Pseudohalide Thiocyanate Research Articles

The Use of Thiocyanate Formulations to Create Manganese Porphyrin Antioxidants That Supplement Innate Immunity.

The innate immune response to infection results in inflammation and oxidative damage, creating a paradox where most anti-inflammatory and antioxidant therapies can further suppress an already inadequate immune response. We have previously reported the beneficial effects of the exogenous supplementation of innate immunity with small pseudohalide thiocyanate (−SCN) in a mouse model of a cystic fibrosis (CF) lung infection and inflammation. The object of this study was to evaluate the use of −SCN as a counter anion for cationic manganese porphyrin (MnP) catalytic antioxidants, which could increase the parent compound’s antioxidant spectrum against hypohalous acids while supplementing innate immunity. The antioxidant activities of the parent compound were examined, as its chloride salt was compared with the −SCN-anion exchanged compound, (MnP(SCN) versus MnP(Cl)). We measured the superoxide dismutase activity spectrophotometrically and performed hydrogen peroxide scavenging using oxygen and hydrogen peroxide electrodes. Peroxidase activity was measured using an amplex red assay. The inhibition of lipid peroxidation was assessed using a thiobarbituric acid reactive species (TBARS) assay. The effects of the MnP compounds on macrophage phagocytosis were assessed by flow cytometry. The abilities of the MnP(Cl) formulations to protect human bronchiolar epithelial cells against hypochlorite (HOCl) and glycine chloramine versus their MnP(SCN) formulations were assessed using a cell viability assay. We found that anions exchanging out the chloride for −SCN improved the cellular bioavailability but did not adversely affect the cell viability or phagocytosis and that they switched hydrogen-peroxide scavenging from a dismutation reaction to a peroxidase reaction. In addition, the −SCN formulations improved the ability of MnPs to protect human bronchiolar epithelial cells against hypochlorous acid (HOCl) and glycine chloramine toxicity. These novel types of antioxidants may be more beneficial in treating lung disease that is associated with chronic infections or acute infectious exacerbations.

Pseudohalide substitution and potassium doping in FA0.98K0.02Pb(SCN)2I for high-stability hole-conductor-free perovskite solar cells

Hybrid organic-inorganic halide perovskite solar cells (PSCs) have achieved intriguingly high photoelectric conversion efficiencies (PCEs), but their operational stability is still not satisfactory owing to their vulnerability to moisture and heat. Herein, we report the introduction of pseudohalide thiocyanate ions and potassium dopants into formamidinium lead triiodide (FAPbI3) to obtain a new class of pseudohalide-substituted lead halide perovskites FAPb(SCN)2I that can be prepared in ambient air and exhibit broad-range light absorption. Through the introduction of K+-cation doping, hole-conductor-free PSCs based on a FA0.98K0.02Pb(SCN)2I perovskite light absorber and a low-cost, screen-printable carbon-based counter electrode exhibited a PCE of 13.39%. Importantly, the FA0.98K0.02Pb(SCN)2I-based hole-conductor-free PSCs exhibited an 88% PCE retention even after continuous heating at 100 °C for 1020 h, indicating excellent thermal stability. This study suggests that the compositional engineering of non-iodine X-site pseudohalide anion substitution and A-site cation doping hold great promise for improving the stability of PSCs.

Rod-shaped thiocyanate-induced abnormal band gap broadening in SCN− doped CsPbBr3 perovskite nanocrystals

In this work, the pseudohalide thiocyanate has been demonstrated as a promising alternative to the halide anion to engineer optoelectronic properties of inorganic/organic hybrid perovskites because it exhibits better chemical stability than the halide anion. Previous reports have suggested that the ionic radii and electronegativity of SCN− is close to that of I−; the SCN− doped CH3NH3PbI3 exhibited similar optical properties as pure CH3NH3PbI3. Consequently, it was expected that doping of CsPbBr3 perovskite with SCN− would result in band gap narrowing. Interestingly, the photoluminescent all-inorganic CsPbBr3 perovskite nanocrystals exhibit an abnormal blue shift in optical properties and improvement of the crystallinity when successfully doped by SCN−. Combined experimental and theoretical investigations revealed that doping of the CsPbBr3 perovskite with the rod-like SCN− anion introduced disorder in the crystal lattice, leading to its expansion, and impacted the electronic structure of the perovskite with band gap broadening.

Pseudohalide-Exchanged Quantum Dot Solids Achieve Record Quantum Efficiency in Infrared Photovoltaics.

Application of pseudohalogens in colloidal quantum dot (CQD) solar-cell active layers increases the solar-cell performance by reducing the trap densities and implementing thick CQD films. Pseudohalogens are polyatomic analogs of halogens, whose chemistry allows them to substitute halogen atoms by strong chemical interactions with the CQD surfaces. The pseudohalide thiocyanate anion is used to achieve a hybrid surface passivation. A fourfold reduced trap state density than in a control is observed by using a suite of field-effect transistor studies. This translates directly into the thickest CQD active layer ever reported, enabled by enhanced transport lengths in this new class of materials, and leads to the highest external quantum efficiency, 80% at the excitonic peak, compared with previous reports of CQD solar cells.

3 - Peroxidasin-Catalysed Oxidative Modifications of Proteins in the Extracellular Matrix

Peroxidasin has been recently identified as a mammalian heme peroxidase that catalyses the formation of an essential crosslink in collagen IV in the extracellular matrix (ECM). This crosslink was identified as sulfilimine bond between methionine and hydroxylysine resuidues in the non-collagenous domain. Heme peroxidases catalyse the oxidation of a variety of substrates by hydrogen peroxide (H2O2). These include bromide (Br–) to give hypobromous acid (HOBr), which is proposed to be responsible for the collagen IV crosslink. However, the specificity of peroxidasin and the exact mechanism of sulfilimine formation are not completely understood. We investigated peroxidasin activity and the cross-linking of collagen IV in isolated ECM from cultured epithelial cells. We found that formation of the sulfilimine crosslink requires both H2O2 and Br–. It did not form in presence of other halides such as chloride, iodide or the pseudohalide thiocyanate, with both of the latter inhibiting the reaction. Acetaminophen (paracetamol), thioxanthine, and 4-aminobenzoic acid hydrazide, which all inhibit myeloperoxidase, had minimal effect on the peroxidasin-dependent crosslink. The specificity and inhibitory profile of peroxidasin is therefore different from myeloperoxidase. HOBr has been shown to form sulfilimine bonds in peptides containing Met and Lys, but it also undergoes other reactions with proteins, including the formation of a specific product bromotyrosine (Br-Tyr)(1). Using LC-MS, we detected Br-Tyr in the noncollagenous domain of collagen IV isolated from cultured cells, and to a lesser extent in the rest of matrix and the cell extract. Br-Tyr levels increased in cells grown in the presence of Br–, and decreased in presence of phloroglucinol, a potent inhibitor of cross-linking. Cross-linking was already maximal without added Br–, suggesting that the reaction is selective only at low peroxidasin activity. These results indicate that cross-linking of collagen IV requires Br–, and is susceptible to modulation by endogenous and exogenous substances. Furthermore, peroxidasin-mediated oxidative modification of proteins in the ECM might not be limited to formation of the sulfilimine crosslink. 1. Ronsein GE, Winterbourn CC, Di Mascio P, Kettle AJ. Cross-linking methionine and amine residues with reactive halogen species. Free radical biology & medicine. 2014;70:278-87.

Eosinophil Peroxidase Catalyzed Protein Carbamylation Participates in Asthma

The biochemical mechanisms through which eosinophils contribute to asthma pathogenesis are unclear. Here we show eosinophil peroxidase (EPO), an abundant granule protein released by activated eosinophils, contributes to characteristic asthma-related phenotypes through oxidative posttranslational modification (PTM) of proteins in asthmatic airways through a process called carbamylation. Using a combination of studies we now show EPO uses plasma levels of the pseudohalide thiocyanate (SCN-) as substrate to catalyze protein carbamylation, as monitored by PTM of protein lysine residues into Nϵ-carbamyllysine (homocitrulline), and contributes to the pathophysiological sequelae of eosinophil activation. Studies using EPO-deficient mice confirm EPO serves as a major enzymatic source for protein carbamylation during eosinophilic inflammatory models, including aeroallergen challenge. Clinical studies similarly revealed significant enrichment in carbamylation of airway proteins recovered from atopic asthmatics versus healthy controls in response to segmental allergen challenge. Protein-bound homocitrulline is shown to be co-localized with EPO within human asthmatic airways. Moreover, pathophysiologically relevant levels of carbamylated protein either incubated with cultured human airway epithelial cells in vitro, or provided as an aerosolized exposure in non-sensitized mice, induced multiple asthma-associated phenotypes including induction of mucin, Th2 cytokines, IFNγ, TGFβ, and epithelial cell apoptosis. Studies with scavenger receptor-A1 null mice reveal reduced IL-13 generation following exposure to aerosolized carbamylated protein, but no changes in other asthma-related phenotypes. In summary, EPO-mediated protein carbamylation is promoted during allergen-induced asthma exacerbation, and can both modulate immune responses and trigger a cascade of many of the inflammatory signals present in asthma.

Pseudohalide (SCN(-))-Doped MAPbI3 Perovskites: A Few Surprises.

Pseudohalide thiocyanate anion (SCN(-)) has been used as a dopant in a methylammonium lead tri-iodide (MAPbI3) framework, aiming for its use as an absorber layer for photovoltaic applications. The substitution of SCN(-) pseudohalide anion, as verified using Fourier transform infrared (FT-IR) spectroscopy, results in a comprehensive effect on the optical properties of the original material. Photoluminescence measurements at room temperature reveal a significant enhancement in the emission quantum yield of MAPbI3-x(SCN)x as compared to MAPbI3, suggestive of suppression of nonradiative channels. This increased intensity is attributed to a highly edge specific emission from MAPbI3-x(SCN)x microcrystals as revealed by photoluminescence microscopy. Fluoresence lifetime imaging measurements further established contrasting carrier recombination dynamics for grain boundaries and the bulk of the doped material. Spatially resolved emission spectroscopy on individual microcrystals of MAPbI3-x(SCN)x reveals that the optical bandgap and density of states at various (local) nanodomains are also nonuniform. Surprisingly, several (local) emissive regions within MAPbI3-x(SCN)x microcrystals are found to be optically unstable under photoirradiation, and display unambiguous temporal intermittency in emission (blinking), which is extremely unusual and intriguing. We find diverse blinking behaviors for the undoped MAPbI3 crystals as well, which leads us to speculate that blinking may be a common phenomenon for most hybrid perovskite materials.

Pseudohalide-induced moisture tolerance in perovskite CH3 NH3 Pb(SCN)2 I thin films.

Two pseudohalide thiocyanate ions (SCN(-) ) have been used to replace two iodides in CH3 NH3 PbI3 , and the resulting perovskite material was used as the active material in solar cells. In accelerated stability tests, the CH3 NH3 Pb(SCN)2 I perovskite films were shown to be superior to the conventional CH3 NH3 PbI3 films as no significant degradation was observed after the film had been exposed to air with a relative humidity of 95 % for over four hours, whereas CH3 NH3 PbI3 films degraded in less than 1.5 hours. Solar cells based on CH3 NH3 Pb(SCN)2 I thin films exhibited an efficiency of 8.3 %, which is comparable to that of CH3 NH3 PbI3 based cells fabricated in the same way.

Pseudohalide‐Induced Moisture Tolerance in Perovskite CH3NH3Pb(SCN)2I Thin Films

AbstractTwo pseudohalide thiocyanate ions (SCN−) have been used to replace two iodides in CH3NH3PbI3, and the resulting perovskite material was used as the active material in solar cells. In accelerated stability tests, the CH3NH3Pb(SCN)2I perovskite films were shown to be superior to the conventional CH3NH3PbI3 films as no significant degradation was observed after the film had been exposed to air with a relative humidity of 95 % for over four hours, whereas CH3NH3PbI3 films degraded in less than 1.5 hours. Solar cells based on CH3NH3Pb(SCN)2I thin films exhibited an efficiency of 8.3 %, which is comparable to that of CH3NH3PbI3 based cells fabricated in the same way.

Tryptophan oxidation in proteins exposed to thiocyanate-derived oxidants

Human defensive peroxidases, including lactoperoxidase (LPO) and myeloperoxidase (MPO), are capable of catalyzing the oxidation of halides (X−) by H2O2 to give hypohalous acids (HOX) for the purpose of cellular defense. Substrate selectivity depends upon the relative abundance of the halides, but the pseudo-halide thiocyanate (SCN−) is a major substrate, and sometimes the exclusive substrate, of all defensive peroxidases in most physiologic fluids. The resulting hypothiocyanous acid (HOSCN) has been implicated in cellular damage via thiol oxidation. While thiols are believed to be the primary target of HOSCN in vivo, Trp residues have also been implicated as targets for HOSCN. However, the mechanism involved in HOSCN-mediated Trp oxidation was not established. Trp residues in proteins appeared to be susceptible to oxidation by HOSCN, whereas free Trp and Trp residues in small peptides were found to be unreactive. We show that HOSCN-induced Trp oxidation is dependent on pH, with oxidation of free Trp, and Trp-containing peptides observed when the pH is below 2. These conditions mimic those employed previously to precipitate proteins after treatment with HOSCN, which accounts for the discrepancy in the results reported for proteins versus free Trp and small peptides. The reactant in these cases may be thiocyanogen ((SCN)2), which is produced by comproportionation of HOSCN and SCN− at low pH. Reaction of thiocyanate-derived oxidants with protein Trp residues at low pH results in the formation of a number of oxidation products, including mono- and di-oxygenated derivatives, which are also formed with other hypohalous acids. Our data suggest that significant modification of Trp by HOSCN in vivo is likely to have limited biological relevance.

TOF-SIMS imaging of halide/thiocyanate anions and hydrogen sulfide in mouse kidney sections using silver-deposited plates

In vivo imaging of reactive small molecule metabolites with high spatial resolution and specificity could give clues to understanding pathophysiology of various diseases. We herein applied time of flight-secondary ion mass spectrometry (TOF-SIMS) to newly developed silver-deposited plates that were stamped on mouse tissues, and succeeded in visualization of halide (Cl(-), Br(-), and I(-)) and pseudohalide thiocyanate (SCN(-)) anions, a class of substrates for neutrophils/eosinophil peroxidases to produce hypohalous acids (HOX/OX(-) mixture; X: (pseudo)halides), as well as hydrogen sulfide (H(2)S). Forty-micrometer frozen mouse kidney sections on cover glasses were attached to 37 °C preheated silver-deposited plates and incubated at -10 °C for 1 h. After sputter cleaning to remove surface contaminants, the plates were analyzed by TOF-SIMS to identify distribution of Br(-), AgBr(2)(-), I(-), AgI(2)(-), SCN(-), as well as S(2-) and AgS(-) as products of tissue-derived H(2)S. Br(-), AgBr(2)(-), I(-), and SCN(-) anions were mainly distributed in core regions including the inner medulla and inner stripe of the outer medulla (except for I(-)), rather than outer regions such as the cortex and outer stripe of the outer medulla. AgI(2)(-) anion was spread over the whole kidney, although its levels were relatively low. In contrast, S(2-) and AgS(-) anions were mainly present in the outer regions. To our knowledge, this is the first imaging study to reveal the distribution of (pseudo)halides and H(2)S in animal tissue sections.

Thiocyanate and Dicyanamide Anion Controlled Nuclearity in Mn, Co, Ni, Cu, and Zn Metal Complexes with Hemilabile Ligand 2-Benzoylpyridine

Two pseudohalides thiocyanate and dicyanamide have been employed to synthesize complexes of MnII, CoII, NiII, CuII, and ZnII in the presence of a hemilabile ligand 2-benzoylpyridine (Phpyk). With thiocyanate all the aforesaid metal ions (except for CoII, of which suitable single crystals for X-ray analysis were not obtained) produce mononuclear complexes having general composition of [MII(NCS)2(Phpyk)2]. The structure determination reveals that Mn and Zn complexes (1, 4) are isomorphous and isostructural (crystallizing in space group C2/c), while Ni and Cu complexes (2, 3) crystallize in space groups P1̅ and P21/n, respectively. Interestingly, no complex has been obtained with a configuration having the N of one Phpyk trans to the O of the other chelating ligand and among the four complexes only in complex 3 the two thiocyanato ligands are in trans-configuration. On the other hand, complexes 5–8 are isomorphous and crystallize in orthorhombic chiral space group P212121. The bridging mode of dicyanamide anions helps to generate a three-dimensional covalently bonded polymeric network of 66 topology for all the polynuclear complexes. By using 8 as sole precursor, we have pyrolytically synthesized triangular shaped ZnO nanoparticles.

Seed-mediated growth of palladium nanocrystals: The effect of pseudo-halide thiocyanate ions

In synthesis in a solution phase, adsorbates such as halides can interact selectively with different metal crystal facets and affect the final morphology of nanocrystals. Pseudo-halide thiocyanate ions (SCN-) can also adsorb on the metal surface, but they have never been used for the synthesis of shape-controlled colloidal metal nanocrystals. In this study, we first investigated the effect of SCN- on the morphology of palladium nanocrystals through a seed-mediated growth method. The presence of 1 µM SCN- in the growth solutions could lead to the formation of palladium polyhedra: truncated rhombic dodecahedra enclosed by twelve {110}, eight {111} and six {100} facets. The products were nanocubes enclosed with six {100} facets if cetyltrimethylammonium bromide (CTAB) was the only capping agent. Meanwhile, the mechanism of the effect of SCN- on the morphology of Pd nanocrystals is discussed.

Euphorbia Peroxidase Catalyzes Thiocyanate Oxidation in Two Different Ways, the Distal Calcium Ion Playing an Essential Role

The oxidation of the pseudohalide thiocyanate (SCN(-)) by Euphorbia peroxidase, in the presence or absence of added calcium, is investigated. After incubation of the native enzyme with hydrogen peroxide, the formation of Compound I occurs and serves to catalyze the thiocyanate oxidation pathways. The addition of a stoichiometric amount of SCN(-) to Compound I leads to the native enzyme spectrum; this process clearly occurs via two electron transfers from pseudohalide to Compound I. In the presence of 10 mM calcium ions, the addition of a stoichiometric amount of SCN(-) to Compound I leads to the formation of Compound II that returns to the native enzyme after addition of a successive stoichiometric amount of SCN(-), indicating that the oxidation occurs via two consecutive one-electron transfer steps. Moreover, different reaction products can be detected when the enzyme-hydrogen peroxide-thiocyanate reaction is performed in the absence or presence of 10 mM Ca(2+) ions. The formation of hypothiocyanous acid is easy demonstrated in the absence of added calcium, whereas in the presence of this ion, CN(-) is formed as a reaction product that leads to the formation of an inactive species identified as the peroxidase-CN(-) complex. Thus, although monomeric, Euphorbia peroxidase is an allosteric enzyme, finely tuned by Ca(2+) ions. These ions either can enhance the catalytic efficiency of the enzyme toward some substrates or can regulate the ability of the enzyme to exploit different metabolic pathways toward the same substrate.

Reactive Sulfur Species: Kinetics and Mechanism of the Reaction of Hypothiocyanous Acid with Cyanide To Give Dicyanosulfide in Aqueous Solution

The chief sources of cyanide (CN(-)) in humans are tobacco and occupationally derived smoke, inflammation [vis-a-vis myeloperoxidase (MPO)-induced chlorination of glycine], and microbial cyanogenesis (including Pseudomonas aeruginosa infection of the cystic fibrosis lung). The human mucosae of healthy individuals are usually protected from infection by innate defense mechanisms that include the defensive peroxidase systems. In the oral cavity, salivary peroxidase and MPO catalyze the oxidation of the pseudohalide thiocyanate (SCN(-)) by hydrogen peroxide to produce the antimicrobial hypothiocyanite (OSCN(-)). Lactoperoxidase carries out the same reaction in the human lung (as does MPO during inflammatory response). In the present study, we show that OSCN(-) and CN(-) react with pH-dependent kinetics to produce SCN(-) and cyanate (OCN(-)) via dicyanosulfide (NCSCN), with the maximum rate occurring near neutral, physiological pH. In addition to presenting a detailed chemical mechanism, we discuss unresolved issues, including the possible biological relevance of the NCSCN intermediate.

Myeloperoxidase Metabolizes Thiocyanate in a Reaction Driven by Nitric Oxide

We examined the potential physiological relevance of myeloperoxidase (MPO)-nitric oxide (NO) interactions as they may relate to the cosubstrate, pseudo halide thiocyanate (SCN(-)), and substrate switching. Direct spectroscopic and rapid kinetics studies revealed that SCN(-) interaction with MPO facilitates formation of the MPO catalytic intermediate Compound II, limiting overall activity. However, a physiological NO concentration (2 microM or less) dramatically influences the build-up, duration, and decay of the steady-state level of MPO Compound II during the metabolism of SCN(-), allowing the enzyme to function at full capacity. At higher NO concentrations, we observed significant increases in the rate of MPO Compound II formation, along with proportional increases in its duration as determined by the time elapsed during catalysis. Surprisingly, the decay rate of MPO Compound II remained unaltered as NO concentrations were increased. Computer simulations were carried out to model the kinetics of MPO Compound II formation, duration, and decay during the metabolism of SCN(-) as a function of NO concentration. These simulation traces closely approximate what was observed experimentally and support the involvement of a conformational intermediate of MPO Compound II complex decay, altering the overall capacity of MPO to promote two electrons versus one-electron oxidation reactions during steady-state catalysis. Collectively, the present studies reveal that (patho)physiologically relevant levels of NO have significant effects on MPO Compound II accumulation. Thus, NO affects the overall rate of peroxidation of substrates and the overall ability of the peroxidase to execute one- versus two-electron oxidation reactions.

Thiocyanate catalyzes myeloperoxidase-initiated lipid oxidation in LDL

There is evidence that LDL oxidation may render the lipoprotein atherogenic. The myeloperoxidase–hydrogen peroxide (MPO/H 2O 2) system of activated phagocytes may be involved in this process. Chloride is supposed to be the major substrate for MPO, generating reactive hypochlorous acid (HOCl), modifying LDL. The pseudo-halide thiocyanate (SCN −) has been shown to be a suitable substrate for MPO, forming reactive HOSCN/SCN ·. As relatively abundant levels of SCN − are found in plasma of smokers—a well-known risk group for cardiovascular disease—the ability of SCN − to act as a catalyst of LDL atherogenic modification by MPO/H 2O 2 was tested. Measurement of conjugated diene and lipid hydroperoxide formation in LDL preparations exposed to MPO/H 2O 2 revealed that SCN − catalyzed lipid oxidation in LDL. Chloride did not diminish the effect of SCN − on lipid oxidation. Surprisingly, SCN inhibited the HOCl-mediated apoprotein modification in LDL. Nitrite—recently found to be a substrate for MPO—showed some competing properties. MPO-mediated lipid oxidation was inhibited by heme poisons (azide, cyanide) and catalase. Ascorbic acid was the most effective compound in inhibiting the SCN −-catalyzed reaction. Bilirubin showed some action, whereas tocopherol was ineffective. When LDL oxidation was performed with activated human neutrophils, which employ the MPO pathway, SCN − catalyzed the cell-mediated LDL oxidation. The MPO/H 2O 2/SCN − system may have the potential to play a significant role in the oxidative modification of LDL—an observation further pointing to the link between the long-recognized risk factors of atherosclerosis: elevated levels of LDL and smoking.

Role of Thiocyanate, Bromide and Hypobromous Acid in Hydrogen Peroxide-induced Apoptosis

We have previously reported that H2O2-induced apoptosis in HL-60 human leukemia cells takes place in the presence of chloride, requires myeloperoxidase (MPO), and occurs through oxidative reactions involving hypochlorous acid and chloramines. We now report that when chloride is replaced by the pseudohalide thiocyanate, there is little or no H2O2-induced apoptosis. Furthermore, thiocyanate inhibits H2O2-induced apoptosis when chloride is present at physiological concentrations, and this occurs at thiocyanate concentrations that are present in human serum and saliva. In contrast, bromide can substitute for chloride in H2O2-induced apoptosis, but results in a lower percent of the cells induced into apoptosis. Hypobromous acid is likely a short-lived intermediate in this H2O2/MPO/bromide apoptosis, and reagent hypobromous acid and bromamines induce apoptosis in HL-60 cells. We conclude that the physiologic concentrations of thiocyanate found in human plasma could modulate the cytototoxicity of H2O2 and its resulting highly toxic MPO-generated hypochlorous acid by competing with chloride for MPO. Furthermore, the oxidative products of the reaction of thiocyanate with MPO are relatively innocuous for human leukemic cells in culture. In contrast, bromide can support H2O2/MPO/halide apoptosis, but is less potent than chloride and it has no effect in the presence of physiological levels of chloride.

Comparison of low-density lipoprotein modification by myeloperoxidase-derived hypochlorous andhypobromous 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 2O 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 μM 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.

Eosinophil Peroxidase Oxidation of Thiocyanate: CHARACTERIZATION OF MAJOR REACTION PRODUCTS AND A POTENTIAL SULFHYDRYL-TARGETED CYTOTOXICITY SYSTEM

Although the pseudohalide thiocyanate (SCN(-)) is the preferred substrate for eosinophil peroxidase (EPO) in fluids of physiologic halide composition, the product(s) of this reaction have not been directly identified, and mechanisms underlying their cytotoxic potential are poorly characterized. We used nuclear magnetic resonance spectroscopy (NMR), electrospray ionization mass spectrometry, and quantitative chemical analysis to identify the principal reaction products of both the EPO/SCN(-)/H(2)O(2) system and activated eosinophils as roughly equimolar amounts of OSCN(-) (hypothiocyanite) and OCN(-) (cyanate). Red blood cells exposed to increasing concentrations of OSCN(-)/OCN(-) are first depleted of glutathione, after which glutathione S-transferase and glyceraldehyde-3-phosphate dehydrogenase then ATPases undergo sulfhydryl (SH) reductant-reversible inactivation before lysing. OSCN(-)/OCN(-) inactivates red blood cell membrane ATPases 10-1000 times more potently than do HOCl, HOBr, and H(2)O(2). Exposure of glutathione S-transferase to [(14)C]OSCN(-)/OCN(-) causes SH reductant-reversible disulfide bonding and covalent isotope labeling. We propose that EPO/SCN(-)/H(2)O(2) reaction products comprise a potential SH-targeted cytotoxic system that functions in striking contrast to HOCl, the highly but relatively indiscriminantly reactive product of the neutrophil myeloperoxidase system.