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Superoxide modulates the activity of myeloperoxidase and optimizes the production of hypochlorous acid.

ABSTRACT: Myeloperoxidase catalyses the conversion of H2O2 and Cl- to hypochlorous acid (HOCl). It also reacts with O2- to form the oxy adduct (compound III). To determine how O2- affects the formation of HOCl, chlorination of monochlorodimedon by myeloperoxidase was investigated using xanthine oxidase and hypoxanthine as a source of O2- and H2O2. Myeloperoxidase was mostly converted to compound III, and H2O2 was essential for chlorination. At pH 5.4, superoxide dismutase (SOD) enhanced chlorination and prevented formation of compound III. However, at pH 7.8, SOD inhibited chlorination and promoted formation of the ferrous peroxide adduct (compound II) instead of compound III. We present spectral evidence for a direct reaction between compound III and H2O2 to form compound II, and for the reduction of compound II by O2- to regenerate native myeloperoxidase. These reactions enable compound III and compound II to participate in the chlorination reaction. Myeloperoxidase catalytically inhibited O2- -dependent reduction of Nitro Blue Tetrazolium. This inhibition is explained by myeloperoxidase undergoing a cycle of reactions with O2-, H2O2 and O2-, with compounds III and II as intermediates, i.e., by myeloperoxidase acting as a combined SOD/catalase enzyme. By preventing the accumulation of inactive compound II, O2- enhances the activity of myeloperoxidase. We propose that, under physiological conditions, this optimizes the production of HOCl and may potentiate oxidant damage by stimulated neutrophils.

SUBMITTER: Kettle AJ

PROVIDER: S-EPMC1149176 | biostudies-other | 1988 Jun

REPOSITORIES: biostudies-other

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Inhibition of myeloperoxidase-mediated hypochlorous acid production by nitroxides.

Project description:Tissue damage resulting from the extracellular production of HOCl (hypochlorous acid) by the MPO (myeloperoxidase)-hydrogen peroxide-chloride system of activated phagocytes is implicated as a key event in the progression of a number of human inflammatory diseases. Consequently, there is considerable interest in the development of therapeutically useful MPO inhibitors. Nitroxides are well established antioxidant compounds of low toxicity that can attenuate oxidative damage in animal models of inflammatory disease. They are believed to exert protective effects principally by acting as superoxide dismutase mimetics or radical scavengers. However, we show here that nitroxides can also potently inhibit MPO-mediated HOCl production, with the nitroxide 4-aminoTEMPO inhibiting HOCl production by MPO and by neutrophils with IC50 values of approx. 1 and 6 microM respectively. Structure-activity relationships were determined for a range of aliphatic and aromatic nitroxides, and inhibition of oxidative damage to two biologically-important protein targets (albumin and perlecan) are demonstrated. Inhibition was shown to involve one-electron oxidation of the nitroxides by the compound I form of MPO and accumulation of compound II. Haem destruction was also observed with some nitroxides. Inhibition of neutrophil HOCl production by nitroxides was antagonized by neutrophil-derived superoxide, with this attributed to superoxide-mediated reduction of compound II. This effect was marginal with 4-aminoTEMPO, probably due to the efficient superoxide dismutase-mimetic activity of this nitroxide. Overall, these data indicate that nitroxides have considerable promise as therapeutic agents for the inhibition of MPO-mediated damage in inflammatory diseases.

| S-EPMC4058678 | biostudies-literature

Fluorescent probes for monitoring myeloperoxidase-derived hypochlorous acid: a comparative study.

Project description:MPO-derived oxidants including HOCl contribute to tissue damage and the initiation and propagation of inflammatory diseases. The search for small molecule inhibitors of myeloperoxidase, as molecular tools and potential drugs, requires the application of high throughput screening assays based on monitoring the activity of myeloperoxidase. In this study, we have compared three classes of fluorescent probes for monitoring myeloperoxidase-derived hypochlorous acid, including boronate-, aminophenyl- and thiol-based fluorogenic probes and we show that all three classes of probes are suitable for this purpose. However, probes based on the coumarin fluorophore turned out to be not reliable indicators of the inhibitors' potency. We have also determined the rate constants of the reaction between HOCl and the probes and they are equal to 1.8 × 104 M-1s-1 for coumarin boronic acid (CBA), 1.1 × 104 M-1s-1 for fluorescein based boronic acid (FLBA), 3.1 × 104 M-1s-1 for 7-(p-aminophenyl)-coumarin (APC), 1.6 × 104 M-1s-1 for 3'-(p-aminophenyl)-fluorescein (APF), and 1 × 107 M-1s-1 for 4-thiomorpholino-7-nitrobenz-2-oxa-1,3-diazole (NBD-TM). The high reaction rate constant of NBD-TM with HOCl makes this probe the most reliable tool to monitor HOCl formation in the presence of compounds showing HOCl-scavenging activity.

| S-EPMC9166712 | biostudies-literature

The myeloperoxidase product hypochlorous acid generates irreversible high-density lipoprotein receptor inhibitors.

Project description:Elevated levels of advanced oxidation protein products have been described in several chronic inflammatory diseases, like chronic renal insufficiency, rheumatoid arthritis, and atherosclerosis. Recent findings revealed that advanced oxidation protein products are inhibitors of the major high-density lipoprotein receptor, scavenger receptor class B, type 1 (SR-BI). Here, we investigated which oxidation-induced structural alterations convert plasma albumin into a high-density lipoprotein-receptor inhibitor.Exposure of albumin to the physiological oxidant, hypochlorous acid, generated high-affinity SR-BI ligands. Protection of albumin-lysine residues before exposure to hypochlorous acid as well as regeneration of N-chloramines after oxidation of albumin completely prevented binding of oxidized albumin to SR-BI, indicating that modification of albumin-lysine residues is required to generate SR-BI ligands. Of particular interest, N-chloramines within oxidized albumin promoted irreversible binding to SR-BI, resulting in permanent receptor blockade. We observed that the SR-BI inhibitory activity of albumin isolated from chronic kidney disease patients correlated with the content of the myeloperoxidase-specific oxidation product 3-chlorotyrosine and was associated with alterations in the composition of high-density lipoprotein.Given that several potential atheroprotective activities of high-density lipoprotein are mediated by SR-BI, the present results raise the possibility that oxidized plasma albumin, through permanent SR-BI blockade, contributes to the pathophysiology of cardiovascular disease.

| S-EPMC4163628 | biostudies-literature

Uric acid disrupts hypochlorous acid production and the bactericidal activity of HL-60 cells.

Project description:Uric acid is the end product of purine metabolism in humans and is an alternative physiological substrate for myeloperoxidase. Oxidation of uric acid by this enzyme generates uric acid free radical and urate hydroperoxide, a strong oxidant and potentially bactericide agent. In this study, we investigated whether the oxidation of uric acid and production of urate hydroperoxide would affect the killing activity of HL-60 cells differentiated into neutrophil-like cells (dHL-60) against a highly virulent strain (PA14) of the opportunistic pathogen Pseudomonas aeruginosa. While bacterial cell counts decrease due to dHL-60 killing, incubation with uric acid inhibits this activity, also decreasing the release of the inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF- α). In a myeloperoxidase/Cl-/H2O2 cell-free system, uric acid inhibited the production of HOCl and bacterial killing. Fluorescence microscopy showed that uric acid also decreased the levels of HOCl produced by dHL-60 cells, while significantly increased superoxide production. Uric acid did not alter the overall oxidative status of dHL-60 cells as measured by the ratio of reduced (GSH) and oxidized (GSSG) glutathione. Our data show that uric acid impairs the killing activity of dHL-60 cells likely by competing with chloride by myeloperoxidase catalysis, decreasing HOCl production. Despite diminishing HOCl, uric acid probably stimulates the formation of other oxidants, maintaining the overall oxidative status of the cells. Altogether, our results demonstrated that HOCl is, indeed, the main relevant oxidant against bacteria and deviation of myeloperoxidase activity to produce other oxidants hampers dHL-60 killing activity.

| S-EPMC5952876 | biostudies-literature

A New and Fast-Response Fluorescent Probe for Monitoring Hypochlorous Acid Derived from Myeloperoxidase.

Project description:Hypochlorous acid (HOCl) has been implicated in numerous pathologies associated with an inflammatory component, but its selective and sensitive detection in biological settings remains a challenge. In this report, imaging of HOCl was realized with a thiomorpholine-based probe as derivative of nitrobenzothiadiazole (NBD-S-TM). The fluorescence is based on photoinduced electron transfer by using nitrobenzothiadiazole core as a donor and thiomorpholine substituent as an acceptor. NBD-S-TM showed high sensitivity and a fast response to HOCl k = (2.6 ± 0.2) × 107 M-1s-1 with a 1:1 stoichiometry. The detection limit for HOCl was determined to be 60 nM. Furthermore, the desirable features of NBD-S-TM for the detection of HOCl in aqueous solutions, such as its reliability at physiological pH, rapid fluorescence response, and biocompatibility, enabled its application in the detection of HOCl in myeloperoxidase enzymatic system. Moreover, NBD-S-TM exhibited excellent selectivity and sensitivity for HOCl over other biologically relevant species, such as hydrogen peroxide and peroxynitrite. The fluorescent S-oxidized product (NBD-S-TSO) is only formed in the presence of HOCl. Probing with NBD-S-TM may be helpful to further the development of high throughput screening assays to monitor the activity of myeloperoxidase.

| S-EPMC10459737 | biostudies-literature

Oxidation of neutrophil glutathione and protein thiols by myeloperoxidase-derived hypochlorous acid.

Project description:Neutrophils, when stimulated, generate reactive oxygen species including myeloperoxidase-derived HOCl. There is an associated decrease in reduced glutathione (GSH) concentration. We have shown that neutrophil GSH levels decrease on exposure to reagent HOCl, whereas the equivalent concentration of H2O2 had no effect. GSH loss occurred without cell lysis, was not reversible, and was accompanied by the loss of an equivalent proportion of the total protein thiols. No glutathione disulphide was formed. Studies with 35S-labelled cells indicated that much of the GSH lost was accounted for by mixed disulphides with protein and a product that co-migrated on HPLC with a novel compound formed in the reaction of HOCl and pure GSH. The properties of this compound are consistent with an intramolecular sulphonamide. Neutrophils stimulated with PMA lost 30-40% of their GSH and a similar proportion of protein thiols. Little glutathione disulphide was formed and the products were the same as seen with HOCl-treated cells. From the results and studies with inhibitors and scavengers, we conclude that HOCl was responsible for the GSH loss. Propargylglycine and buthionine sulphoximine, inhibitors of glutathione synthesis, enhanced GSH loss, but their effects were due to the production of long-lived chloramines that oxidized GSH with greater efficiency than HOCl, rather than to the inhibition of GSH synthesis. The lack of thiol selectivity by HOCl and irreversibility of oxidation means that GSH will provide limited antioxidant protection for thiol enzymes in stimulated neutrophils.

| S-EPMC1218791 | biostudies-other

Blue Laser Irradiation Decreases the ATP Level in Mouse Skin and Increases the Production of Superoxide Anion and Hypochlorous Acid in Mouse Fibroblasts.

Project description:Photobiomodulation studies have reported that blue light irradiation induces the production of reactive oxygen species. We investigated the effect of blue laser (405 nm) irradiation on the ATP levels in mouse skin and determined the types of reactive oxygen species and reactive nitrogen species using cultured mouse fibroblasts. Blue laser irradiation caused a decrease in the ATP level in the mouse skin and triggered the generation of superoxide anion and hypochlorous acid, whereas nitric oxide and peroxynitrite were not detected. Moreover, blue laser irradiation resulted in reduced cell viability. It is believed that the decrease in the skin ATP level due to blue light irradiation results from the increased levels of oxidative stress due to the generation of reactive oxygen species. This method of systematically measuring the levels of reactive oxygen species and reactive nitrogen species may be useful for understanding the effects of irradiation conditions.

| S-EPMC8869339 | biostudies-literature

Chlorination and oxidation of the extracellular matrix protein laminin and basement membrane extracts by hypochlorous acid and myeloperoxidase.

Project description:Basement membranes are specialized extracellular matrices that underlie arterial wall endothelial cells, with laminin being a key structural and biologically-active component. Hypochlorous acid (HOCl), a potent oxidizing and chlorinating agent, is formed in vivo at sites of inflammation via the enzymatic action of myeloperoxidase (MPO), released by activated leukocytes. Considerable data supports a role for MPO-derived oxidants in cardiovascular disease and particularly atherosclerosis. These effects may be mediated via extracellular matrix damage to which MPO binds. Herein we detect and quantify sites of oxidation and chlorination on isolated laminin-111, and laminin in basement membrane extracts (BME), by use of mass spectrometry. Increased modification was detected with increasing oxidant exposure. Mass mapping indicated selectivity in the sites and extent of damage; Met residues were most heavily modified. Fewer modifications were detected with BME, possibly due to the shielding effects. HOCl oxidised 30 (of 56 total) Met and 7 (of 24) Trp residues, and chlorinated 33 (of 99) Tyr residues; 3 Tyr were dichlorinated. An additional 8 Met and 10 Trp oxidations, 14 chlorinations, and 18 dichlorinations were detected with the MPO/H2O2/Cl- system when compared to reagent HOCl. Interestingly, chlorination was detected at Tyr2415 in the integrin-binding region; this may decrease cellular adhesion. Co-localization of MPO-damaged epitopes and laminin was detected in human atherosclerotic lesions. These data indicate that laminin is extensively modified by MPO-derived oxidants, with structural and functional changes. These modifications, and compromised cell-matrix interactions, may promote endothelial cell dysfunction, weaken the structure of atherosclerotic lesions, and enhance lesion rupture.

| S-EPMC6260226 | biostudies-literature

Macrophage migration inhibitory factor (MIF) enhances hypochlorous acid production in phagocytic neutrophils.

Project description:BackgroundMacrophage migration inhibitory factor (MIF) is an important immuno-regulatory cytokine and is elevated in inflammatory conditions. Neutrophils are the first immune cells to migrate to sites of infection and inflammation, where they generate, among other mediators, the potent oxidant hypochlorous acid (HOCl). Here, we investigated the impact of MIF on HOCl production in neutrophils in response to phagocytic stimuli.MethodsProduction of HOCl during phagocytosis of zymosan was determined using the specific fluorescent probe R19-S in combination with flow cytometry and live cell microscopy. The rate of phagocytosis was monitored using fluorescently-labeled zymosan. Alternatively, HOCl production was assessed during phagocytosis of Pseudomonas aeruginosa by measuring the oxidation of bacterial glutathione to the HOCl-specific product glutathione sulfonamide. Formation of neutrophil extracellular traps (NETs), an oxidant-dependent process, was quantified using a SYTOX Green plate assay.ResultsExposure of human neutrophils to MIF doubled the proportion of neutrophils producing HOCl during early stages of zymosan phagocytosis, and the concentration of HOCl produced was greater. During phagocytosis of P. aeruginosa, a greater fraction of bacterial glutathione was oxidized to glutathione sulfonamide in MIF-treated compared to control neutrophils. The ability of MIF to increase neutrophil HOCl production was independent of the rate of phagocytosis and could be blocked by the MIF inhibitor 4-IPP. Neutrophils pre-treated with MIF produced more NETs than control cells in response to PMA.ConclusionOur results suggest a role for MIF in potentiating HOCl production in neutrophils in response to phagocytic stimuli. We propose that this newly discovered activity of MIF contributes to its role in mediating the inflammatory response and enhances host defence.

| S-EPMC8047225 | biostudies-literature

Targeting Myeloperoxidase Activity and Neutrophil ROS Production to Modulate Redox Process: Effect of Ellagic Acid and Analogues.

Project description:Malaria is an infectious disease caused by a Plasmodium genus parasite that remains the most widespread parasitosis. The spread of Plasmodium clones that are increasingly resistant to antimalarial molecules is a serious public health problem for underdeveloped countries. Therefore, the search for new therapeutic approaches is necessary. For example, one strategy could consist of studying the redox process involved in the development of the parasite. Regarding potential drug candidates, ellagic acid is widely studied due to its antioxidant and parasite-inhibiting properties. However, its low oral bioavailability remains a concern and has led to pharmacomodulation and the synthesis of new polyphenolic compounds to improve antimalarial activity. This work aimed at investigating the modulatory effect of ellagic acid and its analogues on the redox activity of neutrophils and myeloperoxidase involved in malaria. Overall, the compounds show an inhibitory effect on free radicals as well as on the enzyme horseradish peroxidase- and myeloperoxidase (HRP/MPO)-catalyzed oxidation of substrates (L-012 and Amplex Red). Similar results are obtained with reactive oxygen species (ROS) produced by phorbol 12-mystate acetate (PMA)-activated neutrophils. The efficiency of ellagic acid analogues will be discussed in terms of structure-activity relationships.

| S-EPMC10254444 | biostudies-literature

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