Photo-oxidation and Photoreduction of Catechols by Chlorophyll Metabolites and Methylene Blue.

While plant-derived oxidants can protect cells from oxidative damage, limited research has examined the role of dietary chlorophyll. Photoreduction of ubiquinone by chlorophyll metabolites and red light has been reported in vitro and in animal models. Herein we examined photo-oxidation and photoreduction reactions of catechols, dopamine and hydrocaffeic acid. Photo-oxidation of dopamine by methylene blue and the chlorophyll metabolites pheophorbide A, chlorin e6 and sodium copper chlorophyllin was studied by monitoring aminochrome, the cyclized product of the dopamine o-quinone with its amine. Singlet oxygen scavengers including sodium azide, ascorbate and glutathione decreased aminochrome formation by methylene blue and pheophorbide A. Addition of EDTA, a tertiary amine electron donor, to the reaction of dopamine, photosensitizer and red light decreased aminochrome formation. Photoreduction of the dopamine o-quinone produced by mushroom tyrosinase was achieved by both methylene blue and pheophorbide A only when an electron donor was included. Due to limited solubility, photo-oxidation and photoreduction reactions by pheophorbide A required 5-7.5% dimethylformamide for optimal reactivity. Catalytic photoreduction of 2,3-dimethoxy-5-methyl-p-benzoquinone by methylene blue or pheophorbide A and tertiary amine electron donors was observed. Among the chlorophyll metabolites, pheophorbide A was more effective than chlorin e6 or sodium copper chlorophyllin in photo-oxidation of dopamine and photoreduction reactions. Singlet oxygen inhibited lactate dehydrogenase A activity, and higher molecular weight protein cross-links were observed on SDS-PAGE. Hydrocaffeic acid competed with lactate dehydrogenase A for reaction with singlet oxygen produced by methylene blue; however, no protection by hydrocaffeic acid (HCA) was observed when pheophorbide A was used. Cysteine modification of lactate dehydrogenase A by the o-quinone of hydrocaffeic acid was detected using a redox cycling stain. Inclusion of an electron donor decreased protein labeling.

Evidence for thiol/disulfide exchange reactions between tubulin and glyceraldehyde-3-phosphate dehydrogenase.

While thiol redox reactions are a common mechanism to regulate protein structure and function, protein disulfide bond formation is a marker of oxidative stress that has been linked to neurodegeneration. Both tubulin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) contain multiple cysteines that have been identified as targets for oxidation to disulfides, S-nitrosation and S-glutathionylation. We show that GAPDH is one of three prominent brain microtubule-associated proteins (MAPs), in addition to MAP-2 and tau, with reactive cysteines. We detected a threefold to fourfold increase in tubulin cysteine oxidation by hydrogen peroxide in the presence of rabbit muscle GAPDH by 5-iodoacetamidofluorescein labeling and by Western blot detection of higher molecular weight inter-chain tubulin disulfides. In thiol/disulfide exchange experiments, tubulin restored ∼50% of oxidized GAPDH cysteines and the equilibrium favored reduced GAPDH. Further, we report that oxidized GAPDH is repaired by the thioredoxin reductase system (TRS). Restoration of GAPDH activity after reduction by both tubulin and the TRS was time-dependent suggesting conformational changes near the active site cysteine149. The addition of brain MAPs to oxidized tubulin reduced tubulin disulfides and labeling of MAP-2 and of GAPDH decreased. Because the extent of tubulin repair of oxidized GAPDH was dependent on buffer strength, we conclude that electrostatics influence thiol/disulfide exchange between the two proteins. The novel interactions presented herein may protect GAPDH from inhibition under oxidative stress conditions.

Hypothiocyanous acid oxidation of tubulin cysteines inhibits microtubule polymerization.

Thiol oxidation is a probable outcome of cellular oxidative stress and is linked to degenerative disease progression. In addition, protein thiol redox reactions are increasingly identified as a mechanism to regulate protein structure and function. We assessed the effect of hypothiocyanous acid on the cytoskeletal protein tubulin. Total cysteine oxidation by hypothiocyanous and hypochlorous acids was monitored by labeling tubulin with 5-iodoacetamidofluorescein and by detecting higher molecular weight inter-chain tubulin disulfides by Western blot under nonreducing conditions. Hypothiocyanous acid induced nearly stoichiometric oxidation of tubulin cysteines (1.9 mol cysteine/mol oxidant) and no methionine oxidation was observed. Because disulfide reducing agents restored all the polymerization activity that was lost due to oxidant treatment, we conclude that cysteine oxidation of tubulin inhibits microtubule polymerization. Hypothiocyanous acid oxidation of tubulin cysteines was markedly decreased in the presence of 4% glycerol, a component of the tubulin purification buffer. Due to its instability and buffer- and pH-dependent reactivity, hypothiocyanous acid studies require careful consideration of reaction conditions.

Inhibition of tubulin polymerization by hypochlorous acid and chloramines.

Protein thiol oxidation and modification by nitric oxide and glutathione are emerging as common mechanisms to regulate protein function and to modify protein structure. Also, thiol oxidation is a probable outcome of cellular oxidative stress and is linked to degenerative disease progression. We assessed the effect of the oxidants hypochlorous acid and chloramines on the cytoskeletal protein tubulin. Total cysteine oxidation by the oxidants was monitored by labeling tubulin with the thiol-selective reagent 5-iodoacetamidofluorescein; by reaction with Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoic acid); and by detecting interchain tubulin disulfides by Western blot under nonreducing conditions. Whereas HOCl induced both cysteine and methionine oxidation of tubulin, chloramines were predominantly cysteine oxidants. Cysteine oxidation of tubulin, rather than methionine oxidation, was associated with loss of microtubule polymerization activity, and treatment of oxidized tubulin with disulfide reducing agents restored a considerable portion of the polymerization activity that was lost after oxidation. By comparing the reactivity of hypochlorous acid and chloramines with the previously characterized oxidants, peroxynitrite and the nitroxyl donor Angeli's salt, we have identified tubulin thiol oxidation, not methionine oxidation or tyrosine nitration, as a common outcome responsible for decreased polymerization activity.

Fluorescein-labeled glutathione to study protein S-glutathionylation.

Numerous studies of S-glutathionylation of cysteine thiols indicate that this protein modification plays a key role in redox regulation of proteins. To facilitate the study of protein S-glutathionylation, we developed a synthesis and purification to produce milligram quantities of fluorescein-labeled glutathione. The amino terminus of the glutathione tripeptide reacted with fluorescein isothiocyanate readily in ammonium bicarbonate. Purification by solid phase extraction on C8 and C18 columns separated excess reactants from desired products. Both oxidized and reduced fluorescein-labeled glutathione reacted with a variety of thiol-containing proteins to yield fluorescent proteins.

Protein thiol modification by peroxynitrite anion and nitric oxide donors.

Oxidation and modification of protein cysteines can have profound effects on protein structure and function. Using tubulin and microtubule-associated proteins (MAP) tau and MAP2 as examples, this chapter summarizes methods employed to characterize total cysteine modification using thiol-specific reagent 5-iodoacetamido-fluorescein labeling. Western blot analysis of peroxynitrite-damaged tubulin under nonreducing conditions reveals the formation of higher molecular weight dimers and tetramers. Disulfides in microtubule proteins are substrates for both the thioredoxin reductase system and the glutaredoxin/glutathione reductase system. The yield of disulfides formed by peroxynitrite anion is quantitated by monitoring the oxidation of NADPH, a cofactor required by the thioredoxin reductase system. Treatment of proteins with S-nitrosothiols, including S-nitrosoglutathione and S-nitroso-N-acetyl penicillamine, can yield either disulfides or protein S-nitrosation. In the case of tubulin, both types of cysteine modification were detected.

Oxidation of 5-thio-2-nitrobenzoic acid, by the biologically relevant oxidants peroxynitrite anion, hydrogen peroxide and hypochlorous acid.

The modification of protein and non-protein thiols by oxidants including hydrogen peroxide (H(2)O(2)), peroxynitrite anion (ONOO(-)) and hypochlorous acid (HOCl) is well documented. Using an aromatic thiol, 5-thio-2-nitrobenzoic acid, and biologically relevant oxidants, we have identified higher oxidation states of sulfur including the sulfonic acid derivative and the disulfide S-oxide, a thiosulfinate, by HPLC and mass spectrometry. The initial reaction of ONOO(-) with 5-thio-2-nitrobenzoic acid yielded a transient red intermediate, the sulfenate anion. The red intermediate was observed when ONOO(-) and H(2)O(2) were used to oxidize 5-thio-2-nitrobenzoic acid and it persisted for several seconds at pH 7. HOCl oxidized the disulfide, 5,5'dithiobis(2-nitrobenzoic acid) to the corresponding sulfonic acid and no additional products were detected. Using this system, we can directly compare the thiol-oxidizing abilities of several oxidants. Because 5-thio-2-nitrobenzoic acid is the product of the reaction of Ellman's reagent with protein thiols, a detailed study of its stability in biological matrices where oxidants may be generated is warranted.

Modification of tubulin cysteines by nitric oxide and nitroxyl donors alters tubulin polymerization activity.

The modification of reduced cysteines of proteins by nitric oxide alters protein function, structure, and potentially, interactions with downstream signaling targets. We assessed the effect of the S-nitroso compounds S-nitrosoglutathione and S-nitroso-N-acetyl-penicillamine, the NO donor 2-(N,N-diethylamino)-diazenolate 2-oxide, and the nitroxyl donor Angeli's salt on the cysteines of the abundant cytoskeletal protein, tubulin. Total cysteine modification by each compound was quantitated and compared to peroxynitrite anion, an oxidant that we have studied previously. Angeli's salt was most effective at modifying the cysteines of tubulin and at inducing the formation of tubulin interchain disulfide bonds followed by peroxynitrite anion, S-nitrosoglutathione, S-nitroso-N-acetyl-penicillamine, and 2-(N,N-diethylamino)-diazenolate 2-oxide. S-nitrosation of tubulin by S-nitrosoglutathione and S-nitroso-N-acetyl-penicillamine was detected by the Saville assay. Our data show that tubulin interchain disulfide bond formation by these molecules correlated with inhibition of tubulin polymerization. Closer examination of the reaction of tubulin with S-nitrosoglutathione showed a concentration-dependent shift in the type of cysteine modification detected. More tubulin disulfides were detected at lower concentrations of S-nitrosoglutathione than at higher concentrations, suggesting that reduced glutathione, generated by the reaction of S-nitrosoglutathione with tubulin cysteines, reduced disulfides initially formed by S-nitrosoglutathione.

Interactions between nitric oxide and peroxynitrite during prostaglandin endoperoxide H synthase-1 catalysis: a free radical mechanism of inactivation.

Peroxynitrite (ONOO(-)) can serve either as a peroxide substrate or as an inactivator of prostaglandin endoperoxide H synthase-1 (PGHS-1). Herein, the mechanism of PGHS-1 inactivation by ONOO(-) and the modulatory role that nitric oxide (*NO) plays in this process were studied. PGHS-1 reacted with ONOO(-) with a second-order rate constant of 1.7 x 10(7) M(-1) s(-1) at pH 7.0 and 8 degrees C. In the absence of substrates, the enzyme was dose-dependently inactivated by ONOO(-) in parallel with 3-nitrotyrosine formation. However, when PGHS-1 was incubated with ONOO(-) in the presence of substrates, the direct reaction with ONOO(-) was less relevant and ONOO(-)-derived radicals became involved in enzyme inactivation. Bicarbonate at physiologically relevant concentrations enhanced PGHS-1 inactivation and nitration by ONOO(-), further supporting a free radical mechanism. Importantly, *NO (0.4-1.5 microM min(-1)) was able to spare the peroxidase activity of PGHS-1 but it enhanced ONOO(-)-mediated inactivation of cyclooxygenase. The observed differential effects of *NO on ONOO(-)-mediated PGHS-1 inactivation emphasize a novel aspect of the complex modulatory role that *NO plays during inflammatory processes. We conclude that ONOO(-)-derived radicals inactivate both peroxidase and cyclooxygenase activities of PGHS-1 during enzyme turnover. Finally, our results reconcile the proposed alternative effects of ONOO(-) on PGHS-1 (activation versus inactivation).

Ascorbic acid reduction of microtubule protein disulfides and its relevance to protein S-nitrosylation assays.

The biotin switch assay was developed to aid in the identification of S-nitrosylated proteins in different cell types. However, our work with microtubule proteins including tubulin and its associated proteins tau and microtubule-associated protein-2 shows that ascorbic acid is not a selective reductant of protein S-nitrosothiols as described in the biotin switch assay. Herein we show that ascorbic acid reduces protein disulfides in tubulin, tau, and microtubule-associated protein-2 that are formed by peroxynitrite anion. Reduction of microtubule-associated protein disulfides by ascorbic acid following peroxynitrite treatment restores microtubule polymerization kinetics to control levels. We also show that ascorbic acid reduces the disulfide dithiobis(2-nitrobenzoic acid), a reagent commonly used to detect protein thiols. Not only do we describe a new reactivity of ascorbic acid with microtubule proteins but we expose an important limitation when using the biotin switch assay to detect protein S-nitrosylation.

Redox modulation of tau and microtubule-associated protein-2 by the glutathione/glutaredoxin reductase system.

Alterations in the redox status of proteins have been implicated in the pathology of several neurodegenerative diseases including Alzheimer's and Parkinson's. We report that peroxynitrite and H2O2-induced disulfides in the porcine brain microtubule-associated proteins tau and microtubule-associated protein-2 are substrates for the glutaredoxin reductase system composed of glutathione reductase, human or Escherichia coli glutaredoxin, reduced glutathione, and NADPH. Oxidation and reduction of cysteines in tau and microtubule-associated protein-2 were quantitated by monitoring the incorporation of 5-iodoacetamido-fluorescein, a thiol-specific labeling reagent. Reduction of disulfide bonds in the microtubule-associated proteins by the glutaredoxin reductase system restored their ability to promote the assembly of microtubules composed of purified porcine tubulin. Thiol-disulfide exchange between oxidized glutathione and the microtubule-associated proteins was detected by monitoring protein oxidation and was quantitated by measuring reduced glutathione by HPLC.

Cysteine oxidation of tau and microtubule-associated protein-2 by peroxynitrite: modulation of microtubule assembly kinetics by the thioredoxin reductase system.

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.

Repair of peroxynitrite damage to tubulin by the thioredoxin reductase system.

Cumulative oxidative damage to proteins coupled with a decrease in repair has been implicated in the pathology of several neurodegenerative diseases. Herein we report that peroxynitrite-induced disulfides in porcine brain tubulin are repaired by the thioredoxin reductase system composed of rat liver thioredoxin reductase, human or Escherichia coli thioredoxin, and NADPH. Disulfide bonds between the alpha-tubulin and the beta-tubulin subunits were repaired by thioredoxin reductase as determined by Western blot under nonreducing conditions. Total disulfide repair by thioredoxin reductase was assessed using a sulfhydryl-specific labeling reagent, 5-iodoacetamido-fluorescein. Treatment of tubulin with 1.0 mM peroxynitrite anion decreased 5-iodoacetamido-fluorescein labeling by 48%; repair of peroxynitrite-damaged tubulin with thioredoxin reductase restored sulfhydryl labeling to control levels. Tubulin disulfide reduction by thioredoxin reductase restored tubulin polymerization activity that was lost after peroxynitrite was added. The extent of activity restored by thioredoxin reductase and by the nonspecific disulfide-reducing agent tris(2-carboxyethyl)phosphine hydrochloride was identical; however, activity was not restored to control levels. Tyrosine nitration of tubulin was detected at all concentrations of peroxynitrite tested; thus, tubulin nitration may be responsible for the fraction of activity that could not be restored. Thiol-disulfide exchange between tubulin and thioredoxin was detected by Western blot, thereby providing further support for our observations that optimal repair of tubulin disulfides required thioredoxin.

Modulation of the redox state of tubulin by the glutathione/glutaredoxin reductase system.

Alterations in the redox status of proteins have been implicated in the pathology of several neurodegenerative diseases. We report that peroxynitrite-induced disulfides in porcine brain tubulin are repaired by the glutaredoxin reductase system composed of glutathione reductase, human or Escherichia coli glutaredoxin, reduced glutathione, and NADPH. Reduction of disulfide bonds between the alpha- and beta-tubulin subunits by the glutathione reductase system was assessed by Western blot. Tubulin cysteine oxidation and reduction was quantitated by monitoring the incorporation of 5-iodoacetamido-fluorescein, a thiol-specific labeling reagent. Tubulin disulfide bond reduction by the glutaredoxin reductase system restored tubulin polymerization activity that was lost following peroxynitrite addition. In support of redox modulations of tubulin by glutathione, thiol-disulfide exchange between tubulin and oxidized glutathione was detected and quantitated by HPLC. In addition, glutathionylation of tubulin was detected by dot blot using an anti-GSH antibody.

Peroxynitrite oxidation of tubulin sulfhydryls inhibits microtubule polymerization.

Considerable evidence both in vitro and in vivo implicates protein damage by peroxynitrite as a probable mechanism of cell death. Herein, we report that treatment of bovine brain microtubule protein, composed of tubulin and microtubule-associated proteins, with peroxynitrite led to a dose-dependent inhibition of microtubule polymerization. The extent of cysteine oxidation induced by peroxynitrite correlated well with inhibition of microtubule polymerization. Disulfide bonds between the subunits of the tubulin heterodimer were detected by Western blot as a result of peroxynitrite-induced cysteine oxidation. Addition of disulfide reducing agents including dithiothreitol and beta-mercaptoethanol restored a significant portion of the polymerization activity that was lost following peroxynitrite addition. Thus, peroxynitrite-induced disulfide bonds are at least partially responsible for the observed inhibition of polymerization. Sodium bicarbonate protected microtubule protein from the peroxynitrite-induced inhibition of polymerization. Tyrosine nitration of microtubule protein by 1 mM peroxynitrite increased approximately twofold when sodium bicarbonate was present whereas the extent of cysteine oxidation decreased from 7.5 to 6.3 mol cysteine/mol tubulin. These results indicate that cysteine oxidation of tubulin by peroxynitrite, rather than tyrosine nitration, is the primary mechanism of inhibition of microtubule polymerization.