined the oxidized MAPs with purified porcine tubulin, and monitored both the rate and extent of microtubule polymerization. Fig. 3 shows overlays of several microtubule polymerization curves in which MAPs were pretreated with oxidants and then combined with tubulin.
Effect of MAP oxidation and reduction on microtubule polymerization. MAPs (1.2 μg/μl, 20 μl) were treated with NaOH and TCEP (Control, open triangles), 0.5 mm ONOO- (open squares), 0.5 mm ONOO-, 1.5 mm TCEP (circles), 1.0 mm H2O2 (closed triangles), or 1.0 mm H2O2, 1.5 mm TCEP (+) as described under “Experimental Procedures.” Following oxidation and repair, MAPs were combined with purified porcine brain tubulin (80 μl, 2.0 μg/μl) and GTP (1.25 mm). Microtubule polymerization was monitored at 340 nm in a 96-well plate thermostated to 37 °C. These results are representative of at least three independent experiments.
A comparison of control MAPs with ONOO--treated MAPs shows a very distinct difference in the kinetics of microtubule polymerization. An extended lag phase is observed for ONOO--treated MAPs, and the maximal rate of polymerization is also reduced by ONOO- treatment. Notably, H2O2 treatment of MAPs induced the same lag and reduced polymerization rate as ONOO- treatment. Because ONOO- decomposes completely within 2 min at neutral pH, and oxidant-treated MAPs were prepared in a reaction separate from tubulin, the observed lag phase cannot be attributed to direct ONOO- oxidation of tubulin (7). Likewise, we performed control experiments to assess any effect of millimolar H2O2 or ONOO- degradation products on the ability of purified tubulin to polymerize, and none was observed (data not shown) (8).
Another key observation to be gleaned from Fig. 3 is that the extended lag and reduced rate caused by both oxidants is fully reversed by the action of TCEP. Thus, it is clear that cysteine oxidation of tau and MAP2 to disulfide species is responsible for the changes in microtubule polymerization kinetics. Likewise, we treated ONOO--damaged MAPs with the TRS in the same manner as in Fig. 2B and then combined the repaired MAPs with tubulin. The TRS, like TCEP, reversed the effects of ONOO- and H2O2 on the kinetics of microtubule polymerization (data not shown).
These findings are noteworthy for several reasons. Clearly, cysteine oxidation of tau and MAP2 affects the ability of the proteins to promote the assembly of microtubules. In fact, the assembly curves shown for ONOO-- and H2O2-damaged MAPs are nearly identical to tubulin polymerization curves obtained in the absence of MAPs (data not shown). The observation that both ONOO- and H2O2 induce the same change in assembly kinetics, coupled with the fact that the effect is reversed by both TCEP and TRS, points to cysteine oxidation as the culprit. This is particularly important because ONOO- can damage several amino acids in proteins, whereas H2O2 can oxidize only cysteines and perhaps methionine (24). Tyrosine nitration of tau has been reported in the brains from Alzheimer patients; however, our work with H2O2 shows that tyrosine nitration of MAPs cannot be responsible for the change in polymerization kinetics that we observe (25).
Although the lag phase for assembly is clearly affected, the extent of microtubule polymerization changes only slightly for samples containing oxidant-treated MAPs. Monitoring polymerization for longer times (up to 25 min) shows that the oxidant-treated MAP samples plateau at nearly the same absorbance value. Following polymerization assays, microtubules were collected by centrifugation, and the concentration of protein in the supernatant (unassembled protein) was measured. Supernatant protein concentrations obtained for the oxidant-treated MAP samples were consistently 10–15% higher than for control MAPs suggesting that less tubulin polymerized. This is in good agreement with the polymerization curves shown in Fig. 3 and with those where assembly was monitored for longer time periods.
Also of note, the absolute absorbance changes shown in Fig. 3 are lower than those typically seen for microtubule assembly with the concentration of tubulin used. Absorbance changes at 360 nm in a cuvette were also monitored, and the changes in absorbance were consistently 2-fold higher. The lower absorbance changes are attributed to the assay format using the microtiter plate, a smaller reaction volume, and detection at 340 nm (8).
The difference in supernatant protein concentration could also be partially attributed to greater amounts of tau and MAP2 present in the supernatant. MAP2 binding to microtubules was assessed by separation of the microtubule pellet from the supernatant after polymerization and analysis of the supernatants and pellets for MAP2 using a monoclonal antibody against porcine MAP2. The binding of MAP2 and tau, two abundant and neuron-specific MAPs, to microtubules is dependent on charge interactions between the very acidic C termini of α- and β-tubulin and the positively charged microtubule-binding domains of MAP2 and tau (26).
Western blot analysis of porcine MAP2 in supernatants and pellets consistently showed a modest increase (10–20% greater than control) in MAP2 in the supernatant fraction for oxidant-treated MAP samples. Treatment of oxidized MAPs with TCEP or the TRS reduced the amount of MAP2 in the supernatants to control levels (data not shown). Similar experiments could not be performed to analyze tau in supernatants and pellets because commercially available antibodies against tau do not recognize the porcine protein. Protein analyses were also complicated because tubulin (present in both fractions) and tau have nearly identical molecular weights.
Some experiments were performed using purified bovine tau in conjunction with porcine tubulin. Western blot analysis of supernatants and pellets obtained following assembly detected a modest increase in tau in the supernatant fraction, a result that was consistent with the results obtained for MAP2.
To rule out nonspecific aggregation and precipitation, control and ONOO--damaged MAPs were treated under conditions identical to those for the polymerization assays, except for the absence of tubulin. Although there was a slight (∼10%) increase in pelletable MAPs for the ONOO--treated sample relative to control, that is not sufficient to explain the levels of MAPs detected in the microtubule pellets following assembly. Thus, it appears that disulfide-linked tau and MAP2 are capable of binding to microtubules, but the ability of the proteins to promote assembly is altered. Mouse tau contains only one cysteine residue, Cys-222, which is located in the microtubule-binding domain (21). Because mammalian tau proteins contain only one (mouse) or two cysteines (human and bovine), it is likely that tau forms disulfides between tau protein chains rather than within a single protein chain (16). For a tau oligomer to form involving more than 2 tau proteins, it is expected that at least 2 cysteines would have to be present per tau protein; otherwise only dimers could form. No sequence data are available for porcine tau.
To assess the type of structure formed following ONOO- addition, we treated purified bovine tau with increasing concentrations of ONOO- and analyzed the reaction products by SDS-PAGE under both reducing and nonreducing conditions. As shown in Fig. 4, analysis of tau by Western blot showed a dose-dependent decrease in the amount of tau detected at 50–60 kDa under nonreducing conditions (Fig. 4, lanes 5–8). No dimers at ∼100 kDa or tetramers (200 kDa) were detected even at the lowest concentrations of ONOO- tested. Analysis under reducing conditions (lanes 1–4) shows identical amounts of tau detected at 50–60 kDa; thus tau oligomer formation is fully reversible in the presence of the disulfide reductant, βME.
Western blot of ONOO--treated tau proteins. Bovine tau (0.5 μg/μl, 20 μl) was treated with increasing concentrations of ONOO- and subjected to SDS-PAGE under reducing and nonreducing conditions on 7.5% polyacrylamide gels. Proteins were transferred to nitrocellulose and incubated for 2 h with an anti-α-tubulin antibody (1:1000), and tau-antibody complexes were visualized by chemiluminescence. Lanes 1 and 5, control tau. Lanes 2 and 6, 100 μm ONOO-. Lanes 3 and 7, 250 μm ONOO-. Lanes 4 and 8, 500 μm ONOO-.
We conclude that ONOO- induced the formation of higher molecular weight tau oligomers that did not enter the separating gel. In support of this, tau immunoreactivity was detected in the sample wells of the SDS-PAGE gels for those samples treated with ONOO-. These observations suggest the formation of a tau oligomer that is capable of binding to microtubules rather than an insoluble aggregate. The only other report of oxidized tau species binding to microtubules concerned the binding of tau-tau dimers to microtubules; however, in that case, engineered portions of the tau protein were used instead of native tau (27).
The results presented herein show that oxidation of native mammalian tau and MAP2 proteins to form disulfide-linked species correlates with an extended lag phase observed during microtubule polymerization experiments. This work is the first to report a link between tau and MAP2 and the TRS, a ubiquitous reductase system. In neurons, tubulin comprises 10–15% of the total protein and the neuron-specific proteins tau and MAP2 bind with a stoichiometry of ∼1 per 10 tubulin dimers (22, 28). Trx and TrxR are both ubiquitous cytosolic proteins expressed in neuronal cells (29, 30). Approximate physiologic concentrations of Trx in mammalian liver and brain are 10–12 and 2 μm, respectively, whereas TrxR concentrations are estimated to be 10–12 nm in liver and 2 nm in brain (29, 31). NADPH concentrations in liver range from 13–15 μm but are considerably higher in brain at 340–390 μm (32).
Although the concentrations of Trx, TrxR, and NADPH used herein (see Fig. 2) are higher than the physiologically relevant estimates provided, the concentration of the substrate, i.e. MAP disulfide, was also much greater than would be expected in vivo. This model study was designed to assess whether oxidative damage to MAPs induces alterations in microtubule polymerization kinetics and whether reversal of that damage by the TRS restores MAP function. Thus, these findings serve as a starting point to propose that the cysteines of tau and MAP2 may be oxidized to disulfides and then reduced by the TRS to modulate microtubule polymerization kinetics in vivo.
Footnotes
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↵1 The abbreviations used are: MAP, microtubule-associated protein; βME, β-mercaptoethanol; IAF, 5-iodoacetamidofluorescein; ONOO-, peroxynitrite anion; TCEP, (tris(2-carboxyethyl)phosphine hydrochloride); Trx, thioredoxin; TrxR, thioredoxin reductase; TRS, thioredoxin reductase system; PIPES, 1,4-piperazinediethanesulfonic acid.
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↵* This work was supported by NINDS, National Institutes of Health Grant R15-NS38885 (to L. M. L.) and Grant J-670 from the Jeffress Memorial Trust (to L. M. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Received May 17, 2004.
- Revision received May 27, 2004.
- The American Society for Biochemistry and Molecular Biology, Inc.
