Comparison of low-density lipoprotein modification by myeloperoxidase-derived hypochlorous and hypobromous acids.

Myeloperoxidase (MPO), a heme enzyme secreted by activated phagocytes, catalyzes the oxidation of halides to hypohalous acids. At plasma concentrations of halides, hypochlorous acid (HOCl) is the major strong oxidant produced. In contrast, the related enzyme eosinophil peroxidase preferentially generates hypobromous acid (HOBr). Since reagent and MPO-derived HOCl converts low-density lipoprotein (LDL) to a potentially atherogenic form, we investigated the effects of HOBr on LDL modification. Compared to HOCl, HOBr caused 2-3-fold greater oxidation of tryptophan and cysteine residues of the protein moiety (apoB) of LDL and 4-fold greater formation of fatty acid halohydrins from the lipids in LDL. In contrast, HOBr was 2-fold less reactive than HOCl with lysine residues and caused little formation of N-bromamines. Nevertheless, HOBr caused an equivalent increase in the relative electrophoretic mobility of LDL as HOCl, which was not reversed upon subsequent incubation with ascorbate, in contrast to the shift in mobility caused by HOCl. Similar apoB modifications were observed with HOBr generated by MPO/H(2)O(2)/Br(-). In the presence of equivalent concentrations of Cl(-) and Br(-), modifications of LDL by MPO resembled those seen in the presence of Br(-) alone. Interestingly, even at physiological concentrations of the two halides (100 mM Cl(-), 100 microM Br(-)), MPO utilized a portion of the Br(-) to oxidize apoB cysteine residues. MPO also utilized the pseudohalide thiocyanate to oxidize apoB cysteine residues. Our data show that even though HOBr has different reactivities than HOCl with apoB, it is able to alter the charge of LDL, converting it into a potentially atherogenic particle.     

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.     

Kinetic analysis of the reactions of hypobromous acid with protein components: implications for cellular damage and use of 3-bromotyrosine as a marker of oxidative stress

Hypohalous acids (HOX, X = Cl, Br) are produced by activated neutrophils, monocytes, eosinophils, and possibly macrophages. These oxidants react readily with biological molecules, with amino acids and proteins being major targets. Elevated levels of halogenated Tyr residues have been detected in proteins isolated from patients with atherosclerosis, asthma, and cystic fibrosis, implicating the production of HOX in these diseases. The quantitative significance of these findings requires knowledge of the kinetics of reaction of HOX with protein targets, and such data have not been previously available for HOBr. In this study, rate constants for reaction of HOBr with protein components have been determined. The second-order rate constants (22 degrees C, pH 7.4) for reaction with protein sites vary by 8 orders of magnitude and decrease in the order Cys > Trp approximately Met approximately His approximately alpha-amino > disulfide > Lys approximately Tyr > Arg > backbone amides > Gln/Asn. For most residues HOBr reacts 30-100 fold faster than HOCl, though Cys and Met residues are approximately 10-fold less reactive, and ring halogenation of Tyr is approximately 5000-fold faster. Thus, Tyr residues are more, and Cys and Met much less, important targets for HOBr than HOCl. Kinetic models have been developed to predict the targets of HOX attack on proteins and free amino acids. Overall, these results shed light on the mechanisms of cell damage induced by HOX and indicate, for example, that the 3-chloro-Tyr:3-bromo-Tyr ratio does not reflect the relative roles of HOCl and HOBr in disease processes.     

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.     

Cyclophosphamide suppresses thioredoxin reductase in bladder tissue and its adaptive response via inductions of thioredoxin reductase and glutathione peroxidase

Mammalian thioredoxin reductase (TrxR) catalyzes the reduction of oxidized thioredoxin in a NADPH-dependent manner, and contains a selenocysteine residue near the C-terminus. Glutathione peroxidase (GPx) is one of the primary antioxidant enzymes that scavenge hydrogen peroxide and organic hydroperoxides. Both TrxR and GPx play an important role in protecting against oxidative stress. Cyclophosphamide (CTX), one of the most widely prescribed antineoplastic drugs, could cause cystitis. We found that 4 h after a bolus dose of CTX (30, 90, 150, 300 and 450 mg/kg) were administrated intraperitoneally, TrxR activity was significantly decreased in a dose-dependent manner, by 32%, 44%, 68%, 87% and 99%, respectively, in comparison with control group. When fixing CTX dose at 150 mg/kg, TrxR activity changed over time, significantly reduced to 68% of the activity in comparison with control tissue at 2 h, and gradually recovered to normal level within 24 h. In addition, we found that GPx activity was induced significantly after 4h. The results of the present study suggest that marked suppression of TrxR activity could be involved in the mechanism of CTX-induced cystitis, bladder may have a protective system against tissue damage by CTX via upregulation of TrxR and GPx, which is an adaptive response to oxidative stress.     

Truncated mutants of human thioredoxin reductase 1 do not exhibit glutathione reductase activity

The substrate spectrum of human thioredoxin reductase (hTrxR) is attributed to its C-terminal extension of 16 amino acids carrying a selenocysteine residue. The concept of an evolutionary link between thioredoxin reductase and glutathione reductase (GR) is presently discussed and supported by the fact that almost all residues at catalytic and substrate recognition sites are identical. Here, we addressed the question if a deletion of the C-terminal part of TrxR leads to recognition of glutathione disulfide (GSSG), the substrate of GR. We introduced mutations at the putative substrate binding site to enhance GSSG binding and turnover. However, none of these enzyme species accepted GSSG as substrate better than the full length cysteine mutant of TrxR, excluding a role of the C-terminal extension in preventing GSSG binding. Furthermore, we show that GSSG binding at the N-terminal active site of TrxR is electrostatically disfavoured.     

Identification of proteins containing cysteine residues that are sensitive to oxidation by hydrogen peroxide at neutral pH

A procedure for detecting proteins that contain H(2)O(2)-sensitive cysteine (or selenocysteine) residues was developed as a means with which to study protein oxidation by H(2)O(2) in cells. The procedure is based on the facts that H(2)O(2) and biotin-conjugated iodoacetamide (BIAM) selectively and competitively react with cysteine residues that exhibit a low pK(a), and that the decrease in the labeling of cell lysate proteins with BIAM caused by prior exposure of cells to H(2)O(2) or to an agent that induces H(2)O(2) production can be monitored by streptavidin blot analysis. This procedure was applied to rat pheochromocytoma PC12 cells directly treated with H(2)O(2), mouse hippocampal HT22 cells in which H(2)O(2) production was induced by glutamate, and human erythroleukemia K562 cells in which H(2)O(2) production was induced by phorbol myristate acetate. It revealed that several cell proteins contain cysteine or selenocysteine residues that are selectively oxidized by H(2)O(2). Three of these H(2)O(2)-sensitive proteins were identified as a member of the protein disulfide isomerase family, thioredoxin reductase, and creatine kinase, all of which were previously known to contain at least one reactive cysteine or selenocysteine at their catalytic sites. This procedure should thus prove useful for the identification of proteins that are oxidized by H(2)O(2) generated in response to a variety of extracellular agents.     

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.     

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.     

Caffeine induces Ca2+ release by reducing the threshold for luminal Ca2+ activation of the ryanodine receptor

Myeloperoxidase, released by activated phagocytes, forms reactive oxidants by catalysing the reaction of halide and pseudo-halide ions with H(2)O(2). These oxidants have been linked to tissue damage in a range of inflammatory diseases. With physiological levels of halide and pseudo-halide ions, similar amounts of HOCl (hypochlorous acid) and HOSCN (hypothiocyanous acid) are produced by myeloperoxidase. Although the importance of HOSCN in initiating cellular damage via thiol oxidation is becoming increasingly recognized, there are limited data on the reactions of HOSCN with other targets. In the present study, the products of the reaction of HOSCN with proteins has been studied. With albumin, thiols are oxidized preferentially forming unstable sulfenyl thiocyanate derivatives, as evidenced by the reversible incorporation of (14)C from HOS(14)CN. On consumption of the HSA (human serum albumin) free thiol group, the formation of stable (14)C-containing products and oxidation of tryptophan residues are observed. Oxidation of tryptophan residues is observed on reaction of HOSCN with other proteins (including myoglobin, lysozyme and trypsin inhibitor), but not free tryptophan, or tryptophan-containing peptides. Peptide mass mapping studies with HOSCN-treated myoglobin, showed the addition of two oxygen atoms on either Trp(7) or Trp(14) with equimolar or less oxidant, and the addition of a further two oxygen atoms to the other tryptophan with higher oxidant concentrations (> or = 2-fold). Tryptophan oxidation was observed on treating myoglobin with HOSCN in the presence of glutathione and ascorbate. Thus tryptophan residues are likely to be favourable targets for the reaction in biological systems, and the oxidation products formed may be useful biomarkers of HOSCN-mediated protein oxidation.     

Hypochlorite- and hypobromite-mediated radical formation and its role in cell lysis.

Activated leukocytes generate the potent oxidants HOCl and HOBr via the formation of H(2)O(2) and the release of peroxidase enzymes (myeloperoxidase, eosinophil peroxidase). HOCl and HOBr are potent microbiocidal agents, but excessive or misplaced production can cause tissue damage and cell lysis. In this study it is shown that HOBr induces red blood cell lysis at approximately 10-fold lower concentrations than HOCl, whereas with monocyte (THP1) and macrophage (J774) cells HOCl and HOBr induce lysis at similar concentrations. The role of radical formation during lysis has been investigated by EPR spin trapping, and it is shown that reaction of both oxidants with each cell type generates cell-derived radicals. Red blood cells exposed to nonlytic doses of HOCl generate novel nitrogen-centered radicals whose formation is GSH dependent. In contrast, HOBr gives rise to nitrogen-centered, membrane-derived protein radicals. With lytic doses of either oxidant, protein (probably hemoglobin)-derived, nitrogen-centered radicals are observed. Unlike the red blood cells, treatment of monocytes and macrophages with HOCl gives significant radical formation only under conditions where cell lysis occurs concurrently. These radicals are nitrogen-centered, cell-protein-derived species and have parameters identical to those detected with red blood cells and HOBr. Exposure of these cells to HOBr did not give detectable radicals. Overall these experiments demonstrate that HOCl and HOBr react with different selectivity with cellular targets, and that this can result in radical formation. This radical generation can precede, and may play a role in, cell lysis.     

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.     

Myeloperoxidase inactivates TIMP-1 by oxidizing its N-terminal cysteine residue: an oxidative mechanism for regulating proteolysis during inflammation

An imbalance between the proteolytic activity of matrix metalloproteinases (MMPs) and the activity of tissue inhibitors of metalloproteinases (TIMPs) is implicated in tissue injury during inflammation. The N-terminal cysteine of TIMP-1 plays a key role in the inhibitory activity of the protein because it coordinates the essential catalytic Zn2+ of the MMP, preventing the metal ion from functioning. An important mechanism for controlling the interaction of TIMPs with MMPs might involve hypochlorous acid (HOCl), a potent oxidant produced by the myeloperoxidase (MPO) system of phagocytes. Here, we show that HOCl generated by the MPO-H2O2-chloride system inactivates TIMP-1 by oxidizing its N-terminal cysteine. The product is a novel 2-oxo acid. Liquid chromatography-mass spectrometry and tandem mass spectrometry analyses demonstrated that methionine and N-terminal cysteine residues were rapidly oxidized by MPO-derived HOCl but only oxidation of the N-terminal cysteine of TIMP-1 correlated well with loss of inhibitory activity. Importantly, we detected the signature 2-oxo-acid N-terminal peptide in tryptic digests of bronchoalveolar lavage fluid from patients with acute respiratory distress syndrome, demonstrating that TIMP-1 oxidation occurs in vivo. Loss of the N-terminal amino group and disulfide structure are crucial for preventing TIMP-1 from inhibiting MMPs. Our findings suggest that pericellular production of HOCl by phagocytes is a pathogenic mechanism for impairing TIMP-1 activity during inflammation.     

Dissociation of DNA double strand by hypohalous acids

Calf thymus DNA was treated with authentic HOCl, and hypohalous acid-generating systems. This caused a decrease in fluorescence of ethidium-DNA complexes when ethidium bromide was subsequently added to the DNA. The fluorescence continued to decrease up to 30 min after adding HOCl. Loss in fluorescence was proportional to the concentration of HOCl and was complete when a 3-fold excess of HOCl was added to the DNA. No significant decrease in the fluorescence was observed when the chlorination was carried out in the presence of a concentration of monochlorodimedone (MCD) equivalent to that of HOCl. MCD is known to react stoichiometrically with HOCl. The decrease in fluorescence was completely inhibited by H2O2, ascorbate and glutathione (GSH). We have estimated the rate constant for the reaction of HOCl with H2O, to be 1-2 x 10(5) M(-1)s(-1). When compared with authentic HOCl, HOCl-generating systems (Cl + H2O2 + MPO or chloroperoxidase) were found to be inefficient in damaging DNA. This result most likely arises because the rate constant for reaction of HOCl with H2O2 is about 1000-fold faster than that for the reaction with DNA. HOBr and HOI generating systems also had a limited ability to damage DNA. We conclude that good chlorine acceptors and antioxidants protect DNA from hypohalous acid-induced oxidative damage.     

A synthetic cysteine oxidase based on a ferrocene-cyclodextrin conjugate.

We report a novel synthetic cysteine oxidase consisting of a ferrocene-beta-cyclodextrin conjugate in which the ferrocene moiety is bound to the secondary hydroxyl side of the cyclodextrin cavity through an ethylenediamine linker. Cysteine oxidation occurs after the ferrocene group is electrochemically oxidized to the ferricinium form, and this generates a voltammetric electrocatalytic wave, the magnitude of which is related to the rate constant for cysteine oxidation. Comparison of cysteine oxidation rates for the primary and secondary beta-cyclodextrin derivatives (105 and 1470 M-1 s-1, respectively) shows that the secondary derivatives are more effective synthetic enzymes. Substrate selectivity of the secondary derivative is demonstrated by comparison of oxidation rates for cysteine (1470 M-1 s-1) and glutathione (260 M-1 s-1) at pH 7.0. The rate constant for cysteine oxidation was 3-fold higher at pH 8.0. With a constant synthetic enzyme concentration, electrocatalytic limiting currents increased linearly with increasing cysteine concentration to a maximum at 6 mM cysteine; above this concentration, the current decreased significantly. These and other results suggest that product inhibition of the catalytic cycle occurs as a result of cystine binding more strongly to the cyclodextrin than cysteine.     

cDNA cloning, expression and chromosomal localization of the mouse mitochondrial thioredoxin reductase gene(1).

Cytosolic thioredoxin (Trx) and thioredoxin reductase (TrxR) comprise a ubiquitous system that uses the reducing power of NADPH to act as a general disulfide reductase system as well as a potent antioxidant system. Human and rat mitochondria contain a complete thioredoxin system different from the one present in the cytosol. The mitochondrial system is involved in the oxidative stress protection through a mitochondrial thioredoxin-dependent peroxidase. We report here the cDNA cloning and chromosomal localization of the mouse mitochondrial thioredoxin reductase gene (TrxR2). The mouse TrxR2 cDNA encodes for a putative protein of 527 amino acid residues with a calculated molecular mass of 57 kDa, that displays high homology with the human and rat counterparts. The N-terminus of the protein displays typical features of a mitochondrial targeting sequence with absence of acidic residues and abundance of basic residues. Mouse TrxR2 also contains a stop codon in frame at the C-terminus of the protein, necessary for the incorporation of selenocysteine that is required for enzymatic activity. The typical stem-loop structure (SECIS element) that drives the incorporation of selenocysteine is identified in the 3'-UTR. Northern analysis of the mouse TrxR2 mRNA shows a similar pattern of expression with the human homologue, with higher expression in liver, heart and kidney. Finally, we have assigned the mouse TrxR2 gene to chromosome 16 mapping at 11.2 cM from the centromer and linked to the catechol-o-methyltransferase (comt) gene.     

Essential role of selenium in the catalytic activities of mammalian thioredoxin reductase revealed by characterization of recombinant enzymes with selenocysteine mutations

Mammalian thioredoxin reductases (TrxR) are dimers homologous to glutathione reductase with a selenocysteine (SeCys) residue in the conserved C-terminal sequence -Gly-Cys-SeCys-Gly. We removed the selenocysteine insertion sequence in the rat gene, and we changed the SeCys(498) encoded by TGA to Cys or Ser by mutagenesis. The truncated protein having the C-terminal SeCys-Gly dipeptide deleted, expected in selenium deficiency, was also engineered. All three mutant enzymes were overexpressed in Escherichia coli and purified to homogeneity with 1 mol of FAD per monomeric subunit. Anaerobic titrations with NADPH rapidly generated the A(540 nm) absorbance resulting from the thiolate-flavin charge transfer complex characteristic of mammalian TrxR. However, only the SeCys(498) --> Cys enzyme showed catalytic activity in reduction of thioredoxin, with a 100-fold lower k(cat) and a 10-fold lower K(m) compared with the wild type rat enzyme. The pH optimum of the SeCys(498) --> Cys mutant enzyme was 9 as opposed to 7 for the wild type TrxR, strongly suggesting involvement of the low pK(a) SeCys selenol in the enzyme mechanism. Whereas H(2)O(2) was a substrate for the wild type enzyme, all mutant enzymes lacked hydroperoxidase activity. Thus selenium is required for the catalytic activities of TrxR explaining the essential role of this trace element in cell growth.     

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.