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.     

Taurine chloramine is more selective than hypochlorous acid at targeting critical cysteines and inactivating creatine kinase and glyceraldehyde-3-phosphate dehydrogenase

Hypochlorous acid (HOCl) and chloramines are produced by the neutrophil enzyme, myeloperoxidase. Both react readily with thiols, although chloramines differ from HOCl in discriminating between low molecular weight thiols on the basis of their pKa. Here, we have compared the reactivity of HOCl and taurine chloramine with thiol proteins by examining inactivation of creatine kinase (CK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). With both enzymes, loss of activity paralleled thiol loss. For CK both were complete at a 1:1 taurine chloramine:thiol mole ratio. For GAPDH each chloramine oxidized two thiols. Three times more HOCl than taurine chloramine was required for inactivation, indicating that HOCl is less thiol specific. Competition studies showed that thiols of CK were 4 times more reactive with taurine chloramine than thiols of GAPDH (rate constants of 1200 and 300 M-1s-1 respectively). These compare with 205 M-1s-1 for cysteine and are consistent with their lower pKa's. Both enzymes were equally susceptible to HOCl. GSH competed directly with the enzyme thiols for taurine chloramine and protected against oxidative inactivation. At lower GSH concentrations, mixed disulfides were formed. We propose that chloramines should preferentially attack proteins with low pKa thiols and this could be important in regulatory processes.     

Relative reactivities of N-chloramines and hypochlorous acid with human plasma constituents

Hypochlorous acid (HOCl), the major strong oxidant produced by the phagocyte enzyme myeloperoxidase, reacts readily with free amino groups to form N-chloramines. Since different N-chloramines have different stabilities and reactivities depending on their structures, we investigated the relative reactivities of three model N-chloramines and HOCl with human plasma constituents. TheN-chloramines studied were N(alpha)-acetyl-lysine chloramine (LysCA, a model of protein-associated N-chloramines), taurine chloramine (TaurCA, the primary N-chloramine produced by activated neutrophils), and monochloramine (MonoCA, a lipophilic N-chloramine). Addition of these chlorine species (100--1000 microM each) to plasma resulted in rapid loss of thiols, with the extent of thiol oxidation decreasing in the order TaurCA = LysCA > MonoCA = HOCl. The single reduced thiol of albumin was the major target. Loss of plasma ascorbate also occurred, with the extent decreasing in the order HOCl > LysCA > TaurCA > MonoCA. Experiments comparing equimolar albumin thiols and ascorbate showed that while HOCl caused equivalent loss of thiols and ascorbate, theN-chloramines reacted preferentially with thiols. The chlorine species also inactivated alpha(1)-antiproteinase, implicating oxidation of methionine residues, and ascorbate provided variable protection depending on the chlorine species involved. Together, our data indicate that in biological fluids N-chloramines react more readily with protein thiols than with methionine residues or ascorbate, and thus may cause biologically relevant, selective loss of thiol groups.     

Evidence for rapid inter- and intramolecular chlorine transfer reactions of histamine and carnosine chloramines: implications for the prevention of hypochlorous-acid-mediated damage

Hypochlorous acid (HOCl) is a powerful oxidant generated from H(2)O(2) and Cl(-) by the heme enzyme myeloperoxidase, which is released from activated leukocytes. HOCl possesses potent antibacterial properties, but excessive production can lead to host tissue damage that is implicated in a wide range of human diseases (e.g., atherosclerosis). Histamine and carnosine have been proposed as protective agents against such damage. However, as recent studies have shown that histidine-containing compounds readily form imidazole chloramines that can rapidly chlorinate other targets, it was hypothesized that similar reactions may occur with histamine and carnosine, leading to propagation, rather than prevention, of HOCl-mediated damage. In this study, the reactions of HOCl with histamine, histidine, carnosine, and other compounds containing imidazole and free amine sites were examined. In all cases, rapid formation (k, 1.6 x 10(5) M(-)(1) s(-)(1)) of imidazole chloramines was observed, followed by chlorine transfer to yield more stable, primary chloramines (R-NHCl). The rates of most of these secondary reactions are dependent upon substrate concentrations, consistent with intermolecular mechanisms (k, 10(3)-10(4) M(-)(1) s(-)(1)). However, for carnosine, the imidazole chloramine transfer rates are independent of the concentration, indicative of intramolecular processes (k, 0.6 s(-)(1)). High-performance liquid chromatography studies show that in all cases the resultant R-NHCl species can slowly chlorinate N-alpha-acetyl-Tyr. Thus, the current data indicate that the chloramines formed on the imidazole and free amine groups of these compounds can oxidize other target molecules but with limited efficiency, suggesting that histamine and particularly carnosine may be able to limit HOCl-mediated oxidation in vivo.     

Regulation of apoptosis by vitamin C. Specific protection of the apoptotic machinery against exposure to chlorinated oxidants

We have investigated the ability of intracellular vitamin C to protect human umbilical vein endothelial cells from exposure to hypochlorous acid (HOCl) and a range of derived chloramines. Ascorbate provided minimal protection against the cytotoxicity induced by these oxidants, as measured by propidium iodide uptake. In contrast, there was a marked effect on apoptosis, monitored by caspase-3 activation and phosphatidylserine exposure. Extended incubation of the cells with glycine chloramine or histamine chloramine completely blocked apoptosis initiated in the cells by serum withdrawal. This effect was significantly abrogated by ascorbate. Inhibition of apoptosis required the oxidant to be present for an extended period after serum withdrawal and occurred prior to caspase-3 activation. General protection of thiols by ascorbate was not responsible for the protection of apoptosis, because intracellular oxidation by HOCl or chloramines was not prevented in supplemented cells. The results suggest a new role for vitamin C in the regulation of apoptosis. We propose that, by protection of an oxidant-sensitive step in the initiation phase, ascorbate allows apoptosis to proceed in endothelial cells under sustained oxidative stress.     

IkappaB is a sensitive target for oxidation by cell-permeable chloramines: inhibition of NF-kappaB activity by glycine chloramine through methionine oxidation

Hypochlorous acid (HOCl) is produced by the neutrophil enzyme, myeloperoxidase, and reacts with amines to generate chloramines. These oxidants react readily with thiols and methionine and can affect cell-regulatory pathways. In the present study, we have investigated the ability of HOCl, glycine chloramine (Gly-Cl) and taurine chloramine (Tau-Cl) to oxidize IkappaBalpha, the inhibitor of NF-kappaB (nuclear factor kappaB), and to prevent activation of the NF-kappaB pathway in Jurkat cells. Glycine chloramine (Gly-Cl) and HOCl were permeable to the cells as determined by oxidation of intracellular GSH and inactivation of glyceraldehyde-3-phosphate dehydrogenase, whereas Tau-Cl showed no detectable cell permeability. Both Gly-Cl (20-200 muM) and HOCl (50 microM) caused oxidation of IkappaBalpha methionine, measured by a shift in electrophoretic mobility, when added to the cells in Hanks buffer. In contrast, a high concentration of Tau-Cl (1 mM) in Hanks buffer had no effect. However, Tau-Cl in full medium did modify IkappaBalpha. This we attribute to chlorine exchange with other amines in the medium to form more permeable chloramines. Oxidation by Gly-Cl prevented IkappaBalpha degradation in cells treated with TNFalpha (tumour necrosis factor alpha) and inhibited nuclear translocation of NF-kappaB. IkappaBalpha modification was reversed by methionine sulphoxide reductase, with both A and B forms required for complete reduction. Oxidized IkappaBalpha persisted intracellularly for up to 6 h. Reversion occurred in the presence of cycloheximide, but was prevented if thioredoxin reductase was inhibited, suggesting that it was due to endogenous methionine sulphoxide reductase activity. These results show that cell-permeable chloramines, either directly or when formed in medium, could regulate NF-kappaB activation via reversible IkappaBalpha oxidation.     

Protein thiol oxidation and formation of S-glutathionylated cyclophilin A in cells exposed to chloramines and hypochlorous acid

Neutrophil oxidants, including the myeloperoxidase products, HOCl and chloramines, have been linked to endothelial dysfunction in inflammatory diseases such as atherosclerosis. As they react preferentially with sulfur centers, thiol proteins are likely to be cellular targets. Our objectives were to establish whether there is selective protein oxidation in vascular endothelial cells treated with HOCl or chloramines, and to identify sensitive proteins. Cells were treated with HOCl, glycine chloramine and monochloramine, reversibly oxidized cysteines were labeled and separated by 1D or 2D SDS-PAGE, and proteins were characterized by mass spectrometry. Selective protein oxidation was observed, with chloramines and HOCl causing more changes than H(2)O(2). Cyclophilin A was one of the most sensitive targets, particularly with glycine chloramine. Cyclophilin A was also oxidized in Jurkat T cells where its identity was confirmed using a knockout cell line. The product was a mixed disulfide with glutathione, with glutathionylation at Cys-161. Glyceraldehyde-3-phosphate dehydrogenase, peroxiredoxins and cofilin were also highly sensitive to HOCl/chloramines. Cyclophilins are becoming recognized as redox regulatory proteins, and glutathionylation is an important mechanism for redox regulation. Cells lacking Cyclophilin A showed more glutathionylation of other proteins than wild-type cells, suggesting that cyclophilin-regulated deglutathionylation could contribute to redox changes in HOCl/chloramine-exposed cells.     

Chlorine transfer between glycine, taurine, and histamine: reaction rates and impact on cellular reactivity

Hypochlorous acid formed by activated neutrophils reacts with amines to produce chloramines. Chloramines vary in stability, reactivity, and cell permeability. We have examined whether chloramine exchange occurs between physiologically important amines or amino acids and if this affects interactions of chloramines with cells. We have demonstrated transchlorination reactions between histamine, glycine, and taurine chloramines by measuring chloramine decay rates with mixtures as well as by mass spectrometry. Kinetic analysis suggested the formation of an intermediate complex with a high K(m). Apparent second-order rate constants, determined for concentrations 相似文献   

Chlorine transfer between glycine, taurine, and histamine: reaction rates and impact on cellular reactivity

Hypochlorous acid formed by activated neutrophils reacts with amines to produce chloramines. Chloramines vary in stability, reactivity, and cell permeability. We have examined whether chloramine exchange occurs between physiologically important amines or amino acids and if this affects interactions of chloramines with cells. We have demonstrated transchlorination reactions between histamine, glycine, and taurine chloramines by measuring chloramine decay rates with mixtures as well as by mass spectrometry. Kinetic analysis suggested the formation of an intermediate complex with a high Km. Apparent second-order rate constants, determined for concentrations 相似文献   

Oxidation of NADH by chloramines and chloramides and its activation by iodide and by tertiary amines

Irreversible oxidation of reduced nicotinamide nucleotides by neutrophil-derived halogen oxidants (HOCl, chloramines, HOBr, etc.) is likely to be a highly lethal process, because of the essential role of NAD(P)H in important cell functions such as mitochondrial electron transport, and control of the cellular thiol redox state by NADPH-dependent glutathione reductase. Chloramines (chloramine-T, NH(2)Cl, etc.) and N-chloramides (N-chlorinated cyclopeptides) react with NADH to generate the same products as HOCl, i.e., pyridine chlorohydrins, as judged from characteristic changes in the NADH absorption spectrum. Compared with the fast oxidation of NADH by HOCl, k approximately 3 x 10(5) M(-1) s(-1) at pH 7.2, the oxidation by chloramines is about five orders of magnitude slower; that by chloramides is about four orders of magnitude slower. Apparent rate constants for oxidation of NADH by chloramines increase with increasing proton or buffer concentration, consistent with general acid catalysis, but oxidation by chloramides proceeds with pH-independent kinetics. In presence of iodide the oxidation of NADH by chloramines or chloramides is faster by at least two orders of magnitude; this is due to reaction of iodide with the N-halogen to give HOI/I(2), the most reactive and selective oxidant for NADH among HOX species. Quinuclidine derivatives (QN) like 3-chloroquinuclidine and quinine are capable of catalyzing the irreversible degradation of NADH by HOCl and by chloramines; QN(+)Cl, the chain carrier of the catalytic cycle, is even more reactive toward NADH than HOCl/ClO(-) at physiological pH. Oxidation of NADH by NH(2)Br proceeds by fast, but complex, biphasic kinetics. A compilation of rate constants for interactions of reactive halogen species with various substrates is presented and the concept of selective reactivity of N-halogens is discussed.     

On the irreversible destruction of reduced nicotinamide nucleotides by hypohalous acids

Degradation of the reduced pyridine nucleotides NMNH and NADH by HOCl involves two distinct stages: a fast reaction, k = 4.2 x 10(5) M(-1) s(-1), leads to generation of stable pyridine products (Py/Cl) with a strong absorption band at 275 nm (epsilon = 12.4 x 10(3) M(-1) cm(-1) in the case of NMNH); secondarily, a subsequent reaction of HOCl, k = 3.9 x 10(3) M(-1) s(-1), leads to a complete loss of the aromatic absorption band of the pyridine ring. HOBr and HOI(I(2)) react similarly. Apparent rate constants of the primary reactions of HOX species with NMNH at pH 7.2 increase in the order HOCl (3 x 10(5) M(-1) s(-1)) < HOBr( approximately 4 x 10(6) M(-1) s(-1)) < HOI(I(2))( approximately 6.5 x 10(7) M(-1) s(-1)). HOBr reacts fast also with the primary product Py/Br, k approximately 9 x 10(5) M(-1) s(-1), while the reactions of HOI and I(2) with Py/I are slower, approximately 1.4 x 10(3) M(-1) s(-1) and >6 x 10(3) M(-1) s(-1), respectively. Halogenation of the amide group of NMN(+) by HOX species is many orders of magnitude slower than oxidation of NMNH. Taurine inhibits HOCl-induced oxidation of NADH, but HOBr-induced oxidation is not inhibited because the taurine monobromamine rapidly oxidizes NADH, and oxidation by HOI(I(2)) is not inhibited because taurine is inert toward HOI(I(2)). Also sulfur compounds (GSH, GSSG, and methionine) are less efficient in protecting NADH against oxidation by HOBr and HOI(I(2)) than against oxidation by HOCl. The results suggest that reactions of HOBr and HOI(I(2)) in a cellular environment are much more selectively directed toward irreversible oxidation of NADH than reactions of HOCl. It is noteworthy that the rather inert N-chloramines react with iodide to generate HOI(I(2)), i.e., the most reactive and selective oxidant of reduced pyridine nucleotides. NMR investigations show that the primary stable products of the reaction between NMNH and HOCl are various isomeric chlorohydrins originating from a nonstereospecific electrophilic addition of HOCl to the C5&dbond;C6 double bond of the pyridine ring. The primary products (Py/X) of NMNH all exhibit similar absorption bands around 275 nm and are hence likely to result from analogous addition of HOX to the C5&dbond;C6 bond of the pyridine ring. Since the Py/X species are stable and inert toward endogeneous reductants like ascorbate and GSH, they may generally be useful markers for assessing the contribution of hypohalous acids to inflammatory injury.     

Chloramines and hypochlorous acid oxidize erythrocyte peroxiredoxin 2

Peroxiredoxin 2 (Prx2) is an abundant thiol protein that is readily oxidized in erythrocytes exposed to hydrogen peroxide. We investigated its reactivity in human erythrocytes with hypochlorous acid (HOCl) and chloramines, relevant oxidants in inflammation. Prx2 was oxidized to a disulfide-linked dimer by HOCl, glycine chloramine (GlyCl), and monochloramine (NH₂Cl) in a dose-dependent manner. In the absence of added glucose, Prx2 and GSH showed similar sensitivities. Second-order rate constants for the reactions of Prx2 with NH₂Cl and GlyCl were 1.5 × 10⁴ and 8 M⁻¹ s⁻¹, respectively. The NH₂Cl value is 10 times higher than that for GSH, whereas Prx2 is 30 times less sensitive than GSH to GlyCl. Thus, the relative sensitivity of Prx2 to GlyCl is greater in the erythrocyte. Oxidation of erythrocyte Prx2 and GSH was less in the presence of glucose, probably because of recycling. High doses of NH₂Cl resulted in incomplete regeneration of reduced Prx2, suggesting impairment of the recycling mechanism. Our results show that, although HOCl and chloramines are less selective than H₂O₂, they nevertheless oxidize Prx2. Exposure to these inflammatory oxidants will result in Prx2 oxidation and could compromise the erythrocyte's ability to resist damaging oxidative insult.     

The vinyl ether linkages of plasmalogens are favored targets for myeloperoxidase-derived oxidants: a kinetic study

Plasmalogens, which contain a vinyl ether bond, are major phospholipids of the plasma membranes of endothelial and vascular smooth muscle cells and cardiac myocytes. These lipids, in contrast to other phospholipids, have been reported to be targets of HOCl/HOBr generated by myeloperoxidase, with elevated levels of the products of these reactions (alpha-chloro/alpha-bromo aldehydes and unsaturated lysophospholipids) having been detected in human atherosclerotic lesions. The reason(s) for the targeting of this lipid class, over other phospholipids, is poorly understood, and is examined here. It is shown that HOCl and HOBr react with a model vinyl ether (ethylene glycol vinyl ether) 200-300-fold faster ( k = 1.6 x 10 (3) and 3.5 x 10 (6) M (-1) s (-1), respectively) than with aliphatic alkenes (models of phospholipids). True plasmalogens react ca. 20-fold slower than the models. Chloramines and bromamines (from reaction of HOCl/HOBr with primary amines and alpha-amino groups) also react with vinyl ethers, unlike aliphatic alkenes, with k = 10 (-3)-10 (2) M (-1) s (-1) for chloramines (with the His side chain chloramine being the most reactive, k = 172 M (-1) s (-1)) and k = 10 (3)-10 (4) M (-1) s (-1) for bromamines. The bromamine rate constants are typically 10 (5)-10 (6) larger than those of the chloramines. Intermolecular vinyl ether oxidation by phospholipid headgroup bromamines can also occur. These kinetic data indicate that plasmalogens are significantly more susceptible to oxidation than the aliphatic alkenes of phospholipids, thereby rationalizing the detection of products from the former, but not the latter, in human atherosclerotic lesions.     

Glutaredoxin 2 catalyzes the reversible oxidation and glutathionylation of mitochondrial membrane thiol proteins: implications for mitochondrial redox regulation and antioxidant DEFENSE

The redox poise of the mitochondrial glutathione pool is central in the response of mitochondria to oxidative damage and redox signaling, but the mechanisms are uncertain. One possibility is that the oxidation of glutathione (GSH) to glutathione disulfide (GSSG) and the consequent change in the GSH/GSSG ratio causes protein thiols to change their redox state, enabling protein function to respond reversibly to redox signals and oxidative damage. However, little is known about the interplay between the mitochondrial glutathione pool and protein thiols. Therefore we investigated how physiological GSH/GSSG ratios affected the redox state of mitochondrial membrane protein thiols. Exposure to oxidized GSH/GSSG ratios led to the reversible oxidation of reactive protein thiols by thiol-disulfide exchange, the extent of which was dependent on the GSH/GSSG ratio. There was an initial rapid phase of protein thiol oxidation, followed by gradual oxidation over 30 min. A large number of mitochondrial proteins contain reactive thiols and most of these formed intraprotein disulfides upon oxidation by GSSG; however, a small number formed persistent mixed disulfides with glutathione. Both protein disulfide formation and glutathionylation were catalyzed by the mitochondrial thiol transferase glutaredoxin 2 (Grx2), as were protein deglutathionylation and the reduction of protein disulfides by GSH. Complex I was the most prominent protein that was persistently glutathionylated by GSSG in the presence of Grx2. Maintenance of complex I with an oxidized GSH/GSSG ratio led to a dramatic loss of activity, suggesting that oxidation of the mitochondrial glutathione pool may contribute to the selective complex I inactivation seen in Parkinson's disease. Most significantly, Grx2 catalyzed reversible protein glutathionylation/deglutathionylation over a wide range of GSH/GSSG ratios, from the reduced levels accessible under redox signaling to oxidized ratios only found under severe oxidative stress. Our findings indicate that Grx2 plays a central role in the response of mitochondria to both redox signals and oxidative stress by facilitating the interplay between the mitochondrial glutathione pool and protein thiols.     

Radiosensitization, thiol oxidation, and inhibition of DNA repair by SR 4077

The mechanism of radiosensitization by diazenedicarboxylic acid bis(N),N-piperidide (SR 4077), a less toxic analog of diamide, was studied using Chinese hamster ovary cells. SR 4077 gave an average SER of 1.58 for postirradiation incubations of 0.5, 1.0, or 2.0 h. Intracellular GSH and protein thiols decreased rapidly following drug addition and GSSG increased. The GSH/GSSG ratio shifted to 1/1.6 after SR 4077 addition but returned to greater than 10/1 between 0.5 and 1.0 h. After 4 h, total intracellular GSH was only 58% of pretreatment level and extracellular GSSG increased. Protein thiols decreased to 18% of pretreatment values, recovered most rapidly between 0.5 and 1.0 h, and reached 87% of pretreatment level after 4 h. A decrease in DNA single-strand break repair as measured by alkaline filter elution rate over 0.5 h was seen, and the initial rate of repair was slower than in cells not treated with SR 4077. DNA double-strand break repair as measured by neutral filter elution rate was delayed during the first hour after irradiation when cells were treated with SR 4077. The times for maximum radiosensitization, GSH and protein thiol oxidation and recovery, and DNA strand break repair kinetics were closely linked. We propose that a protein thiol(s) required in repair processes was reversibly oxidized during SR 4077 treatment.     

Hypochlorous Acid Converts the ��-Glutamyl Group of Glutathione Disulfide to 5-Hydroxybutyrolactam, a Potential Marker for Neutrophil Activation

In healthy cells, glutathione disulfide (GSSG) is rapidly reduced back to glutathione (GSH) by glutathione reductase to maintain redox status. The ratio of GSH/GSSG has been used as an indicator of oxidative stress. However, hypochlorous acid (HOCl) generated by the myeloperoxidase-H₂O₂-Cl⁻ system of neutrophils converts GSH to irreversible oxidation products. Although several such products have been identified, yields of these compounds are very low in biological systems, and they cannot account quantitatively for thiol loss. In the current studies, we use liquid chromatography-mass spectrometry (LC-MS) to demonstrate that HOCl and chloramines oxidize GSSG to two irreversible products in high yield. The products, termed M-45 and M-90, are, respectively, 45 or 90 atomic mass units lighter than GSSG. The reaction pathway involves chloramine and aldehyde intermediates, and converts the γ-glutamyl residues of GSSG to 5-hydroxybutyrolactam. Importantly, M-45 and M-90 were resistant to reduction by glutathione reductase. Moreover, the monohydroxylbutyrolactam M-45 accounted for >90% of the endogenous GSH oxidation products generated by activated neutrophils. Because the reaction pathway involves chlorinating intermediates, hydroxylbutyrolactams are likely to be specific products of HOCl, which is generated only by myeloperoxidase. Therefore, our observations implicate M-45 as a potential biomarker for myeloperoxidase activity in vivo.Glutathione (GSH), a tripeptide synthesized in the cytosol from glutamate, cysteine, and glycine, is the predominant antioxidant in mammalian cells. Its concentration ranges from millimolar inside cells to micromolar in plasma (1, 2). In many cells, GSH accounts for >90% of total nonprotein thiol (3, 4). The free thiol group in GSH is responsible for biological activity. As a nucleophilic scavenger, GSH can directly react with electrophilic substances, such as reactive oxygen/nitrogen species, or be oxidized by GSH peroxidase to glutathione disulfide (GSSG). Therefore, it is essential for maintaining intracellular redox status and defending against oxidative injury. Under normal circumstances, GSSG is rapidly reduced back to GSH by glutathione reductase and NADPH. Thus, most of the GSH remains in the reduced form. Under oxidative stress, however, GSH is converted to GSSG, which potentially accumulates (2, 5). Indeed, the GSH/GSSG ratio has been used to evaluate oxidative stress in biological systems. Alterations of this ratio associate with a variety of diseases, including atherosclerosis, cancer, and human immunodeficiency virus infection (6–10).One important source of oxidative stress in humans is myeloperoxidase (MPO),² a heme protein expressed by neutrophils, monocytes, and certain populations of macrophages (11–13). Activation of these inflammatory white blood cells results in the secretion of MPO, which uses hydrogen peroxide (H₂O₂, produced by NADPH oxidase) and chloride anion to generate hypochlorous acid (HOCl) (14). HOCl rapidly reacts with a wide range of functional groups (15–19). At physiological pH, thiol groups and free amino groups are its main targets, and the initial products are oxidized thiols and chloramines.HOCl generates other products in addition to GSSG when it reacts with GSH. Chesney et al. (20) suggested that it oxidizes GSH to a higher oxidation state than the disulfide form because the molar ratio of HOCl consumed to GSH oxidized was 4:1 instead of 1:1 in Escherichia coli. Winterbourn (21) reported that approximately half of the GSH oxidized by HOCl could not be regenerated. These researchers have identified glutathione sulfonamide (GSA), glutathione thiosulfonate, and dehydroglutathione as irreversible higher oxidation products (22, 23). Their observations suggest that the formation of higher order GSSG oxidation products might account in part for the irreversible loss of GSH induced by HOCl. However, activated neutrophils (the source of MPO and therefore of HOCl) generate only low yields of these higher oxidation products, suggesting that the major products of GSH oxidation by MPO remain to be identified.The disulfide and α-amino groups of GSSG are also potential targets of HOCl (17). Disulfides can be oxidized to sulfonic acid via a sulfenyl chloride intermediate (16). α-Amino groups yield chloramines, which undergo decarboxylation, intramolecular H-abstraction, or other reaction pathways to form various products, such as aldehydes and carboxymethyllysine (16, 24). These reactions may be biologically relevant, because carboxymethyllysine production is impaired in mice deficient in the phagocyte NADPH oxidase (25). These observations suggest that GSSG is a potential scavenger of HOCl. Indeed, GSSG reportedly competes for HOCl with its rate constant expected to be 2 × 10⁵ m⁻¹ s⁻¹ (26, 27). Studies from Bast et al. (28) demonstrated that GSSG protects acetylcholinesterase from oxidative inactivation by HOCl. Nagy and Ashby (29) studied the kinetics and mechanism of GSSG oxidation by HOCl. They proposed that HOCl generates the bis-N-chloro-γ-l-glutamyl derivative of GSSG. These studies suggest that GSSG itself may function as an antioxidant.In the current study, we investigated the reaction of GSSG with HOCl and other oxidants. Using liquid chromatography in concert with mass spectrometry (LC-MS), we identified two groups of novel oxidation products, which we termed M-45 and M-90. We characterized their structures and potential reaction pathways. Our results indicate that HOCl and chloramines oxidize the γ-glutamyl moiety of GSSG to 5-hydroxybutyrolactam in high yield.     

Human neutrophils oxidize low-density lipoprotein by a hypochlorous acid-dependent mechanism: the role of vitamin C

Oxidatively modified low-density lipoprotein (LDL) has been strongly implicated in the pathogenesis of atherosclerosis. Peripheral blood leukocytes, such as neutrophils, can oxidize LDL by processes requiring superoxide and redox-active transition metal ions; however, it is uncertain whether such catalytic metal ions are available in the artery wall. Stimulated leukocytes also produce the reactive oxidant hypochlorous acid (HOCl) via the heme enzyme myeloperoxidase. Since myeloperoxidase-derived HOCl may be a physiologically relevant oxidant in atherogenesis, we investigated the mechanisms of neutrophil-mediated LDL modification and its possible prevention by the antioxidant ascorbate (vitamin C). As a sensitive marker of LDL oxidation, we measured LDL thiol groups. Stimulated human neutrophils (5x10(6) cells/ml) incubated with human LDL (0.25 mg protein/ml) time-dependently oxidized LDL thiols (33% and 79% oxidized after 10 and 30 min, respectively). Supernatants from stimulated neutrophils also oxidized LDL thiols (33% oxidized after 30 min), implicating long-lived oxidants such as N-chloramines. Experiments using specific enzyme inhibitors and oxidant scavengers showed that HOCl, but not hydrogen peroxide nor superoxide, plays a critical role in LDL thiol oxidation by neutrophils. Ascorbate (200 microM) protected against neutrophil-mediated LDL thiol oxidation for up to 15 min of incubation, after which LDL thiols became rapidly oxidized. Although stimulated neutrophils accumulated ascorbate during oxidation of LDL, pre-loading of neutrophils with ascorbate did not attenuate oxidant production by the cells. Thus, activated neutrophils oxidize LDL thiols by HOCl- and N-chloramine-dependent mechanisms and physiological concentrations of vitamin C delay this process, most likely due to scavenging of extracellular oxidants, rather than by attenuating neutrophil oxidant production.     

Role of ascorbate in oxidative protein folding

Both in prokaryotic and eukaryotic cells, disulfide bond formation (oxidation and isomerization steps) are catalyzed exclusively in extracytoplasmic compartments. In eukaryotes, protein folding and disulfide bond formation are coupled processes that occur both co- and posttranslationally in the endoplasmic reticulum (ER), which is the main site of the synthesis and posttranslational modification of secretory and membrane proteins. The formation of a disulfide bond from the thiol groups of two cysteine residues requires the removal of two electrons, consequently, these bonds cannot form spontaneously; an oxidant is needed to accept the electrons. In aerobic conditions the ultimate electron acceptor is usually oxygen; however, oxygen itself is not effective in protein thiol oxidation. Therefore, a small molecular weight membrane permeable compound should be supposed for the transfer of electrons from the ER lumen. The aim of the present study was the investigation of the role of ascorbate/dehydroascorbate redox couple in oxidative folding of proteins. We demonstrated that ascorbate addition or its in situ synthesis from gulonolactone results in protein thiol (and/or glutathione; GSH) oxidation in rat liver microsomes. Since microsomal membrane is hardly permeable to ascorbate, the existence of a transport metabolon was hypothesized. Three components of the system have been described and partially characterized: (i) A microsomal metalloenzyme is responsible for ascorbate oxidation on the outer surface of the ER. Ascorbate oxidation results in ascorbate free radical and dehydroascorbate production. (ii) Facilitated diffusion of dehydroascorbate is present in microsomal vesicles. The transport is presumably mediated by a GLUT-type transporter. On the contrary, the previously hypothesized glutathione disulfide (GSSG) transport is practically absent, while GSH is transported with a moderate velocity. (iii) Protein disulfide isomerase catalyzes the reduction of dehydroascorbate in the ER lumen. Both GSH and protein thiols can be electron donors in the process. Intraluminal dehydroascorbate reduction and the consequent ascorbate accumulation strictly correlate with protein disulfide isomerase activity and protein thiol concentration. The concerted action of the three components of the system results in the intraluminal accumulation of ascorbate, protein disulfide and GSSG. In fact, intraluminal ascorbate and GSSG accumulation could be observed upon dehydroascorbate and GSH uptake. In conclusion, ascorbate is able to promote protein disulfide formation in an in vitro system. Further work is needed to justify its role in intact cellular and in vivo systems, as well as to explore the participation of other antioxidants (e.g. tocopherol, ubiquinone, and vitamin K) in the electron transfer chain responsible for oxidative protein folding in the ER.     

Hypochlorite-induced oxidation of thiols: Formation of thiyl radicals and the role of sulfenyl chlorides as intermediates

Activated phagocytic cells generate hypochlorite (HOCl) via release of hydrogen peroxide and the enzyme myeloperoxidase. HOCl plays an important role in bacterial cell killing, but excessive or misplaced production of HOCl is also known to cause tissue damage. Studies have shown that low-molecular-weight thiols such as reduced glutathione (GSH), and sulfur-containing amino acids in proteins, are major targets for HOCl. Radicals have not generally been implicated as intermediates in thiol oxidation by HOCl, though there is considerable literature evidence for the involvement of radicals in the metal ion-, thermal- or UV light-catalysed decomposition of sulfenyl or sulfonyl chlorides which are postulated intermediates in thiol oxidation. In this study we show that thiyl radicals are generated on reaction of a number of low-molecular-weight thiols with HOCl. With sub-stoichiometric amounts of HOCl, relative to the thiol, thiyl radicals are the major species detected by EPR spin trapping. When the HOCl is present in excess over the thiol, additional radicals are detected with compounds which contain amine functions; these additional radicals are assigned to nitrogen-centered species. Evidence is presented for the involvement of sulfenyl chlorides (RSCl) in the formation of these radicals, and studies with an authentic sulfenyl chloride have demonstrated that this compound readily decomposes in thermal-, metal-ion- or light-catalysed reactions to give thiyl radicals. The formation of thiyl radicals on oxidation of thiols with HOCl appears to compete with non-radical reactions. The circumstances under which radical formation may be important are discussed.     

Hypochlorite-induced oxidation of thiols: formation of thiyl radicals and the role of sulfenyl chlorides as intermediates

Activated phagocytic cells generate hypochlorite (HOCl) via release of hydrogen peroxide and the enzyme myeloperoxidase. HOCl plays an important role in bacterial cell killing, but excessive or misplaced production of HOCl is also known to cause tissue damage. Studies have shown that low-molecular-weight thiols such as reduced glutathione (GSH), and sulfur-containing amino acids in proteins, are major targets for HOCl. Radicals have not generally been implicated as intermediates in thiol oxidation by HOCl, though there is considerable literature evidence for the involvement of radicals in the metal ion-, thermal- or UV light-catalysed decomposition of sulfenyl or sulfonyl chlorides which are postulated intermediates in thiol oxidation. In this study we show that thiyl radicals are generated on reaction of a number of low-molecular-weight thiols with HOCl. With sub-stoichiometric amounts of HOCl, relative to the thiol, thiyl radicals are the major species detected by EPR spin trapping. When the HOCl is present in excess over the thiol, additional radicals are detected with compounds which contain amine functions; these additional radicals are assigned to nitrogen-centered species. Evidence is presented for the involvement of sulfenyl chlorides (RSCl) in the formation of these radicals, and studies with an authentic sulfenyl chloride have demonstrated that this compound readily decomposes in thermal-, metal-ion- or light-catalysed reactions to give thiyl radicals. The formation of thiyl radicals on oxidation of thiols with HOCl appears to compete with non-radical reactions. The circumstances under which radical formation may be important are discussed.