Determination of glutathionyl hemoglobin in hemodialysis patients using electrospray ionization liquid chromatography–mass spectrometry

We first detected glutathionyl hemoglobin (Hb) β-chain in hemodialysis patients and healthy subjects using electrospray ionization liquid chromatography–mass spectrometry. The ratio of glutathionyl Hb β-chain to total β-chain was markedly increased in the hemodialysis patients as compared with healthy subjects. Glutathionyl Hb will be used as a new clinical marker of oxidative stress.     

Regulation of PTP1B via glutathionylation of the active site cysteine 215.

The reversible regulation of protein tyrosine phosphatase is an important mechanism in processing signal transduction and regulating cell cycle. Recent reports have shown that the active site cysteine residue, Cys215, can be reversibly oxidized to a cysteine sulfenic derivative (Denu and Tanner, 1998; Lee et al., 1998). We propose an additional modification that has implications for the in vivo regulation of protein tyrosine phosphatase 1B (PTP1B, EC 3.1.3.48): the glutathionylation of Cys215 to a mixed protein disulfide. Treatment of PTP1B with diamide and reduced glutathione or with only glutathione disulfide (GSSG) results in a modification detected by mass spectrometry in which the cysteine residues are oxidized to mixed disulfides with glutathione. The activity is recovered by the addition of dithiothreitol, presumably by reducing the cysteine disulfides. In addition, inactivated PTP1B is reactivated enzymatically by the glutathione-specific dethiolase enzyme thioltransferase (glutaredoxin), indicating that the inactivated form of the phosphatase is a glutathionyl mixed disulfide. The cysteine sulfenic derivative can easily oxidize to its irreversible sulfinic and sulfonic forms and hinder the regulatory efficiency if it is not converted to a more stable and reversible end product such as a glutathionyl derivative. Glutathionylation of the cysteine sulfenic derivative will prevent the enzyme from further oxidation to its irreversible forms, and constitutes an efficient regulatory mechanism.     

Different types of glutathionylation of hemoglobin can exist in intact erythrocytes

Glutathionylation of hemoglobin (Hb) was studied by incubation of intact human erythrocytes with 1 mM tert-butylhydroperoxide (tBHP). Electrophoresis of the membranes showed a time dependent increase of membrane-bound Hb alpha chain until 10 min, and immunoblotting study showed that membrane-bound Hb alpha chain reacted with anti-glutathione antibody only after 10 min. Concomitant with the Hb alpha chain, membrane associated actin, spectrin, and glyceraldehyde 3-phosphate dehydrogenase reacted with the antibody. Cytosolic Hb of the control erythrocytes reacted with anti-glutathione antibody. Together with our previous paper, the present study indicates that at least three different types of glutathionylation of Hb can exist in erythrocytes. The first type is a mixed disulfide bond between reduced glutathione (GSH) and normal Hb. The second type is a disulfide bond between the cysteine 93 of metHb beta chain and oxidized glutathione (GSSG), and the third type is a disulfide bond between the other cysteine residues of metHb alpha chain and/or metHb beta chain and GSSG.     

Chromatographic and mass spectrometric analysis of glutathione in biological samples

Biological thiol compounds are classified into high-molecular-mass protein thiols and low-molecular-mass free thiols. Endogenous low-molecular-mass thiol compounds, namely, reduced glutathione (GSH) and its corresponding disulfide, glutathione disulfide (GSSG), are very important molecules that participate in a variety of physiological and pathological processes. GSH plays an essential role in protecting cells from oxidative and nitrosative stress and GSSG can be converted into the reduced form by action of glutathione reductase. Measurement of GSH and GSSG is a useful indicator of oxidative stress and disease risk. Many publications have reported successful determination of GSH and GSSG in biological samples. In this article, we review newly developed techniques, such as liquid chromatography coupled with mass spectrometry and tandem mass spectrometry, for identifying GSH bound to proteins, or for localizing GSH in bound or free forms at specific sites in organs and in cellular locations.     

Detection and mapping of widespread intermolecular protein disulfide formation during cardiac oxidative stress using proteomics with diagonal electrophoresis

Regulation of protein function by reversible cysteine-targeted oxidation can be achieved by multiple mechanisms, such as S-glutathiolation, S-nitrosylation, sulfenic acid, sulfinic acid, and sulfenyl amide formation, as well as intramolecular disulfide bonding of vicinal thiols. Another cysteine oxidation state with regulatory potential involves the formation of intermolecular protein disulfides. We utilized two-dimensional sequential non-reducing/reducing SDS-PAGE (diagonal electrophoresis) to investigate intermolecular protein disulfide formation in adult cardiac myocytes subjected to a series of interventions (hydrogen peroxide, S-nitroso-N-acetylpenicillamine, doxorubicin, simulated ischemia, or metabolic inhibition) that alter the redox status of the cell. More detailed experiments were undertaken with the thiol-specific oxidant diamide (5 mm), a concentration that induces a mild non-injurious oxidative stress. This increase in cellular oxidation potential caused global intermolecular protein disulfide formation in cytosolic, membrane, and myofilament/cytoskeletal compartments. A large number of proteins that undergo these associations were identified using liquid chromatography-mass spectrometry/mass spectrometry. These associations, which involve metabolic and antioxidant enzymes, structural proteins, signaling molecules, and molecular chaperones, were confirmed by assessing "shifts" on non-reducing immunoblots. The observation of widespread protein-protein disulfides indicates that these oxidative associations are likely to be fundamental in how cells respond to redox changes.     

Proteomics analysis of cellular response to oxidative stress. Evidence for in vivo overoxidation of peroxiredoxins at their active site

The proteomics analysis reported here shows that a major cellular response to oxidative stress is the modification of several peroxiredoxins. An acidic form of the peroxiredoxins appeared to be systematically increased under oxidative stress conditions. Peroxiredoxins are enzymes catalyzing the destruction of peroxides. In doing so, a reactive cysteine in the peroxiredoxin active site is weakly oxidized (disulfide or sulfenic acid) by the destroyed peroxides. Cellular thiols (e.g. thioredoxin) are used to regenerate the peroxiredoxins to their active state. Tandem mass spectrometry was carried out to characterize the modified form of the protein produced in vivo by oxidative stress. The cysteine present in the active site was shown to be oxidized into cysteic acid, leading to an inactivated form of peroxiredoxin. This strongly suggested that peroxiredoxins behave as a dam upon oxidative stress, being both important peroxide-destroying enzymes and peroxide targets. Results obtained in a primary culture of Leydig cells challenged with tumor necrosis factor alpha suggested that this oxidized/native balance of peroxiredoxin 2 may play an active role in resistance or susceptibility to tumor necrosis factor alpha-induced apoptosis.     

Metallothionein disulfides are present in metallothionein-overexpressing transgenic mouse heart and increase under conditions of oxidative stress

Metallothionein (MT) releases zinc under oxidative stress conditions in cultured cells. The change in the MT molecule after zinc release in vivo is unknown although in vitro studies have identified MT disulfide bond formation. The present study was undertaken to test the hypothesis that MT disulfide bond formation occurs in vivo. A cardiac-specific MT-overexpressing transgenic mouse model was used. Mice were administered saline as a control or doxorubicin (20 mg/kg), which is an effective anticancer drug but with severe cardiac toxicity at least partially because of the generation of reactive oxygen species. A differential alkylation of cysteine residues in MT of the heart extracts was performed. Free and metal-bound cysteines were first trapped by N-ethylmaleimide and the disulfide bonds were reduced by dithiothreitol followed by alkylation with radiolabeled iodoacetamide. Analyses of the differentially alkylated MTs in the heart extract by high performance liquid chromatography, SDS-PAGE, Western blot, and mass spectrometry revealed that disulfide bonds were present in MT in vivo under both physiological and oxidative stress conditions. More disulfide bonds were found in MT under the oxidative stress conditions. The MT disulfide bonds were likely intramolecular and both alpha- and beta-domains were involved in the disulfide bond formation, although the alpha-domain appeared to be more easily oxidized than the beta-domain. The results suggest that under physiological conditions, the formation of MT disulfide bonds is involved in the regulation of zinc homeostasis. Additional zinc release from MT under oxidative stress conditions is accompanied by more MT disulfide bond formation.     

In vivo targets of S-thiolation in Chlamydomonas reinhardtii

Glutathionylation is the major form of S-thiolation in cells. This reversible redox post-translational modification consists of the formation of a mixed disulfide between a free thiol on a protein and a molecule of glutathione. This recently described modification, which is considered to occur under oxidative stress, can protect cysteine residues from irreversible oxidation, and alter positively or negatively the activity of diverse proteins. This modification and its targets have been mainly studied in non-photosynthetic organisms so far. We report here the first proteomic approach performed in vivo on photosynthetically competent cells, using the eukaryotic unicellular green alga Chlamydomonas reinhardtii with radiolabeled [(35)S]cysteine to label the glutathione pool and diamide as oxidant. This method allowed the identification of 25 targets, mainly chloroplastic, involved in various metabolic processes. Several targets are related to photosynthesis, such as the Calvin cycle enzymes phosphoglycerate kinase and ribose-5-phosphate isomerase. A number of targets, such as chaperones and peroxiredoxins, are related to stress responses. The glutathionylation of HSP70B, chloroplastic 2-Cys peroxiredoxin and isocitrate lyase was confirmed in vitro on purified proteins and the targeted residues were identified.     

Structural perturbation of human hemoglobin on glutathionylation probed by hydrogen-deuterium exchange and MALDI mass spectrometry

Glutathionyl hemoglobin, an example of post-translationally modified hemoglobin, has been studied as a marker of oxidative stress in various diseased conditions. Compared to normal hemoglobin, glutathionyl hemoglobin has been found to have increased oxygen affinity and reduced cooperativity. However, detailed information concerning the structural perturbation of hemoglobin associated with glutathionylation is lacking. In the present study, we report structural changes associated with glutathionylation of deoxyhemoglobin by hydrogen/deuterium (H/D) exchange coupled to matrix assisted laser desorption ionization (MALDI) mass spectrometry. We analyzed isotope exchange kinetics of backbone amide hydrogen of eleven peptic peptides in the deoxy state of both hemoglobin and glutathionyl hemoglobin molecules. Analysis of the deuterium incorporation kinetics for both molecules showed structural changes associated with the following peptides: α34-46, α1-29, β32-41, β86-102, β115-129, and β130-146. H/D exchange experiments suggest that glutathionylation of hemoglobin results in a change in conformation located at the above-mentioned regions of the hemoglobin molecule. MALDI mass spectrometry based H/D exchange experiment might be a simple way of monitoring structural changes associated with post-translational modification of protein.     

Regulation of mitochondrial glutathione redox status and protein glutathionylation by respiratory substrates

Brain and liver mitochondria isolated by a discontinuous Percoll gradient show an oxidized redox environment, which is reflected by low GSH levels and high GSSG levels and significant glutathionylation of mitochondrial proteins as well as by low NAD(P)H/NAD(P) values. The redox potential of brain mitochondria isolated by a discontinuous Percoll gradient method was calculated to be -171 mV based on GSH and GSSG concentrations. Immunoblotting and LC/MS/MS analysis revealed that succinyl-CoA transferase and ATP synthase (F(1) complex, α-subunit) were extensively glutathionylated; S-glutathionylation of these proteins resulted in a substantial decrease of activity. Supplementation of mitochondria with complex I or complex II respiratory substrates (malate/glutamate or succinate, respectively) increased NADH and NADPH levels, resulting in the restoration of GSH levels through reduction of GSSG and deglutathionylation of mitochondrial proteins. Under these conditions, the redox potential of brain mitochondria was calculated to be -291 mV. Supplementation of mitochondria with respiratory substrates prevented GSSG formation and, consequently, ATP synthase glutathionylation in response to H(2)O(2) challenges. ATP synthase appears to be the major mitochondrial protein that becomes glutathionylated under oxidative stress conditions. Glutathionylation of mitochondrial proteins is a major consequence of oxidative stress, and respiratory substrates are key regulators of mitochondrial redox status (as reflected by thiol/disulfide exchange) by maintaining mitochondrial NADPH levels.     

Hemoglobin S-thiolation during peroxide-induced oxidative stress in chicken blood

Protein S-thiolation or protein-glutathione mixed disulfide (PSSG) occurs when cells are exposed to oxidative stress, and has been implicated in several cellular functions. The S-thiolation of hemoglobin as well as other abundant proteins is proposed to participate as a redox buffer, being part of the antioxidant protection system of the cell during the oxidative challenge. We studied the oxidative stress caused by peroxides (H(2)O(2), cumene and tert-butyl hydroperoxide) on chicken blood by measuring the thiol/disulfide status. Chicken blood under peroxide treatment showed a time- and concentration-dependent increase in glutathione disulfide (GSSG) and PSSG. GSSG peaked immediately after treatment (1 min), while PSSG increased progressively over time, showing a maximum after about 30 min. The system recovered after 140 min of incubation, with GSSG and PSSG then barely reaching control values. The S-thiolation of hemoglobin was monitored under nondenaturing PAGE, and the fraction of S-thiolated hemoglobin, or Hb A1, rose in a dose-dependent fashion and was proportional to total S-thiolation, measured as PSSG. This significant correlation indicates that hemoglobin is the major S-thiolated protein in chicken erythrocytes treated with peroxides. The present work shows the behavior of chicken blood under peroxide treatment; it anticipated that chicken hemoglobin thiol groups can actively participate in the redox processes of erythrocytes exposed to oxidative stress, and that hemoglobin is the major S-thiolated protein. This further corroborates the hypothesis that abundant proteins, such as hemoglobin, may take part in the cellular antioxidant defense system.     

Regulation of mitochondrial NADP+-dependent isocitrate dehydrogenase activity by glutathionylation

Recently, we demonstrated that the control of mitochondrial redox balance and oxidative damage is one of the primary functions of mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDPm). Because cysteine residue(s) in IDPm are susceptible to inactivation by a number of thiol-modifying reagents, we hypothesized that IDPm is likely a target for regulation by an oxidative mechanism, specifically glutathionylation. Oxidized glutathione led to enzyme inactivation with simultaneous formation of a mixed disulfide between glutathione and the cysteine residue(s) in IDPm, which was detected by immunoblotting with anti-GSH IgG. The inactivated IDPm was reactivated enzymatically by glutaredoxin2 in the presence of GSH, indicating that the inactivated form of IDPm is a glutathionyl mixed disulfide. Mass spectrometry and site-directed mutagenesis further confirmed that glutathionylation occurs to a Cys(269) of IDPm. The glutathionylated IDPm appeared to be significantly less susceptible than native protein to peptide fragmentation by reactive oxygen species and proteolytic digestion, suggesting that glutathionylation plays a protective role presumably through the structural alterations. HEK293 cells and intact respiring mitochondria treated with oxidants inducing GSH oxidation such as H(2)O(2) or diamide showed a decrease in IDPm activity and the accumulation of glutathionylated enzyme. Using immunoprecipitation with anti-IDPm IgG and immunoblotting with anti-GSH IgG, we were also able to purify and positively identify glutathionylated IDPm from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice, a model for Parkinson's disease. The results of the current study indicate that IDPm activity appears to be modulated through enzymatic glutathionylation and deglutathionylation during oxidative stress.     

Proline and glycinebetaine enhance antioxidant defense and methylglyoxal detoxification systems and reduce NaCl-induced damage in cultured tobacco cells

Salt stress impairs reactive oxygen species (ROS) and methylglyoxal (MG) detoxification systems, and causes oxidative damage to plants. Up-regulation of the antioxidant and glyoxalase systems provides protection against NaCl-induced oxidative damage in plants. Thiol–disulfide contents, glutathione content and its associated enzyme activities involved in the antioxidant defense and glyoxalase systems, and protein carbonylation in tobacco Bright Yellow-2 cells grown in suspension culture were investigated to assess the protection offered by proline and glycinebetaine against salt stress. Salt stress increased protein carbonylation, contents of thiol, disulfide, reduced (GSH) and oxidized (GSSG) forms of glutathione, and the activity of glutathione-S-transferase and glyoxalase II enzymes, but decreased redox state of both thiol–disulfide and glutathione, and the activity of glutathione peroxidase and glyoxalase I enzymes involved in the ROS and MG detoxification systems. Exogenous application of proline or glycinebetaine resulted in a reduction of protein carbonylation, and in an increase in glutathione redox state and activity of glutathione peroxidase, glutathione-S-transferase and glyoxalase I under salt stress. Neither proline nor glycinebetaine, however, had any direct protective effect on NaCl-induced GSH-associated enzyme activities. The present study, therefore, suggests that both proline and glycinebetaine provide a protective action against NaCl-induced oxidative damage by reducing protein carbonylation, and enhancing antioxidant defense and MG detoxification systems.     

Corynebacterium diphtheriae Methionine Sulfoxide Reductase A Exploits a Unique Mycothiol Redox Relay Mechanism

Methionine sulfoxide reductases are conserved enzymes that reduce oxidized methionines in proteins and play a pivotal role in cellular redox signaling. We have unraveled the redox relay mechanisms of methionine sulfoxide reductase A of the pathogen Corynebacterium diphtheriae (Cd-MsrA) and shown that this enzyme is coupled to two independent redox relay pathways. Steady-state kinetics combined with mass spectrometry of Cd-MsrA mutants give a view of the essential cysteine residues for catalysis. Cd-MsrA combines a nucleophilic cysteine sulfenylation reaction with an intramolecular disulfide bond cascade linked to the thioredoxin pathway. Within this cascade, the oxidative equivalents are transferred to the surface of the protein while releasing the reduced substrate. Alternatively, MsrA catalyzes methionine sulfoxide reduction linked to the mycothiol/mycoredoxin-1 pathway. After the nucleophilic cysteine sulfenylation reaction, MsrA forms a mixed disulfide with mycothiol, which is transferred via a thiol disulfide relay mechanism to a second cysteine for reduction by mycoredoxin-1. With x-ray crystallography, we visualize two essential intermediates of the thioredoxin relay mechanism and a cacodylate molecule mimicking the substrate interactions in the active site. The interplay of both redox pathways in redox signaling regulation forms the basis for further research into the oxidative stress response of this pathogen.     

Role of curcuminoids in ameliorating oxidative modification in β-thalassemia/Hb E plasma proteome

Thalassemic patients often exhibit high levels of oxidative stress and iron overload, which can lead to hazardous complications. Curcuminoids, extracted from the spice turmeric, are known to have antioxidant and iron-chelating properties and have been proposed as a potential upstream therapy of thalassemia. Here we have applied proteomic techniques to study the protein profile and oxidative damage in the plasma of β-thalassemia/Hb E patients before and after treatment with curcuminoids. In this study, 10 β-thalassemia/Hb E patients were treated with 500 mg curcuminoids daily for 12 months. The plasma protein profile and protein carbonyl content were determined at baseline, 6 and 12 months using two-dimensional fluorescence difference gel electrophoresis and carbonyl immunoblotting, respectively. Other hematological, clinical, and biochemical parameters were also analyzed. Twenty-six spots, identified as coagulation factors and proteins involved in iron homeostasis, showed significantly decreased intensity in thalassemic plasma, compared to those of normal subjects. Treatment with curcuminoids up-regulated the plasma levels of these proteins and reduced their oxidative damage. Serum non-transferrin bound iron, platelet factor-3 like activity, oxidative stress parameters and antioxidant enzymes were also improved after curcuminoids treatment. This study is the first proteomic study of plasma in the thalassemic state and also shows the ameliorating role of curcuminoids towards oxidative stress and iron overload in the plasma proteome.     

Oxidative stress-induced modifications of histidyl-tRNA synthetase affect its tRNA aminoacylation activity but not its immunoreactivity

The aminoacyl-tRNA synthetases are ubiquitously expressed enzymes that catalyze the esterification of amino acids to their cognate tRNAs. Autoantibodies against several aminoacyl-tRNA synthetases are found in autoimmune polymyositis and dermatomyositis patients. Because necrosis is often found in skeletal muscle biopsies of these patients, we hypothesized that cell-death-induced protein modifications may help in breaking immunological tolerance. Since cell death is associated with oxidative stress, the effect of oxidative stress on the main myositis-specific autoantibody target Jo-1 (histidyl-tRNA synthetase; HisRS) was studied in detail. The exposure of Jurkat cells to hydrogen peroxide resulted in the detection of several oxidized methionines and one oxidized tryptophan residue in the HisRS protein, as demonstrated by mass spectrometry. Unexpectedly, the tRNA aminoacylation activity of HisRS appeared to be increased upon oxidative modification. The analysis of myositis patient sera did not lead to the detection of autoantibodies that are specifically reactive with the modified HisRS protein. The results of this study demonstrate that the Jo-1/HisRS autoantigen is modified under oxidative stress conditions. The consequences of these modifications for the function of HisRS and its autoantigenicity are discussed.     

Carboplatin-induced alteration of the thiol homeostasis in the isolated perfused rat kidney

Carboplatin elicits minor side effects with respect to its first generation analogue cisplatin. Nevertheless, a dose-dependent nephrotoxicity of the drug has been reported to occur both in patients and in rats and a possible pathogenic role have been attributed to oxidative stress. We have studied the effect of carboplatin administration on the thiol/disulfide balance, on other biomarkers of oxidative stress and on antioxidant enzymes in the isolated perfused rat kidney. A 5-500 μM carboplatin dose range did not alter renal function but significantly decreased levels of cysteine, glutathione and exposed protein sulfhydryl groups. Only a minimal increment in disulfides was observed, whereas malonyldialdehyde and protein carbonyls did not increase significantly. Among the antioxidant enzymes studied, only thioltransferase was inhibited by the treatment. Our data suggest that a minimal oxidative stress is present under our experimental conditions, thus indicating that platinum-based drugs do not produce significant amount of reactive oxygen species.     

Protein glutathionylation in health and disease

Background

It is now recognized that protein cysteines exist not only as free thiols or intramolecular disulfides, that help maintain the 3D structure of proteins, but can also undergo different types of oxidation, one of which is glutathionylation, or the formation of mixed disulfides with glutathione (GSH).

Scope of the review

We will discuss how proteins can undergo glutathionylation and how this can affect the protein characteristics/function. Glutathionylation is reversible and de-glutathionylation can be catalysed by protein thiol–disulfide oxidoreductases. Genetic modification of the expression of these enzymes, particularly glutaredoxin, using overexpression, knockout mice or siRNA, is becoming an important tool to study the role of protein glutathionylation. While in the past this post-translational modification was mainly known in the context of oxidative stress, measurement of glutathionylated proteins in patients is pointing out a potential importance if this modification in pathogenesis and could identify new biomarkers. We also wanted to point out the main findings in the role of glutathionylation in diseases and drug action.

Major conclusions

We identify two major open problems in the field, namely the complexity of the mechanisms responsible for glutathionylation and de-glutathionylation, as well as what makes a protein susceptible to glutathionylation.

General significance

This review underlines the peculiarities of this post-translational modification and their biological role. This article is part of a Special Issue entitled Cellular functions of glutathione.     

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.     

Oxidative stress-induced posttranslational modifications of human hemoglobin in erythrocytes

Posttranslational modifications (PTMs) have been reported in hemoglobin (Hb) treated with ROS/RNS in cell-free experiments. However, little is known about oxidative PTMs of Hb occurring within the erythrocytes. The aim of this study is to characterize the patterns of Hb PTMs in erythrocytes under oxidative stress. Using mass spectrometry, we investigated specifically methionine/tryptophan oxidation, tyrosine nitration, and the modification via 4-hydroxynonenal (HNE), a product of lipid-peroxidation, on Hb. We demonstrated that the treatment with H₂O₂/nitrite induced higher levels of Hb oxidation/nitration in purified Hb preparations than in unpurified hemolysates and erythrocytes, indicating that ROS/RNS are primarily removed by antioxidative mechanisms. We further studied Hb from erythrocytes exposed to γ-irradiation. An irradiation of 30–100 Gy triggered a remarkable increase of intracellular ROS. However, 30 Gy did not induce apparent changes in Hb oxidation/nitration and hemolysis, while Hb oxidation/nitration and hemolysis were significantly enhanced by 100 Gy, suggesting that Hb oxidation/nitration are the consequence of overwhelmed antioxidative mechanisms after oxidative attack and reflect the severity of the oxidative damage of erythrocytes. Although irradiation was known to induce lipid-peroxidation, we could not detect HNE-Hb adducts in irradiated erythrocytes. Analyzing PTM patterns suggests Hb nitration as a more suitable indicator of the oxidative damage of erythrocytes.