A colorimetric assay for sulfiredoxin activity using inorganic phosphate measurement

2-Cys peroxiredoxin (Prx) is the major subgroup of a family of Prx enzymes that reduce peroxide molecules such as hydrogen peroxide (H₂O₂). 2-Cys Prxs are inactivated when their active site cysteine residue is hyperoxidized to sulfinic acid. Sulfiredoxin (Srx) is an enzyme that catalyzes reduction of hyperoxidized 2-Cys Prxs in the presence of ATP, Mg²⁺, and thiol equivalent. Therefore, Srx activity is crucial for cellular function of 2-Cys Prxs. The method currently available for the determination of Srx activity relies on immunoblot detection using antibodies to hyperoxidized enzymes. Here we introduce a simple quantitative assay for Srx activity based on the colorimetric determination of inorganic phosphate released in Srx-dependent reduction of hyperoxidized Prx using the malachite green. The colorimetric assay was used for high-throughput screening of 25,000 chemicals to find Srx inhibitors.     

Irreversible oxidation of the active-site cysteine of peroxiredoxin to cysteine sulfonic acid for enhanced molecular chaperone activity

The thiol (-SH) of the active cysteine residue in peroxiredoxin (Prx) is known to be reversibly hyperoxidized to cysteine sulfinic acid (-SO(2)H), which can be reduced back to thiol by sulfiredoxin/sestrin. However, hyperoxidized Prx of an irreversible nature has not been reported yet. Using an antibody developed against the sulfonylated (-SO(3)H) yeast Prx (Tsa1p) active-site peptide (AFTFVCPTEI), we observed an increase in the immunoblot intensity in proportion to the H(2)O(2) concentrations administered to the yeast cells. We identified two species of hyperoxidized Tsa1p: one can be reduced back (reversible) with sulfiredoxin, and the other cannot (irreversible). Irreversibly hyperoxidized Tsa1p was identified as containing the active-site cysteine sulfonic acid (Tsa1p-SO(3)H) by mass spectrometry. Tsa1p-SO(3)H was not an autoxidation product of Tsa1p-SO(2)H and was maintained in yeast cells even after two doubling cycles. Tsa1p-SO(3)H self-assembled into a ring-shaped multimeric form was shown by electron microscopy. Although the Tsa1p-SO(3)H multimer lost its peroxidase activity, it gained approximately 4-fold higher chaperone activity compared with Tsa1p-SH. In this study, we identify an irreversibly hyperoxidized Prx, Tsa1p-SO(3)H, with enhanced molecular chaperone activity and suggest that Tsa1p-SO(3)H is a marker of cumulative oxidative stress in cells.     

The cloning,expression, and comparative analysis of peroxiredoxin 6 from various sources

Human, rat, Xenopus, and Drosophila (DPx2540 and DPx6005) peroxiredoxin cDNAs were cloned and expressed in Escherichia coli. The recombinant enzymes were compared with respect to enzymatic activity toward various substrates and protection of plasmid DNA from the Fenton reaction products. The activity toward H₂O₂ decreased in the following order: DPx2540 > human Prx6 > Xenopus Prx6 > rat Prx6 > DPx6005. The activity toward tret-butyl hydroperoxide decreased in the following order: DPx2540 = DPx6005 > rat Prx6 > Xenopus Prx6 > human Prx6. The efficiency of plasmid DNA protection from oxidative damage mediated by the Fenton reaction decreased in the order of DPx2540 > DPx6005 = rat Prx6 = human Prx6 > Xenopus Prx6. The optimal temperature for activity of all enzymes was 37°C. Peroxiredoxins from rat, Xenopus, and Drosophila (DPx6005) retained no less than 50% of their activity in a wider temperature range (10–50°C) as compared with the human and Drosophila (DPx2540) enzymes (25–45°C). The thermostability of the enzymes decreased in the following order: DPx6005 = rat > human > Xenopus > DPx2540. The results confirmed a negative correlation between the activity and stability of peroxiredoxin 6, especially in the case of the Xenopus and Drosophila enzymes.     

Structural and functional regulation of eukaryotic 2-Cys peroxiredoxins including the plant ones in cellular defense-signaling mechanisms against oxidative stress

The ubiquitously distributed peroxiredoxins (Prxs) have been shown to have diverse functions in cellular defense‐signaling pathways. They have been largely classified into three Prx classes, 2‐Cys Prx, atypical 2‐Cys Prx and 1‐Cys Prx, which can be distinguished by how many Cys residues they possess and by their catalytic mechanisms. Proteins belonging to the typical 2‐Cys Prx group containing the N‐terminal peroxidatic Cys residue undergo a cycle of peroxide‐dependent oxidation to sulfenic acid and thiol‐dependent reduction during H₂O₂ catalysis. However, in the presence of high concentrations of H₂O₂ and catalytic components, including thioredoxin (Trx), Trx reductase and NADPH, the sulfenic acid can be hyperoxidized to cysteine sulfinic acid. The overoxidized 2‐Cys Prxs are slowly reduced by the action of the adenosine 5′‐triphosphate‐dependent enzyme, sulfiredoxin. Upon exposure of cells to strong oxidative or heat‐shock stress conditions, 2‐Cys Prxs change their protein structures from low‐molecular weight to high‐molecular weight complexes, which trigger their functional switching from peroxidases to molecular chaperones. The C‐terminal region of 2‐Cys Prx also plays an essential role in this structural conversion. Thus, proteins with truncated C‐termini are resistant to overoxidation and cannot regulate their structures or functions. These reactions are primarily guided by the active site peroxidatic Cys residue, which serves as an ‘H₂O₂‐sensor’ in cells. The reversible structural and functional switching of 2‐Cys Prxs provides cells with a means to adapt to external stresses by presumably activating intracellular defense‐signaling systems. In particular, plant 2‐Cys Prxs localized in chloroplasts have dynamic protein structures that undergo major conformational changes during catalysis, forming super‐complexes and reversibly attaching to thylakoid membranes in a redox‐dependent manner.     

Sulfiredoxin protein is critical for redox balance and survival of cells exposed to low steady-state levels of H2O2

Sulfiredoxin (Srx) is an enzyme that catalyzes the reduction of cysteine sulfinic acid of hyperoxidized peroxiredoxins (Prxs). Having high affinity toward H2O2, 2-Cys Prxs can efficiently reduce H2O2 at low concentration. We previously showed that Prx I is hyperoxidized at a rate of 0.072% per turnover even in the presence of low steady-state levels of H2O2. Here we examine the novel role of Srx in cells exposed to low steady-state levels of H2O2, which can be achieved by using glucose oxidase. Exposure of low steady-state levels of H2O2 (10-20 μm) to A549 or wild-type mouse embryonic fibroblast (MEF) cells does not lead to any significant change in oxidative injury because of the maintenance of balance between H2O2 production and elimination. In contrast, loss-of-function studies using Srx-depleted A549 and Srx-/- MEF cells demonstrate a dramatic increase in extra- and intracellular H2O2, sulfinic 2-Cys Prxs, and apoptosis. Concomitant with hyperoxidation of mitochondrial Prx III, Srx-depleted cells show an activation of mitochondria-mediated apoptotic pathways including mitochondria membrane potential collapse, cytochrome c release, and caspase activation. Furthermore, adenoviral re-expression of Srx in Srx-depleted A549 or Srx-/- MEF cells promotes the reactivation of sulfinic 2-Cys Prxs and results in cellular resistance to apoptosis, with enhanced removal of H2O2. These results indicate that Srx functions as a novel component to maintain the balance between H2O2 production and elimination and then protects cells from apoptosis even in the presence of low steady-state levels of H2O2.     

Evolution of Eukaryotic Cysteine Sulfinic Acid Reductase,Sulfiredoxin (Srx), from Bacterial Chromosome Partitioning Protein ParB

Sufiredoxin (Srx) is a sulfinic acid reductase, a recently identified eukaryotic enzyme, which is involved in the reduction of the hyperoxidized sulfinic acid form of the catalytic cysteine of 2-Cys peroxiredoxins (Prx). This reaction contributes to the oxidative stress response and H202 mediated signaling. We show that Srx has significant sequence and structural similarity to a functionally unrelated protein, ParB, a DNA-binding protein with a helix-turn-helix (HTH) domain which is involved in chromosome partitioning in bacteria. Sequence comparison and phylogenetic analysis of the Srx and ParB protein families suggest that Srx evolved via truncation of ParB, which removed the entire C-terminal half of the protein, including the HTH domain, and a substitution of cysteine for a glutamic acid in a highly conserved structural motif of ParB. The latter substitution apparently created the sulfinic acid reductase catalytic site. Evolution of a redox enzyme from a DNA-binding protein, with retention of highly significant sequence similarity, is unusual, even when compared to functional switches accompanying recruitment of other prokaryotic proteins for new functions in eukaryotes.     

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.     

Backbone chemical shift assignments for <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> peroxiredoxin Q in the reduced and oxidized states: a dramatic change in backbone dynamics

Peroxiredoxins (Prx) are ubiquitous enzymes that reduce peroxides as part of antioxidant defenses and redox signaling. While Prx catalytic activity and sensitivity to hyperoxidative inactivation depend on their dynamic properties, there are few examples where their dynamics has been characterized by NMR spectroscopy. Here, we provide a foundation for studies of the solution properties of peroxiredoxin Q from the plant pathogen Xanthomonas campestris (XcPrxQ) by assigning the observable ¹H^N, ¹⁵N, ¹³C^α, ¹³C^β, and ¹³C′ chemical shifts for both the reduced (dithiol) and oxidized (disulfide) states. In the reduced state, most of the backbone amide resonances (149/152, 98 %) can be assigned in the XcPrxQ ¹H–¹⁵N HSQC spectrum. In contrast, a remarkable 51 % (77) of these amide resonances are not visible in the ¹H–¹⁵N HSQC spectrum of the disulfide state of the enzyme, indicating a substantial change in backbone dynamics associated with the formation of an intramolecular C48–C84 disulfide bond.     

Endophytic <Emphasis Type="Italic">Streptomyces</Emphasis> sp. AC35, a producer of bioactive isoflavone aglycones and antimycins

In this research, a microbial endophytic strain obtained from the rhizosphere of the conifer Taxus baccata and designated as Streptomyces sp. AC35 (FJ001754.1 Streptomyces, GenBank) was investigated. High 16S rDNA gene sequence similarity suggests that this strain is closely related to S. odorifer. The major fatty acid profile of intracellular lipids was also carried out to further identify this strain. Atomic force microscopy and scanning acoustic microscopy were used to image our strain. Its major excreted substances were extracted, evaluated for antimicrobial activity, purified, and identified by ultraviolet–visible spectroscopy (UV–vis), liquid chromatography–mass spectrometry (LC–MS/MS) and nuclear magnetic resonance as the bioactive isoflavone aglycones—daidzein, glycitein and genistein. Batch cultivation, performed under different pH conditions, revealed enhanced production of antimycin components when the pH was stable at 7.0. Antimycins were detected by HPLC and identified by UV–vis and LC–MS/MS combined with the multiple reaction monitoring. Our results demonstrate that Streptomyces sp. AC35 might be used as a potential source of effective, pharmaceutically active compounds.     

Molecular Basis for the Resistance of Human Mitochondrial 2-Cys Peroxiredoxin 3 to Hyperoxidation

Peroxiredoxins (Prxs) detoxify peroxides and modulate H₂O₂-mediated cell signaling in normal and numerous pathophysiological contexts. The typical 2-Cys subclass of Prxs (human Prx1–4) utilizes a Cys sulfenic acid (Cys-SOH) intermediate and disulfide bond formation across two subunits during catalysis. During oxidative stress, however, the Cys-SOH moiety can react with H₂O₂ to form Cys sulfinic acid (Cys-SO₂H), resulting in inactivation. The propensity to hyperoxidize varies greatly among human Prxs. Mitochondrial Prx3 is the most resistant to inactivation, but the molecular basis for this property is unknown. A panel of chimeras and Cys variants of Prx2 and Prx3 were treated with H₂O₂ and analyzed by rapid chemical quench and time-resolved electrospray ionization-TOF mass spectrometry. The latter utilized an on-line rapid-mixing setup to collect data on the low seconds time scale. These approaches enabled the first direct observation of the Cys-SOH intermediate and a putative Cys sulfenamide (Cys-SN) for Prx2 and Prx3 during catalysis. The substitution of C-terminal residues in Prx3, residues adjacent to the resolving Cys residue, resulted in a Prx2-like protein with increased sensitivity to hyperoxidation and decreased ability to form the intermolecular disulfide bond between subunits. The corresponding Prx2 chimera became more resistant to hyperoxidation. Taken together, the results of this study support that the kinetics of the Cys-SOH intermediate is key to determine the probability of hyperoxidation or disulfide formation. Given the oxidizing environment of the mitochondrion, it makes sense that Prx3 would favor disulfide bond formation as a protection mechanism against hyperoxidation and inactivation.     

Current development in non-enzymatic lipid peroxidation products,isoprostanoids and isofuranoids,in novel biological samples

Abstract

Isoprostanoids and isofuranoids are lipid mediators that can be formed from omega-3 and omega-6 polyunsaturated fatty acids (PUFAs). F₂-isoprostanes formed from arachidonic acid, especially 15-F_2t-isoprostane, are commonly measured in biological tissues for decades as the biomarker for oxidative stress and diseases. Recently, other forms of isoprostanoids derived from adrenic, eicosapentaenoic, and docosahexaenoic acids namely F₂-dihomo-isoprostanes, F₃-isoprostanes, and F₄-neuroprostanes respectively, and isofuranoids including isofurans, dihomo-isofurans, and neurofurans are reported as oxidative damage markers for different metabolisms. The most widely used samples in measuring lipid peroxidation products include but not limited to the blood and urine; other biological fluids, specialized tissues, and cells can also be determined. In this review, measurement of isoprostanoids and isofuranoids in novel biological samples by gas chromatography (GC)–mass spectrometry (MS), GC–MS/MS, liquid chromatography (LC)–MS, and LC–MS/MS will be discussed.     

Reduction of cysteine sulfinic acid by sulfiredoxin is specific to 2-cys peroxiredoxins

Cysteine residues of certain peroxiredoxins (Prxs) undergo reversible oxidation to sulfinic acid (Cys-SO2H) and the reduction reaction is catalyzed by sulfiredoxin (Srx). Specific Cys residues of various other proteins are also oxidized to sulfinic acid, suggesting that formation of Cys-SO2H might be a novel posttranslational modification that contributes to regulation of protein function. To examine the susceptibility of sulfinic forms of proteins to reduction by Srx, we prepared such forms of all six mammalian Prx isoforms and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Purified sulfiredoxin reduced the sulfinic forms of the four 2-Cys members (Prx I to Prx IV) of the Prx family in vitro, but it did not affect those of Prx V, Prx VI, or GAPDH. Furthermore, Srx bound specifically to the four 2-Cys Prxs in vitro and in cells. Sulfinic forms of Prx I and Prx II, but not of Prx VI or GAPDH, present in H2O2-treated A549 cells were gradually reduced after removal of H2O2; overexpression of Srx increased the rate of the reduction of Prx I and Prx II but did not induce that of Prx VI or GAPDH. These results suggest that reduction of Cys-SO2H by Srx is specific to 2-Cys Prx isoforms. For proteins such as Prx VI and GAPDH, sulfinic acid formation might be an irreversible process that causes protein damage.     

Molecular cloning,expression and antioxidant activity of a peroxiredoxin 2 homologue from Lampetra japonica

Peroxiredoxin (Prx) is a cellular antioxidant protein family that plays important roles in oxidative stress and immune cytotoxicity. In this study, we cloned a homologue of the Prx2 from the buccal gland of Lampetra japonica (L. japonica). L. japonica Prx2 (Lj-Prx2) contained two highly conserved motifs and shared more than 70% identity with the homologs from other vertebrate species. Phylogenetic analysis revealed that Lj-Prx2 is closely related to other available teleost Prx2. The real-time PCR results demonstrated that the Prx2 gene was widely expressed in adult lamprey. In addition, the expression of Prx2 gene was particularly up-regulated in red blood cells (RBCs) after the experimental animals were challenged with lipopolysaccharide (LPS) in vivo. Lj-Prx2 gene was subcloned into the pET23b vector and expressed in Escherichia coli BL21 (DE3). The recombinant L. japonica Prx2 (rLj-Prx2) was purified by using His Bind affinity chromatography. Polyclonal antibody to rLj-Prx2 was generated in New Zealand Rabbit. Western blot analysis showed that the Lj-Prx2 is present in the buccal gland secretion, suggesting the secretory feature of it. The function assays revealed that rLj-Prx2 has the capability to reduce the H₂O₂ when dithiothreitol (DTT) is used as a reducing equivalent and to protect DNA from oxidative damage. These findings suggested that Lj-Prx2 probably plays an essential role in antioxidant defense in RBCs to keep lamprey alive.     

The oxido-redox potential of albumin: Methodological approach and relevance to human diseases

In humans, an increased synthesis of reactive oxygen species (ROS) may be a relevant cause of amplification of physiologic processes resulting in inflammatory organ damage or neoplasia. Efficient anti-oxidative systems targeting oxidative stress are thus essential to prevent tissue damage. In plasma, proteins proved to be the first line of defence against ROS and albumin, the highest concentration plasma protein, has a key role in this antioxidant function. Recent studies have clearly documented that albumin oxido-redox potential changes upon oxidation by different oxidants thus becoming a deputy biomarker of this process.ROS react primarily with the free ³⁴Cysteine (³⁴Cys) residue of albumin to form two reversible intermediate derivatives, sulfenic-(SOH-alb) and sulfinic acid (SO₂H-alb), resulting in sulfonic acid (SO₃H-alb), the final stable product of the reaction.Upon stable oxidation (SO₃H-alb), albumin properties are altered: the protein becomes more susceptible to trypsin digestion and is degraded faster compared to the non-oxidized counterpart.The present review focuses on the characterization of albumin chemical changes induced by ROS, their relevance in human pathology and the most recent advances in the approach to oxidation adduct analysis.     

Protein Engineering of the Quaternary Sulfiredoxin��Peroxiredoxin Enzyme��Substrate Complex Reveals the Molecular Basis for Cysteine Sulfinic Acid Phosphorylation

Oxidative stress can damage the active site cysteine of the antioxidant enzyme peroxiredoxin (Prx) to the sulfinic acid form, Prx-SO₂⁻. This modification leads to inactivation. Sulfiredoxin (Srx) utilizes a unique ATP-Mg²⁺-dependent mechanism to repair the Prx molecule. Using selective protein engineering that involves disulfide bond formation and site-directed mutagenesis, a mimic of the enzyme·substrate complex has been trapped. Here, we present the 2.1 Å crystal structure of human Srx in complex with PrxI, ATP, and Mg²⁺. The Cys⁵² sulfinic acid moiety was substituted by mutating this residue to Asp, leading to a replacement of the sulfur atom with a carbon atom. Because the Srx reaction cannot occur, the structural changes in the Prx active site that lead to the attack on ATP may be visualized. The local unfolding of the helix containing C52D resulted in the packing of Phe⁵⁰ in PrxI within a hydrophobic pocket of Srx. Importantly, this structural rearrangement positioned one of the oxygen atoms of Asp⁵² within 4.3 Å of the γ-phosphate of ATP bound to Srx. These observations support a mechanism where phosphorylation of Prx-SO₂⁻ is the first chemical step.     

Natively oxidized amino acid residues in the spinach cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript><Emphasis Type="Italic">6</Emphasis></Subscript><Emphasis Type="Italic">f</Emphasis> complex

The cytochrome b ₆ f complex of oxygenic photosynthesis produces substantial levels of reactive oxygen species (ROS). It has been observed that the ROS production rate by b ₆ f is 10–20 fold higher than that observed for the analogous respiratory cytochrome bc₁ complex. The types of ROS produced (O₂^{??, 1}O₂, and, possibly, H₂O₂) and the site(s) of ROS production within the b ₆ f complex have been the subject of some debate. Proposed sources of ROS have included the heme b _p , PQ _p ^?? (possible sources for O₂^??), the Rieske iron–sulfur cluster (possible source of O₂^?? and/or ¹O₂), Chl a (possible source of ¹O₂), and heme c _n (possible source of O₂^?? and/or H₂O₂). Our working hypothesis is that amino acid residues proximal to the ROS production sites will be more susceptible to oxidative modification than distant residues. In the current study, we have identified natively oxidized amino acid residues in the subunits of the spinach cytochrome b ₆ f complex. The oxidized residues were identified by tandem mass spectrometry using the MassMatrix Program. Our results indicate that numerous residues, principally localized near p-side cofactors and Chl a, were oxidatively modified. We hypothesize that these sites are sources for ROS generation in the spinach cytochrome b ₆ f complex.     

Quantitative Analysis of Flavonoids in Chamomile Flowers (<Emphasis Type="Italic">Matricaria chamomilla</Emphasis> L.) by Microcolumn HPLC-UV

A reverse-phase microcolumn HPLC method with UV detection (330 nm) was developed for quantitative analysis of flavonoids of Matricaria chamomilla flowers using a ProntoSIL-120-5-C18 AQ column (60 mm × 1 mm × 5 μm), gradient elution system 0.2 M LiClO₄/0.006 M HClO₄–acetonitrile and cosmosiin as a reference compound. Optimal conditions for the hydrolytic process of flavonoids (KOH concentration 0.25%, extraction time 30 min) and the parameters of flavonoids extraction (particle size 0.25 mm, extraction temperature 60°C, a single extraction step lasting 30 min at a ratio 1: 100) were selected. Validation analysis showed that the proposed method is characterized by satisfactory metrological parameters. The limit of detection (LOD) and limit of quantification (LOQ) of cosmosiin were 84 and 255 ng/mL, respectively. The accuracy for cosmosiin content levels 80–120% was less than 101.93–103.00%. The method was used for the analysis of introduced and commercial samples of M. chamomilla flowers.     

Novel Protective Mechanism against Irreversible Hyperoxidation of Peroxiredoxin: N��-TERMINAL ACETYLATION OF HUMAN PEROXIREDOXIN II*

Peroxiredoxins (Prxs) are a group of peroxidases containing a cysteine thiol at their catalytic site. During peroxidase catalysis, the catalytic cysteine, referred to as the peroxidatic cysteine (C_P), cycles between thiol (C_P-SH) and disulfide (–S–S–) states via a sulfenic (C_P-SOH) intermediate. Hyperoxidation of the C_P thiol to its sulfinic (C_P-SO₂H) derivative has been shown to be reversible, but its sulfonic (C_P-SO₃H) derivative is irreversible. Our comparative study of hyperoxidation and regeneration of Prx I and Prx II in HeLa cells revealed that Prx II is more susceptible than Prx I to hyperoxidation and that the majority of the hyperoxidized Prx II formation is reversible. However, the hyperoxidized Prx I showed much less reversibility because of the formation of its irreversible sulfonic derivative, as verified with C_P-SO₃H-specific antiserum. In an attempt to identify the multiple hyperoxidized spots of the Prx I on two-dimensional PAGE analysis, an N-acetylated Prx I was identified as part of the total Prx I using anti-acetylated Lys antibody. Using peptidyl-Asp metalloendopeptidase (EC 3.4.24.33) peptide fingerprints, we found that N^α-terminal acetylation (N^α-Ac) occurred exclusively on Prx II after demethionylation. N^α-Ac of Prx II blocks Prx II from irreversible hyperoxidation without altering its affinity for hydrogen peroxide. A comparative study of non-N^α-acetylated and N^α-terminal acetylated Prx II revealed that N^α-Ac of Prx II induces a significant shift in the circular dichroism spectrum and elevation of T_m from 59.6 to 70.9 °C. These findings suggest that the structural maintenance of Prx II by N^α-Ac may be responsible for preventing its hyperoxidation to form C_P-SO₃H.Peroxiredoxins (Prxs)⁴ are a family of peroxidases that possess a conserved cysteine residue at the catalytic site for the reduction of peroxide/peroxynitrite. Using thiol-based reducing equivalents, like thioredoxin, Prxs catalyze the reduction of hydrogen peroxide, alkylhydroperoxides, and peroxynitrite to water, corresponding alcohols, and nitrite, respectively (1–8). Based on the number and location of conserved cysteine residue(s) directly involved in peroxide reduction, the six isotypes of mammalian Prx can be grouped into three distinct subgroups as follows: 2-Cys Prx, atypical 2-Cys Prx, and 1-Cys Prx, (1–2, 5). Human Prx I (hPrx I) and Prx II (hPrx II) are members of the 2-Cys Prx subgroup and thus contain two conserved cysteine residues that are directly involved in peroxidase activity. Cys⁵² for hPrx I and Cys⁵¹ for hPrx II are designated the peroxidatic cysteines (C_P). These residues attack the O–O bond of the peroxide (ROOH) substrate to form the product (ROH) and the sulfenic derivative C_P-SOH. This sulfenic derivative then forms a disulfide bond with the other conserved cysteine residue, which is referred to as the resolving cysteine (C_R; Cys¹⁷³ in hPrx I and Cys¹⁷² in hPrx II). In the case of 2-Cys Prxs, the disulfide partners, C_P and C_R, reside within different subunits; therefore, the disulfide bond established between C_P and C_R (C_P-S–S-C_R) is intermolecular. The reduced thioredoxin molecule is responsible for reducing the C_P-S–S-C_R disulfide bond to generate sulfhydryls (1–3, 5, 9).The C_P of eukaryotic 2-Cys Prxs is vulnerable to hyperoxidation, which results in the loss of its peroxidase activity. This feature is referred to as the “floodgate” mechanism, by which Prxs function as a redox sensor for the regulation of cell signaling (10–11). Hyperoxidation of C_P does not occur when the disulfide bond (C_P-S–S-C_R) is formed. However, the thiol (C_P-SH) can be hyperoxidized via the sulfenic (C_P-SOH) derivative intermediate in the absence of C_P-S-S-C_R formation during catalysis (12). Two different hyperoxidation products of C_P, the reversible sulfinic (C_P-SO₂H) derivative and the irreversible sulfonic (C_P-SO₃H) derivative, have been identified. The irreversible C_P-SO₃H was reported in Tsa1p, a yeast 2-Cys Prx, based on in vivo and in vitro regeneration assay results, and a stronger reactivity to an anti-Tsa1p-SO₃H antibody, which exhibits high specificity toward Tsa1p-C_P-SO₃H relative to Tsa1p-C_P-SO₂H (13). Both forms of hyperoxidized Prxs, C_P-SO₂H and C_P-SO₃H, are superimposed on the acidic migrated spot instead of the Prx-SH spot on a two-dimensional polyacrylamide gel because of the introduction of one negative charge by hyperoxidation (12–16). The protein sulfinic acid reductase, sulfiredoxin, is responsible for reversing 2-Cys Prx-SO₂H to Prx-SH in the presence of ATP and thiol-reducing equivalents like thioredoxin or glutathione (17–24). Until now, an intracellular enzymatic regeneration system for Prx-SO₃H has not been reported.Because mammalian Prx I and Prx II have been studied independently in a number of different organisms and cultured cells, the comparative biochemical data supporting their distinctive functional identities is still very limited. Recombinant Prx I (rPrx I) showed a 2.6-fold higher specific activity as a peroxidase than the recombinant Prx II (rPrx II) without any obvious catalytic or mechanistic differences (25, 26). Recent competition kinetics studies of hPrx II revealed a rate constant of 1.3 × 10⁷ m^–1 s^–1, which is fast enough to favor an intracellular hydrogen peroxide target even in competition with catalase or glutathione peroxidase (27, 28). The kinetic parameters of the competition assay for hPrx I are still not available. Mammalian Prx I interacts with and regulates a broad spectrum of proteins, such as the Src homology domain 3 of c-Abl (29), the Myc box II (MBII) domain of c-Myc (30), the macrophage migration inhibitory factor (MIF, 31), the androgen receptor (32), and the apoptosis signal-regulating kinase-1 (ASK-1) (33). The suggested roles of Prx I in interactions with these molecules are those of a tumor repressor, a survival enhancer, and a growth regulator. Although these suggested functions are controversial (34), all of them can be attributed to the peroxide-scavenging capacity of Prx I (at least in part), except for the enhancement of androgen receptor transactivation (32). Prx II interacts with platelet-derived growth factor receptor and functions as a negative regulator for platelet-derived growth factor signaling (35). Prx II also binds to phospholipase D1 (PLD1) and functions as a hydrogen peroxide-stimulated PLD1 signal terminator (36). Both of these suggested Prx II roles are attributable to the peroxidase activity of Prx II. The major phenotypes of Prx I knock-out mice involve the development of a variety of age-related cancers, hemolytic anemia (37), and dramatic shifts in subcellular reactive oxygen species localization (38). Prx II knock-out mice exhibit splenomegaly and a lack of tumor development in any cell type or tissue (39). Until now, the protein molecule that interacts with Prx I and Prx II has not been characterized, and there is no indication of a heterodimer between Prx I and Prx II. Despite their similar peroxide-scavenging capacities, it is reasonable to conclude that the Prx I and Prx II are unable to compensate for each other in terms of physiological roles. There are several examples of tissue- or cell type-specific expression patterns, such as exclusive Prx I expression in astrocytes and Leydig cells and Prx II expression in neurons and Sertoli cells (40, 41); however, Prx I and Prx II are coexpressed in the majority of mammalian cells and tissues, suggesting distinguished biochemical characteristics of their cellular regulatory mechanisms. Recently, the unique presence of Cys⁸³ in hPrx I, which contributes to the stability of the dimer-dimer interface and suppresses local unfolding, has been claimed to be prone to overoxidation of Prx I (42). The contribution of the dimer-decamer interconversion to the regulation of Prx I activity has also been reported (43).In this study, we confirmed that hPrx II was more susceptible to hyperoxidation as well as more prone to regeneration than hPrx I in HeLa cells. We also found that the difficulty in regenerating hPrx I was caused by irreversible sulfonic (C_P-SO₃H) hyperoxidation. Using AspN (EC 3.4.24.33) peptide fingerprints, we identified the N^α-terminal acetylation exclusively on hPrx II. In addition, we provide evidence for the structural maintenance offered by N^α-terminal acetylation of hPrx II, which possibly contributes to preventing irreversible overoxidation of C_P-SO₃H.     

Cyclic voltammetry: A new strategy for the evaluation of oxidative damage to bovine insulin

Research on protein oxidative damage may give insight into the nature of protein functions and pathological conditions. In this work, the oxidative damage of bovine insulin on Au electrode was investigated by cyclic voltammetry (CV). The experimental results show that there are two anodic peaks for the oxidative damage of bovine insulin, which arise from the oxidation of the exposed disulfide bond S? S_CYS7A,CYS7B, forming sulfenic acid RSOH (1.20 V, vs. SCE), sulfinic acid RSO₂H and sulfonic acid RSO₃H (1.35 V, vs. SCE). These in vitro findings not only demonstrate the applicability of CV in simulating/evaluating the oxidative damage of nonredox proteins but also find two promising candidates (two anodic peaks) for measuring insulin.