Myeloperoxidase-derived oxidants inhibit sarco/endoplasmic reticulum Ca2+-ATPase activity and perturb Ca2+ homeostasis in human coronary artery endothelial cells

The sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) plays a critical role in Ca(2+) homeostasis via sequestration of this ion in the sarco/endoplasmic reticulum. The activity of this pump is inhibited by oxidants and impaired in aging tissues and cardiovascular disease. We have shown previously that the myeloperoxidase (MPO)-derived oxidants HOCl and HOSCN target thiols and mediate cellular dysfunction. As SERCA contains Cys residues critical to ATPase activity, we hypothesized that HOCl and HOSCN might inhibit SERCA activity, via thiol oxidation, and increase cytosolic Ca(2+) levels in human coronary artery endothelial cells (HCAEC). Exposure of sarcoplasmic reticulum vesicles to preformed or enzymatically generated HOCl and HOSCN resulted in a concentration-dependent decrease in ATPase activity; this was also inhibited by the SERCA inhibitor thapsigargin. Decomposed HOSCN and incomplete MPO enzyme systems did not decrease activity. Loss of ATPase activity occurred concurrent with oxidation of SERCA Cys residues and protein modification. Exposure of HCAEC, with or without external Ca(2+), to HOSCN or HOCl resulted in a time- and concentration-dependent increase in intracellular Ca(2+) under conditions that did not result in immediate loss of cell viability. Thapsigargin, but not inhibitors of plasma membrane or mitochondrial Ca(2+) pumps/channels, completely attenuated the increase in intracellular Ca(2+) consistent with a critical role for SERCA in maintaining endothelial cell Ca(2+) homeostasis. Angiotensin II pretreatment potentiated the effect of HOSCN at low concentrations. MPO-mediated modulation of intracellular Ca(2+) levels may exacerbate endothelial dysfunction, a key early event in atherosclerosis, and be more marked in smokers because of their higher SCN(-) levels.     

Reactions and reactivity of myeloperoxidase-derived oxidants: differential biological effects of hypochlorous and hypothiocyanous acids

Myeloperoxidase (MPO) is recognised to play important roles both in the immune system and during the development of numerous human pathologies. MPO is released by activated neutrophils, monocytes and some tissue macrophages, where it catalyses the conversion of hydrogen peroxide to hypohalous acids (HOX; X = Cl, Br, SCN) in the presence of halide and pseudo-halide ions. The major reactive species produced by MPO under physiological conditions are hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN), with the ratio of these oxidants critically dependent on the concentration of thiocyanate ions (SCN?). The reactivity and selectivity of HOCl and HOSCN for biological targets are markedly different, indicating that SCN? ions have the potential to modulate both the extent and nature of oxidative damage in vivo. This article reviews recent developments in our understanding of the role of SCN? in modulating the formation of MPO-derived oxidants, particularly in respect to the differences in reaction kinetics and targets of HOCl compared to HOSCN and the ability of these two oxidants to induce damage in biological systems.     

Inactivation of thiol-dependent enzymes by hypothiocyanous acid: role of sulfenyl thiocyanate and sulfenic acid intermediates

Myeloperoxidase (MPO) forms reactive oxidants including hypochlorous and hypothiocyanous acids (HOCl and HOSCN) under inflammatory conditions. HOCl causes extensive tissue damage and plays a role in the progression of many inflammatory-based diseases. Although HOSCN is a major MPO oxidant, particularly in smokers, who have elevated plasma thiocyanate, the role of this oxidant in disease is poorly characterized. HOSCN induces cellular damage by targeting thiols. However, the specific targets and mechanisms involved in this process are not well defined. We show that exposure of macrophages to HOSCN results in the inactivation of intracellular enzymes, including creatine kinase (CK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In each case, the active-site thiol residue is particularly sensitive to oxidation, with evidence for reversible inactivation and the formation of sulfenyl thiocyanate and sulfenic acid intermediates, on treatment with HOSCN (less than fivefold molar excess). Experiments with DAz-2, a cell-permeable chemical trap for sulfenic acids, demonstrate that these intermediates are formed on many cellular proteins, including GAPDH and CK, in macrophages exposed to HOSCN. This is the first direct evidence for the formation of protein sulfenic acids in HOSCN-treated cells and highlights the potential of this oxidant to perturb redox signaling processes.     

Modification of tryptophan and tryptophan residues in proteins by reactive nitrogen species.

Formation of 3-nitrotyrosine by the reaction between reactive nitrogen species (RNS) and tyrosine residues in proteins has been analyzed extensively and it is used widely as a biomarker of pathophysiological and physiological conditions mediated by RNS. In contrast, few studies on the nitration of tryptophan have been reported. This review provides an overview of the studies on tryptophan modifications by RNS and points out the possible importance of its modification in pathophysiological and physiological conditions. Free tryptophan can be modified to several nitrated products (1-, 4-, 5-, 6-, and 7-), 1-N-nitroso product, and several oxidized products by reaction with various RNS, depending on the conditions used. Among them, 1-N-nitrosotryptophan and 6-nitrotryptophan (6-NO(2)Trp) have been found as the abundant products in the reaction with peroxynitrite, and 6-NO(2)Trp has been the most abundant product in the reaction with the peroxidase/hydrogen peroxide/nitrite systems. 6-NO(2)Trp has also been observed as the most abundant nitrated product of the reactions between peroxynitrite or myeloperoxidase/hydrogen peroxide/nitrite and tryptophan residues both in human Cu,Zn-superoxide dismutase and in bovine serum albumin, as well as the reaction of peroxynitrite with myoglobin and hemoglobin. Several oxidized products have also been identified in the modified Cu,Zn-SOD. However, no 1-N-nitrosotryptophan and 1-N-nitrotryptophan has been observed in the proteins reacted with peroxynitrite or the myeloperoxidase/H(2)O(2)/nitrite system. The modification of tryptophan residues in proteins may occur at a more limited number of sites in vivo than that of tyrosine residues, since tryptophan residues are more buried inside proteins and exist less frequently in proteins, generally. However, surface-exposed tryptophan residues tend to participate in the interaction with the other molecules, therefore the modification of those tryptophans may result in modulation of the specific interaction of proteins and enzymes with other molecules.     

Selenium-containing amino acids are targets for myeloperoxidase-derived hypothiocyanous acid: determination of absolute rate constants and implications for biological damage

Elevated MPO (myeloperoxidase) levels are associated with multiple human inflammatory pathologies. MPO catalyses the oxidation of Cl-, Br- and SCN- by H2O2 to generate the powerful oxidants hypochlorous acid (HOCl), hypobromous acid (HOBr) and hypothiocyanous acid (HOSCN) respectively. These species are antibacterial agents, but misplaced or excessive production is implicated in tissue damage at sites of inflammation. Unlike HOCl and HOBr, which react with multiple targets, HOSCN targets cysteine residues with considerable selectivity. In the light of this reactivity, we hypothesized that Sec (selenocysteine) residues should also be rapidly oxidized by HOSCN, as selenium atoms are better nucleophiles than sulfur. Such oxidation might inactivate critical Sec-containing cellular protective enzymes such as GPx (glutathione peroxidase) and TrxR (thioredoxin reductase). Stopped-flow kinetic studies indicate that seleno-compounds react rapidly with HOSCN with rate constants, k, in the range 2.8×10(3)-5.8×10(6) M-1·s-1 (for selenomethionine and selenocystamine respectively). These values are ~6000-fold higher than the corresponding values for H2O2, and are also considerably larger than for the reaction of HOSCN with thiols (16-fold for cysteine and 80-fold for selenocystamine). Enzyme studies indicate that GPx and TrxR, but not glutathione reductase, are inactivated by HOSCN in a concentration-dependent manner; k for GPx has been determined as ~5×105 M-1·s-1. Decomposed HOSCN did not induce inactivation. These data indicate that selenocysteine residues are oxidized rapidly by HOSCN, with this resulting in the inhibition of the critical intracellular Sec-dependent protective enzymes GPx and TrxR.     

The role of hypothiocyanous acid (HOSCN) in biological systems

Abstract

Hypohalous acids (HOX), produced by peroxidase-catalysed reactions of halide and pseudohalide ions with H₂O₂, play an important role in the human immune system. However, there is compelling evidence that these oxidants also mediate host tissue damage and contribute to the progression of a number of inflammatory diseases. Although it is well established that significant amounts of hypothiocyanous acid (HOSCN) are formed under physiological conditions, the reactions of this oxidant with host biological systems are relatively poorly characterized. It is generally accepted that HOSCN is a mild oxidant that reacts selectively with thiols. However, it is becoming increasingly recognized that this selectivity can result in the induction of significant cellular damage, which may contribute to disease. This review will outline the formation and reactivity of HOSCN and the role of this oxidant in biological systems.     

Taurine bromamine: a potent oxidant of tryptophan residues in albumin

Taurine is the most abundant free amino acid in leukocytes and can react with HOBr to produce taurine bromamine (Tau-NHBr). The aim of this study was to assess the ability of Tau-NHBr to oxidize tryptophan, either free or as a residue in albumin. We have demonstrated that Tau-NHBr is a powerful oxidant for tryptophan. Importantly, in comparison to taurine chloramine, HOCl or HOBr, Tau-NHBr exhibits a degree of selectivity for tryptophan. Oxidation of albumin by Tau-NHBr resulted in emission of light, and the quantum yield was more than 10-fold more efficient than that of the other oxidants. The fluorescence band corresponding to oxidized albumin (λ_ex 350/λ_em 450), which is characteristic of the formation of formylkynurenine, was significantly higher in reactions using Tau-NHBr. Excitation of the fluorescent probe 8-anilino-1-naphthalenesulfonate at 295 nm was used to assess the depletion of tryptophan residues in albumin. Results from this experiment further supported a higher efficiency of oxidation of tryptophan residues by Tau-NHBr. Other parameters of protein oxidation, including cysteine depletion and formation of carbonyl groups, were not significantly different between the oxidants tested. In conclusion, these results indicate that Tau-NHBr has a higher affinity for tryptophan residues in proteins.     

Probing the free radicals formed in the metmyoglobin-hydrogen peroxide reaction

The reaction between metmyoglobin and hydrogen peroxide results in the two-electron reduction of H2O2 by the protein, with concomitant formation of a ferryl-oxo heme and a protein-centered free radical. Sperm whale metmyoglobin, which contains three tyrosine residues (Tyr-103, Tyr-146, and Tyr-151) and two tryptophan residues (Trp-7 and Trp-14), forms a tryptophanyl radical at residue 14 that reacts with O2 to form a peroxyl radical and also forms distinct tyrosyl radicals at Tyr-103 and Tyr-151. Horse metmyoglobin, which lacks Tyr-151 of the sperm whale protein, forms an oxygen-reactive tryptophanyl radical and also a phenoxyl radical at Tyr-103. Human metmyoglobin, in addition to the tyrosine and tryptophan radicals formed on horse metmyoglobin, also forms a Cys-110-centered thiyl radical that can also form a peroxyl radical. The tryptophanyl radicals react both with molecular oxygen and with the spin trap 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS). The spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) traps the Tyr-103 radicals and the Cys-110 thiyl radical of human myoglobin, and 2-methyl-2-nitrosopropane (MNP) traps all of the tyrosyl radicals. When excess H2O2 is used, DBNBS traps only a tyrosyl radical on horse myoglobin, but the detection of peroxyl radicals and the loss of tryptophan fluorescence support tryptophan oxidation under those conditions. Kinetic analysis of the formation of the various free radicals suggests that tryptophanyl radical and tyrosyl radical formation are independent events, and that formation of the Cys-110 thiyl radical on human myoglobin occurs via oxidation of the thiol group by the Tyr-103 phenoxyl radical. Peptide mapping studies of the radical adducts and direct EPR studies at low temperature and room temperature support the conclusions of the EPR spin trapping studies.     

A simple method of determination of antigenic determinants in proteins with known primary structure

A simple and rapid method is proposed for the localization of antigenic determinants in proteins of known primary structure exemplified by human myoglobin. The polypeptide chain of myoglobin was cleaved with BrCN (at Met residues) or with bromosuccinimide (at Trp and Tyr residues) under conditions which on average gave less than one scission per myoglobin molecule. The "single-hit" cleavage products were separated by gel electrophoresis and transferred to nitrocellulose by electroblotting. The peptides containing intact antigenic determinants were vizualized by immuno-peroxidase staining with four monoclonal anti-myoglobin antibodies. Comparison of the lengths of the immuno-reactive peptides with the known positions of methionine, tryptophan and tyrosine residues suggested that the four monoclonal antibodies were bound by myoglobin over the region Trp-14 to Met-55. As compared with other methods of localization, the method proposed is much faster and takes much lesser amount of protein.     

Hypochlorous acid oxidizes methionine and tryptophan residues in myoglobin

After acute myocardial infarction (AMI), infiltrating proinflammatory cells generate two-electron oxidants such as hypochlorous acid (HOCl). Myoglobin (Mb) is present at approximately 0.3 mM in cardiomyocytes and, therefore, represents a significant target for oxidation. Exposure of horse Mb (50 microM) to reagent HOCl (0-500 microM) or activated human neutrophils (4-40x10(6) cells/ml) yielded oxidized Mb (Mb(ox)) as judged by amino acid analysis and peptide mass mapping. HOCl/Mb ratios of 1-5 mol/mol gave Mb(ox) with up to four additional oxygen atoms. Hydrolysis of Mb(ox) followed by amino acid analysis indicated that methionine (Met) and tryptophan (Trp) residues were modified by HOCl. Peptide mass mapping revealed that Met55 was oxidized at a lower HOCl/Mb ratio than Met131 and this preceded Trp7/14 modification (susceptibility Met55>Met131>Trp7>Trp14). Incubation of Mb with activated neutrophils and physiological chloride anion yielded Mb(ox) with a composition similar to that determined with HOCl/Mb ratios <2 mol/mol, with oxidation of Met, but not Trp, detected. These data indicate that Mb undergoes site-specific oxidation depending on the HOCl/protein ratio. As Mb is released from necrotic cardiomyocytes into the vasculature after AMI, HOCl-modified Mb may be a useful surrogate marker to gauge the extent of myocardial inflammation.     

Thiol-oxidant monochloramine mobilizes intracellular Ca2+ in parietal cells of rabbit gastric glands

In Helicobacter pylori-induced gastritis, oxidants are generated through the interactions of bacteria in the lumen, activated granulocytes, and cells of the gastric mucosa. In this study we explored the ability of one such class of oxidants, represented by monochloramine (NH(2)Cl), to serve as agonists of Ca(2+) accumulation within the parietal cell of the gastric gland. Individual gastric glands isolated from rabbit mucosa were loaded with fluorescent reporters for Ca(2+) in the cytoplasm (fura-2 AM) or intracellular stores (mag-fura-2 AM). Conditions were adjusted to screen out contributions from metal cations such as Zn(2+), for which these reporters have affinity. Exposure to NH(2)Cl (up to 200 microM) led to dose-dependent increases in intracellular Ca(2+) concentration ([Ca(2+)](i)), in the range of 200-400 nM above baseline levels. These alterations were prevented by pretreatment with the oxidant scavenger vitamin C or a thiol-reducing agent, dithiothreitol (DTT), which shields intracellular thiol groups from oxidation by chlorinated oxidants. Introduction of vitamin C during ongoing exposure to NH(2)Cl arrested but did not reverse accumulation of Ca(2+) in the cytoplasm. In contrast, introduction of DTT or N-acetylcysteine permitted arrest and partial reversal of the effects of NH(2)Cl. Accumulation of Ca(2+) in the cytoplasm induced by NH(2)Cl is due to release from intracellular stores, entry from the extracellular fluid, and impaired extrusion. Ca(2+)-handling proteins are susceptible to oxidation by chloramines, leading to sustained increases in [Ca(2+)](i). Under certain conditions, NH(2)Cl may act not as an irritant but as an agent that activates intracellular signaling pathways. Anti-NH(2)Cl strategies should take into account different effects of oxidant scavengers and thiol-reducing agents.     

Thiocyanate is the major substrate for eosinophil peroxidase in physiologic fluids. Implications for cytotoxicity

The potent cytotoxic capacity of eosinophils for parasites and host tissue has in part been attributed to the catalytic action of eosinophil peroxidase (EPO), which preferentially oxidizes Br- to the powerful bleaching oxidant HOBr in buffers that mimic serum halide composition (100 mM Cl-, 20-100 microM Br-, less than 1 microM I-). However, serum also contains 20-120 microM SCN-, a pseudohalide whose peroxidative product, HOSCN, is a weak, primarily sulfhydryl-reactive oxidant. Because of its relative abundance and high oxidation potential, we hypothesized that SCN-, not Br- or I-, is the major substrate for EPO in physiologic fluids. We find that in Earle's buffer (100 mM Cl-) supplemented with 100 microM Br- and varying concentrations of SCN-, HOBr production by activated eosinophils and purified EPO, assayed by conversion of fluorescein to dibromofluorescein, was 50% inhibited (ID50) by only 1 microM SCN-. SCN- also blocked (ID50 10 microM) EPO oxidation of I- to HOI, assayed as iodofluorescein, despite the presence of 100 microM (i.e. grossly supraphysiologic) I-. Thionitrobenzoic acid oxidation kinetics indicate that SCN- is the initial species oxidized by EPO in equimolar mixtures of SCN- and Br- and in human serum. EPO also catalyzed the covalent incorporation of [14C]SCN- into proteins in buffers regardless of Br- concentration and in human serum. Comparing the cytotoxicity of HOSCN and HOBr for host cells, we find that even subphysiologic concentrations of SCN- (3.3-10 microM) nearly completely abrogate the potent Br(-)-dependent toxicity of EPO for 51Cr-labeled aortic endothelial cells and isolated working rat hearts, recently developed models of eosinophilic endocarditis. Thus, HOSCN, hitherto best known as a bacteriostatic agent in saliva and milk, is likely also the major oxidant produced by EPO in physiologic fluids, and the presence of SCN- averts damage to EPO-coated host tissues that might otherwise accrue as a result of HOBr generation. In view of these findings, the potential role of HOSCN in eosinophil killing of parasitic pathogens deserves close examination.     

Exposure of aconitase to smoking-related oxidants results in iron loss and increased iron response protein-1 activity: potential mechanisms for iron accumulation in human arterial cells

Smokers have an elevated risk of cardiovascular disease, but the origin(s) of this increased risk are incompletely defined. Evidence supports an accumulation of the oxidant-generating enzyme myeloperoxidase (MPO) in the inflamed artery wall, and smokers have high levels of SCN^?, a preferred MPO substrate, with this resulting in HOSCN formation. We hypothesised that HOSCN, a thiol-specific oxidant may target the iron-sulphur cluster of aconitase (both isolated, and within primary human coronary artery endothelial cells; HCAEC) resulting in enzyme dysfunction, release of iron, and conversion of the cytosolic isoform to iron response protein-1, which regulates intracellular iron levels. We show that exposure of isolated aconitase to increasing concentrations of HOSCN releases iron from the aconitase [Fe-S]₄ cluster, and decreases enzyme activity. This is associated with protein thiol loss and modification of specific Cys residues in, and around, the [Fe-S]₄ cluster. Exposure of HCAEC to HOSCN resulted in increased intracellular levels of chelatable iron, loss of aconitase activity and increased iron response protein-1 (IRP-1) activity. These data indicate HOSCN, an oxidant associated with oxidative stress in smokers, can induce aconitase dysfunction in human endothelial cells via Cys oxidation, damage to the [Fe-S]₄ cluster, iron release and generation of IRP-1 activity, which modulates ferritin protein levels and results in dysregulation of iron metabolism. These data may rationalise, in part, the presence of increased levels of iron in human atherosclerotic lesions and contribute to increased oxidative damage and endothelial cell dysfunction in smokers. Similar reactions may occur at other sites of inflammation.     

Halide-stabilizing residues of haloalkane dehalogenases studied by quantum mechanic calculations and site-directed mutagenesis

Haloalkane dehalogenases catalyze cleavage of the carbon-halogen bond in halogenated aliphatic compounds, resulting in the formation of an alcohol, a halide, and a proton as the reaction products. Three structural features of haloalkane dehalogenases are essential for their catalytic performance: (i) a catalytic triad, (ii) an oxyanion hole, and (iii) the halide-stabilizing residues. Halide-stabilizing residues are not structurally conserved among different haloalkane dehalogenases. The level of stabilization of the transition state structure of S(N)2 reaction and halide ion provided by each of the active site residues in the enzymes DhlA, LinB, and DhaA was quantified by quantum mechanic calculations. The residues that significantly stabilize the halide ion were assigned as the primary (essential) or the secondary (less important) halide-stabilizing residues. Site-directed mutagenesis was conducted with LinB enzyme to confirm location of its primary halide-stabilizing residues. Asn38Asp, Asn38Glu, Asn38Phe, Asn38Gln, Trp109Leu, Phe151Leu, Phe151Trp, Phe151Tyr, and Phe169Leu mutants of LinB were constructed, purified, and kinetically characterized. The following active site residues were classified as the primary halide-stabilizing residues: Trp125 and Trp175 of DhlA; Asn38 and Trp109 of LinB; and Asn41 and Trp107 of DhaA. All these residues make a hydrogen bond with the halide ion released from the substrate molecule, and their substitution results in enzymes with significantly modified catalytic properties. The following active site residues were classified as the secondary halide-stabilizing residues: Phe172, Pro223, and Val226 of DhlA; Trp207, Pro208, and Ile211 of LinB; and Phe205, Pro206, and Ile209 of DhaA. The differences in the halide stabilizing residues of three haloalkane dehalogenases are discussed in the light of molecular adaptation of these enzymes to their substrates.     

Oxidation of thiols in the Ca2+-ATPase of sarcoplasmic reticulum microsomes

We recently showed that oxidative stress impairs the function of the sarcoplasmic reticulum to transport and retain calcium. Inhibition results primarily from oxidation of one or more thiol groups in the Ca2+-ATPase. We now report that thiol oxidation does not result in disulfide formation. Oxidative inhibition of Ca2+-ATPase activity was not reversed by dithiothreitol. Also, arsenite, which crosslinks dithiols, only mildly inhibited Ca2+-ATPase activity and protected against inhibition by peroxydisulfate. These data suggest the thiols susceptible to oxidation are not spatially close enough to form a disulfide. Furthermore, these thiols appear to be involved in some aspect of phosphoenzyme formation. ATP, in the presence of calcium and magnesium, protected against inhibition of Ca2+-ATPase activity by both oxidants and thiol-binding agents. Both inhibitors also decreased binding of the nucleotide analogue TNP-AMP after phosphorylation by Pi. Dithiothreitol and arsenite were protective. In conclusion, reversible redox regulation of the Ca2+-ATPase of sarcoplasmic reticulum by thiol-disulfide exchange does not occur. However, some other mechanism of redox regulation may operate because the enzyme is sensitive to oxidants, thiol-binding agents and activity can be enhanced by prolonged exposure to dithiothreitol.     

High plasma thiocyanate levels in smokers are a key determinant of thiol oxidation induced by myeloperoxidase

Smokers have an elevated risk of atherosclerosis but the origins of this elevated risk are incompletely defined, though evidence supports an accumulation of the oxidant-generating enzyme myeloperoxidase (MPO) in the inflamed artery wall. We hypothesized that smokers would have a high level of thiocyanate (SCN(-)), a preferred substrate for MPO, which in turn would predispose to thiol oxidation, an established independent risk factor for atherosclerosis. In this study it is shown that on exposure to MPO/H(2)O(2), thiols on plasma proteins from nonsmokers were increasingly oxidized with increasing added SCN(-) concentrations. Plasma from smokers contained significantly higher endogenous levels of SCN(-) than that from nonsmokers (131±31 vs 40±24 μM, P<0.0001). When plasma from smokers and nonsmokers was exposed to MPO/H(2)O(2)-stimulated oxidation, a strong positive correlation (r=0.8139, P<0.0001) between the extent of thiol oxidation and the plasma SCN(-) concentrations was observed. Computational calculations indicate a changeover from HOCl to HOSCN as the major MPO-generated oxidant in plasma, with increasing SCN(-) levels. These data indicate that plasma SCN(-) levels are a key determinant of the extent of thiol oxidation on plasma proteins induced by MPO, and implicate HOSCN as an important mediator of inflammation-induced oxidative damage to proteins in smokers.     

High plasma thiocyanate levels modulate protein damage induced by myeloperoxidase and perturb measurement of 3-chlorotyrosine

Smokers have an elevated risk of atherosclerosis but the origin of this elevated risk is incompletely defined, though increasing evidence supports a role for the oxidant-generating enzyme myeloperoxidase (MPO). In previous studies we have demonstrated that smokers have elevated levels of thiocyanate ions (SCN(-)), relative to nonsmokers, and increased thiol oxidation, as SCN(-) is a favored substrate for MPO, and the resulting hypothiocyanous acid (HOSCN) targets thiol groups rapidly and selectively. In this study we show that increased HOSCN formation by MPO diminishes damage to nonthiol targets on both model proteins and human plasma proteins. Thus high SCN(-) levels protect against HOCl- and MPO-mediated damage to methionine, tryptophan, lysine, histidine, and tyrosine residues on proteins. Furthermore, levels of the HOCl-mediated marker compound 3-chlorotyrosine and the cross-linked product dityrosine are decreased. Plasma protein 3-chlorotyrosine levels induced by HOCl exposure in nonsmokers are elevated over the levels detected in smokers when exposed to identical oxidative insult (P<0.05), and a strong inverse correlation exists between plasma SCN(-) levels and 3-chlorotyrosine concentrations (r=0.6182; P<0.0001). These correlations were also significant for smokers (r=0.2724; P<0.05) and nonsmokers (r=0.4141; P<0.01) when analyzed as individual groups. These data indicate that plasma SCN(-) levels are a key determinant of the extent and type of protein oxidation induced by MPO on isolated and plasma proteins and that smoking status and resulting high SCN(-) levels can markedly modulate the levels of the widely used biomarker compound 3-chlorotyrosine.     

Peroxynitrite oxidation of sulfhydryls. The cytotoxic potential of superoxide and nitric oxide.

Peroxynitrite anion (ONOO-) is a potent oxidant that mediates oxidation of both nonprotein and protein sulfhydryls. Endothelial cells, macrophages, and neutrophils can generate superoxide as well as nitric oxide, leading to the production of peroxynitrite anion in vivo. Apparent second order rate constants were 5,900 M-1.s-1 and 2,600-2,800 M-1.s-1 for the reaction of peroxynitrite anion with free cysteine and the single thiol of albumin, respectively, at pH 7.4 and 37 degrees C. These rate constants are 3 orders of magnitude greater than the corresponding rate constants for the reaction of hydrogen peroxide with sulfhydryls at pH 7.4. Unlike hydrogen peroxide, which oxidizes thiolate anion, peroxynitrite anion reacts preferentially with the undissociated form of the thiol group. Peroxynitrite oxidizes cysteine to cystine and the bovine serum albumin thiol group to an arsenite nonreducible product, suggesting oxidation beyond sulfenic acid. Peroxynitrous acid was a less effective thiol-oxidizing agent than its anion, with oxidation presumably mediated by the decomposition products, hydroxyl radical and nitrogen dioxide. The reactive peroxynitrite anion may exert cytotoxic effects in part by oxidizing tissue sulfhydryls.     

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.