Alexander V. Peskin

A microtiter plate assay for superoxide dismutase using a water-soluble tetrazolium salt (WST-1).

A simple and reproducible microtiter plate assay for measuring superoxide dismutase (SOD) activity is described. Water-soluble tetrazolium, the sodium salt of 4-[3-(4iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-be nzene disulfonate, was used as a detector of superoxide radical generated by xanthine oxidase and hypoxanthine, in the presence of a range of concentrations of superoxide dismutase. A major advantage of the assay is that one reaction mixture is prepared and aliquotted into wells, avoiding pipetting errors and variable xanthine oxidase activity between samples. Inclusion of standardized SOD solution in each run enables inter-assay comparability without requiring a constant superoxide generation rate under all occasions. The assay is applicable for chloroform-ethanol red cell extracts as well as tissue homogenates without high-speed centrifugation. Fifty percent inhibition of formazan formation was achieved at 2.4+/-0.1 ng of Cu, ZnSOD per well with the coefficient of variation 4.2%.

The High Reactivity of Peroxiredoxin 2 with H2O2 Is Not Reflected in Its Reaction with Other Oxidants and Thiol Reagents

Peroxiredoxin 2 is a member of the mammalian peroxiredoxin family of thiol proteins that is important in antioxidant defense and redox signaling. We have examined its reactivity with various biological oxidants, in order to assess its ability to act as a direct physiological target for these species. Human erythrocyte peroxiredoxin 2 was oxidized stoichiometrically to its disulfide-bonded homodimer by hydrogen peroxide, as monitored electrophoretically under nonreducing conditions. The protein was highly susceptible to oxidation by adventitious peroxide, which could be prevented by treating buffers with low concentrations of catalase. However, this did not protect peroxiredoxin 2 against oxidation by added H2O2. Experiments measuring inhibition of dimerization indicated that at pH 7.4 catalase and peroxiredoxin 2 react with hydrogen peroxide at comparable rates. A rate constant of 1.3 × 107 m-1 s-1 for the peroxiredoxin reaction was obtained from competition kinetic studies with horseradish peroxidase. This is 100-fold faster than is generally assumed. It is sufficiently high for peroxiredoxin to be a favored cellular target for hydrogen peroxide, even in competition with catalase or glutathione peroxidase. Reactions of t-butyl and cumene hydroperoxides with peroxiredoxin were also fast, but amino acid chloramines reacted much more slowly. This contrasts with other thiol compounds that react many times faster with chloramines than with hydrogen peroxide. The alkylating agent iodoacetamide also reacted extremely slowly with peroxiredoxin 2. These results demonstrate that peroxiredoxin 2 has a tertiary structure that facilitates reaction of the active site thiol with hydrogen peroxide while restricting its reactivity with other thiol reagents.

Kinetics of the reactions of hypochlorous acid and amino acid chloramines with thiols, methionine, and ascorbate

Thiol oxidation by hypochlorous acid and chloramines is a favorable reaction and may be responsible for alterations in regulatory or signaling pathways in cells exposed to neutrophil oxidants. In order to establish the mechanism for such changes, it is necessary to appreciate whether these oxidants are selective for different thiols as compared with other scavengers. We have measured rate constants for reactions of amino acid chloramines with a range of thiols, methionine, and ascorbate, using a combination of stopped-flow and competitive kinetics. For HOCl, rate constants are too fast to measure directly by our system and values relative to reduced glutathione were determined by competition with methionine. For taurine chloramine, the rate constants for reaction with 5-thio-2-nitrobenzoic acid, GSH, methionine, and ascorbate at pH 7.4 were 970, 115, 39, and 13 M(-1) s(-1), respectively. Values for 10 thiols varied by a factor of 20 and showed an inverse relationship to the pK(a) of the thiol group. Rate constants for chloramines of glycine and N-alpha-acetyl-lysine also showed these relationships. Rates increased with decreasing pH, suggesting a mechanism involving acid catalysis. For hypochlorous acid, rates of reaction with 5-thio-2-nitrobenzoic acid, GSH, cysteine, and most of the other thiols were very similar. Relative reactivities varied by less than 5 and there was no dependence on thiol pK(a). Chloramines have the potential to be selective for different cellular thiols depending on their pK(a). For HOCl to be selective, other factors must be important, or its reactions could be secondary to chloramine formation.

Redox Potential and Peroxide Reactivity of Human Peroxiredoxin 3

Peroxiredoxins (Prxs) are a ubiquitous family of thiol peroxidases that protect cells from peroxides and have a putative role in redox signaling. In this study, we investigated the redox properties of human Prx 3, a typical 2-Cys Prx that is localized to the mitochondrial matrix. We found that Prx 3 displayed strong reactivity with H(2)O(2), with a competitive kinetic approach generating a second order rate constant of 2 x 10(7) M(-1) s(-1). This is considerably higher than typical thiols and similar to values for other mammalian 2-Cys Prxs. In contrast, Prx 3 reacted very slowly with the thiol alkylating agents iodoacetamide and N-ethylmaleimide. Using dithiothreitol redox buffers, we measured the redox potential of Prx 3 of -290 mV. This is similar to the redox potential of mitochondrial thioredoxin 2 and is consistent with optimal operation of Prx 3 in the mitochondrial matrix.

Model for the exceptional reactivity of peroxiredoxins 2 and 3 with hydrogen peroxide: a kinetic and computational study.

Peroxiredoxins (Prx) are thiol peroxidases that exhibit exceptionally high reactivity toward peroxides, but the chemical basis for this is not well understood. We present strong experimental evidence that two highly conserved arginine residues play a vital role in this activity of human Prx2 and Prx3. Point mutation of either ArgI or ArgII (in Prx3 Arg-123 and Arg-146, which are ∼3–4 Å or ∼6–7 Å away from the active site peroxidative cysteine (Cp), respectively) in each case resulted in a 5 orders of magnitude loss in reactivity. A further 2 orders of magnitude decrease in the second-order rate constant was observed for the double arginine mutants of both isoforms, suggesting a cooperative function for these residues. Detailed ab initio theoretical calculations carried out with the high level G4 procedure suggest strong catalytic effects of H-bond-donating functional groups to the Cp sulfur and the reactive and leaving oxygens of the peroxide in a cooperative manner. Using a guanidinium cation in the calculations to mimic the functional group of arginine, we were able to locate two transition structures that indicate rate enhancements consistent with our experimentally observed rate constants. Our results provide strong evidence for a vital role of ArgI in activating the peroxide that also involves H-bonding to ArgII. This mechanism could explain the exceptional reactivity of peroxiredoxins toward H2O2 and may have wider implications for protein thiol reactivity toward peroxides.

Hyperoxidation of Peroxiredoxins 2 and 3 RATE CONSTANTS FOR THE REACTIONS OF THE SULFENIC ACID OF THE PEROXIDATIC CYSTEINE

Background: H2O2 oxidizes peroxiredoxins (Prxs) to sulfenic acid intermediates which form disulfides or become hyperoxidized. Results: Rate constants for hyperoxidation and disulfide formation were obtained for Prx2 and Prx3. Conclusions: Prx2 is more susceptible than Prx3 to hyperoxidation due to slower disulfide formation. Significance: H2O2 reacts with Prx sulfenic acid faster than with most reduced thiols. Typical 2-Cys peroxiredoxins (Prxs) react rapidly with H2O2 to form a sulfenic acid, which then condenses with the resolving cysteine of the adjacent Prx in the homodimer or reacts with another H2O2 to become hyperoxidized. Hyperoxidation inactivates the Prx and is implicated in cell signaling. Prxs vary in susceptibility to hyperoxidation. We determined rate constants for disulfide formation and hyperoxidation for human recombinant Prx2 and Prx3 by analyzing the relative proportions of hyperoxidized and dimeric products using mass spectrometry as a function of H2O2 concentration (in the absence of reductive cycling) and in competition with catalase at a fixed concentration of H2O2. This gave a second order rate constant for hyperoxidation of 12,000 m−1 s−1 and a rate constant for disulfide formation of 2 s−1 for Prx2. A similar hyperoxidation rate constant for Prx3 was measured, but its rate of disulfide formation was ∼10-fold higher, making it is more resistant than Prx2 to hyperoxidation. There are two active sites within the homodimer, and at low H2O2 concentrations one site was hyperoxidized and the other present as a disulfide. Prx with two hyperoxidized sites formed progressively at higher H2O2 concentrations. Although the sulfenic acid forms of Prx2 and Prx3 are ∼1000-fold less reactive with H2O2 than their active site thiols, they react several orders of magnitude faster than most reduced thiol proteins. This observation has important implications for understanding the mechanism of peroxide sensing in cells.

Antioxidant activity of procyanidin-containing plant extracts at different pHs

Anti-oxidant activity was measured for a Pinus radiata bark extract (bark 1), a Pinus maritima bark extract (bark 2), two grape seed extracts, three grape skin extracts, catechin, trolox, ascorbic acid (vitamin C) and calcium ascorbate. The activity was analyzed as ability to scavenge superoxide radicals in a hypoxanthine-xanthine oxidase system, using NBT and WST-1 assays. The 50% inhibition (IC50) of formazan formation was estimated for the samples studied. This study demonstrated that, in basic solution, bark extracts were 13 times more effective antioxidants than vitamin C; however, in aqueous and acidic solutions, bark1 acted as a more potent antioxidant than the other antioxidants in the NBT assay. Grape seed extracts 1 and 2 acted as moderate antioxidants in aqueous and basic solutions in both NBT and WST-1 assays and, in most cases, were three times as efficient as vitamin C. Activities of grape skin extracts were in the same range as trolox, being about 10 times lower than that of the bark extracts. Statistical analysis showed that there was no significant difference between the two assay methods or pH of the solution.

Oxidant Carcinogenesis and Antioxidant Defensea

Growth promotion by oxidants is observed with cultured human and mouse fibroblasts as well as epidermal cells. It is expected to play a role in inflammation, fibrosis, and tumorigenesis. Indeed, oxidants trigger (patho)physiological reactions that resemble those induced by growth and differentiation factors. For example, active oxygen activates protein kinases. causes DNA breakage, and induces the growth competence‐related protooncogenes c‐fos and c‐myc.

Histamine chloramine reactivity with thiol compounds, ascorbate, and methionine and with intracellular glutathione

Histamine is stored in granules of mast cells and basophils and released by inflammatory mediators. It has the potential to intercept some of the HOCl generated by the neutrophil enzyme, myeloperoxidase, to produce histamine chloramine. We have measured rate constants for reactions of histamine chloramine with methionine, ascorbate, and GSH at pH 7.4, of 91 M(-1)s(-1), 195 M(-1)s(-1), and 721 M(-1)s(-1), respectively. With low molecular weight thiols, the reaction was with the thiolate and rates increased exponentially with decreasing thiol group pK(a). Comparing rate constants for different chloramines reacting with ascorbate or a particular thiol anion, these were higher when there was less negative charge in the vicinity of the chloramine group. Histamine chloramine was the most reactive among biologically relevant chloramines. Consumption of histamine chloramine and oxidation of intracellular GSH were examined for human fibroblasts. At nontoxic doses, GSH loss over 10 min was slightly greater than that with HOCl, but the cellular uptake of histamine chloramine was 5-10-fold less. With histamine chloramine, GSSG was a minor product and most of the GSH was converted to mixed disulfides with proteins. HOCl gave a different profile of GSH oxidation products, with significantly less GSSG and mixed disulfide formation. There was irreversible oxidation and losses to the medium, as observed with HOCl and other cell types. Thus, histamine chloramine shows high preference for thiols both in isolation and in cells, and in this respect is more selective than HOCl.

Glutathionylation of the Active Site Cysteines of Peroxiredoxin 2 and Recycling by Glutaredoxin

Peroxiredoxin 2 (Prx2) is a thiol protein that functions as an antioxidant, regulator of cellular peroxide concentrations, and sensor of redox signals. Its redox cycle is widely accepted to involve oxidation by a peroxide and reduction by thioredoxin/thioredoxin reductase. Interactions of Prx2 with other thiols are not well characterized. Here we show that the active site Cys residues of Prx2 form stable mixed disulfides with glutathione (GSH). Glutathionylation was reversed by glutaredoxin 1 (Grx1), and GSH plus Grx1 was able to support the peroxidase activity of Prx2. Prx2 became glutathionylated when its disulfide was incubated with GSH and when the reduced protein was treated with H2O2 and GSH. The latter reaction occurred via the sulfenic acid, which reacted sufficiently rapidly (k = 500 m−1 s−1) for physiological concentrations of GSH to inhibit Prx disulfide formation and protect against hyperoxidation to the sulfinic acid. Glutathionylated Prx2 was detected in erythrocytes from Grx1 knock-out mice after peroxide challenge. We conclude that Prx2 glutathionylation is a favorable reaction that can occur in cells under oxidative stress and may have a role in redox signaling. GSH/Grx1 provide an alternative mechanism to thioredoxin and thioredoxin reductase for Prx2 recycling.