Involvement of cysteine, serotonin and their analogues in peroxidase-oxidase reactions

Myeloperoxidase-oxidase reactions with close to physiological concentrations of thiols and phenols were studied. Cysteine was shown to be a myeloperoxidase-oxidase substrate when catalytic amounts of serotonin were added as cosubstrate. Penicillamine could be substituted for cysteine and acetaminophen could be substituted for serotonin. The properties of these peroxidase-oxidase reactions, e.g. the dependence on substrate and myeloperoxidase concentration, reduced oxygen species, metal ions and pH, were studied. Also, eosinophil, lacto- and horseradish peroxidase could catalyse these reactions.     

Mechanism of melatonin-induced oscillations in the peroxidase-oxidase reaction

Melatonin induces oscillations in the peroxidase-oxidase (PO) reaction catalyzed by horseradish peroxidase. We present here studies of the effect of pH, enzyme concentration, and concentration of melatonin on the oscillation frequency. We also present a mechanistic model to explain the experimentally observed changes in oscillation frequency. Using the data obtained here we are able to predict that oscillations will also occur in the PO reaction catalyzed by myeloperoxidase. Myeloperoxidase is an important protein in activated neutrophils and we provide evidence that the oscillations of NAD(P)H, superoxide and hydrogen peroxide in these cells may involve this enzyme. Thus, our experimental system can be considered a model system for the nonrespiratory oxygen metabolism in activated neutrophils and other similar cells participating in the defence against invading pathogens.     

Computer simulation of sustained oscillations in peroxidase-oxidase reaction

A system of differential equations of second order exhibiting transitional behaviour and sustained oscillations has been obtained for a complete scheme of the peroxidase-oxidase reaction. The concentrations of hydrogen peroxide and of hydrogen donor radicals are slow variables of the system. The most essential reactions responsible for oscillations have been selected. Analysis of the system in phase plane and in parameter space has been carried out. The dependence of oscillation period and amplitude on the parameter values has been investigated.     

Bromination and chlorination reactions of myeloperoxidase at physiological concentrations of bromide and chloride

Myeloperoxidase and eosinophil peroxidase use hydrogen peroxide to oxidize halides and thiocyanate to their respective hypohalous acids. Myeloperoxidase produces mainly hypochlorous acid and hypothiocyanite. Hypobromous acid and hypothiocyanite are the major products of eosinophil peroxidase. We have investigated the ability of myeloperoxidase to produce hypobromous acid in the presence of physiological concentrations of chloride and bromide. In accord with previous studies, between pH 5 and 7, myeloperoxidase converted about 90% of available hydrogen peroxide to hypochlorous acid and the remainder to hypobromous acid. Above pH 7, there was an abrupt rise in the yield of hypobromous acid. At pH 7.8, it accounted for 40% of the hydrogen peroxide. Bromide, at physiological concentrations, promoted a dramatic increase in bromination of human serum albumin catalyzed by myeloperoxidase. The level of 3-bromotyrosine increased to 16-fold greater than that for 3-chlorotyrosine. Chlorination of tyrosyl residues was not affected by bromide. With reagent hypohalous acids, bromination of tyrosyl residues was considerably more facile than chlorination. Hypochlorous acid promoted bromination to only a limited extent, which ruled out transhalogenation as a substantive route to 3-bromotyrosine. Chloramines and bromamines were also formed on albumin. Bromamines decayed much faster than chloramines and rapidly gave rise to protein carbonyls. We conclude that at physiological concentrations of chloride and bromide, hypobromous acid can be a major oxidant produced by myeloperoxidase. Its production in vivo will depend on pH and the concentration of bromide. Once produced, hypobromous acid will react with proteins to form bromamines, carbonyls, and brominated tyrosine residues. Consequently, 3-bromotyrosine should be considered as an oxidative product of myeloperoxidase and cannot be used as a specific biomarker for eosinophil peroxidase.     

A theoretical three-dimensional model for lactoperoxidase and eosinophil peroxidase,built on the scaffold of the myeloperoxidase X-ray structure

 Lactoperoxidase (LPO), eosinophil peroxidase (EPO) and myeloperoxidase (MPO) belong to the class of haloperoxidases, a group of mammalian enzymes able to catalyze the peroxidative oxidation of halides and pseudohalides, such as thiocyanate. They all play a key role in the development of antibacterial activity. The homology in their functional role is emphasized by the striking similarity of their primary structures. A theoretical model for the three-dimensional structure of LPO and EPO has been developed on the basis of the X-ray structure of MPO, a high degree of similarity having been found in their sequences. Evidence supporting the hypothesis of an ester linkage between heme and apoprotein in LPO and EPO, originally proposed by Hultquist and Morrison is discussed. Received: 2 May 1996 / Accepted: 25 July 1996     

Determination of reduced and oxidized homocysteine and related thiols in plasma by thiol-specific pre-column derivatization and capillary electrophoresis with laser-induced fluorescence detection

A new sensitive and rapid capillary electrophoresis (CE) assay for measuring reduced and oxidized thiols in human plasma has been developed. To prevent oxidation of the thiols, whole blood was immediately centrifuged after collection and the plasma proteins were precipitated with perchloric acid. The reduced thiols in the supernatant were derivatized quantitatively at 25°C, pH 7.5 with a fluorescent reagent, fluorescein-5-maleimide (FM). The total plasma concentration of thiols, including the fraction coupled to proteins, was assayed after an initial reduction of the disulfide linkage in plasma with dithiothreitol. The separation of FM-thiols was performed in an acetonitrile/10 mM sodium phosphate–50 mM SDS buffer [25:75 (v/v); pH 7.0] using a fused-silica capillary (57 cm×75 μm I.D.) at 45°C. A 3-mW argon-ion laser (λ_ex 488 nm/λ_em 520 nm) was employed for FM-thiol detection. With the electric field of 530 V/cm, the time needed for the separation of FM-homocysteine, FM-glutathione and FM-N-acetylcysteine was less than 8 min. The lower limit of detection was 3 μM for the total thiols and 10 nM for the reduced thiols. The method was applied to the determination of homocysteine levels in plasma from patients with end-stage renal disease.     

Formation of reactive halide species by myeloperoxidase and eosinophil peroxidase

The formation of chloro- and bromohydrins from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine following incubation with myeloperoxidase or eosinophil peroxidase in the presence of hydrogen peroxide, chloride and/or bromide was analysed by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. These products were only formed below a certain pH threshold value, that increased with increasing halide concentration. Thermodynamic considerations on halide and pH dependencies of reduction potentials of all redox couples showed that the formation of a given reactive halide species in halide oxidation coupled with the reduction of compound I of heme peroxidases is only possible below a certain pH threshold that depends on halide concentration. The comparison of experimentally derived and calculated data revealed that Cl(2), Br(2), or BrCl will primarily be formed by the myeloperoxidase-H(2)O(2)-halide system. However, the eosinophil peroxidase-H(2)O(2)-halide system forms directly HOCl and HOBr.     

The basics of thiols and cysteines in redox biology and chemistry

Cysteine is one of the least abundant amino acids, yet it is frequently found as a highly conserved residue within functional (regulatory, catalytic, or binding) sites in proteins. It is the unique chemistry of the thiol or thiolate group of cysteine that imparts to functional sites their specialized properties (e.g., nucleophilicity, high-affinity metal binding, and/or ability to form disulfide bonds). Highlighted in this review are some of the basic biophysical and biochemical properties of cysteine groups and the equations that apply to them, particularly with respect to pK_a and redox potential. Also summarized are the types of low-molecular-weight thiols present in high concentrations in most cells, as well as the ways in which modifications of cysteinyl residues can impart or regulate molecular functions important to cellular processes, including signal transduction.     

Determination of cellular thiols and glutathione-related enzyme activities: versatility of high-performance liquid chromatography–spectrofluorimetric detection

A high-performance liquid chromatography (HPLC) method to determine the most important cellular thiols [reduced glutathione (GSH), cysteine, γ-glutamylcysteine and cysteinylglycine] is described. Separation relies upon isocratic ion-pairing reversed-phase chromatography and detection is operated by spectrofluorimetry coupled with post-column derivatization reactions using either N-(1-pyrenyl)maleimide (NPM) or ortho-phthalaldehyde (OPA). When OPA is used without co-reagent, only GSH and γ-glutamylcysteine are detected (heterobifunctional reaction). However, either the OPA reaction in the presence of glycine in the mobile phase (thiol-selective reaction) or NPM allows the detection of all the cited thiols. The HPLC system has been validated as concerning linearity, accuracy and precision. The low detection limits reached (in the pmol range for each thiol injected) allow the screening and the quantification of thiols (as NPM derivatives) in V79cl and V79HGGT cells as well as the measurement of two cytosolic enzymes related to the glutathione synthesis, using the heterobifunctional OPA reaction.     

Fluorescent sensors based on [12]aneN3-modified BODIPY: Discrimination of different biological thiols in aqueous solution and living cells

Two fluorescent probes, 1 and 2, derived from borondipyrromethene (BODIPY) modified with macrocyclic polyamine [12]aneN₃, were synthesized and applied in the discrimination of cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) with absorption and fluorescent spectroscopy in comparison. It was found that Boc-protected 1 showed highly sensitive and selective recognition of GSH over Cys and Hcy; while probe 2 was able to distinguish the three different thiols due to their different reactivities. With its water-solubility, rapid responsiveness, high sensitivity and low cytotoxicity, probe 2 was successfully applied in the fast detection of three biothiols in living cells.     

Influence of pH on the reactivity of diphenyl ditelluride with thiols and anti-oxidant potential in rat brain

Thiol oxidation by diphenyl ditelluride is a favorable reaction and may be responsible for alteration in regulatory or signaling pathways. We have measured rate constants for reactions of diphenyl ditelluride with cysteine, dimercaptosuccinic acid, glutathione and dithiothreitol in phosphate buffer. The relative reactivities of the different thiols with diphenyl ditelluride were independent of the pK_a of the thiol group, such that at pH 7.4, cysteine and dithiothreitol were the most reactive and low reactivity was observed with glutathione and dimercaptosuccinic acid. The reactivity of diphenyl ditelluride was not modified by change in pH. Rate of oxidation increased with increasing pH for all thiols except dimercaptosuccinic acid, where the rate of oxidation was faster at low pH. The lipid peroxidation product malonaldehyde (MDA) was measured in rat brain homogenate and phospholipids extract from egg yolk after incubation in phosphate buffer at various pHs ranging from 7.4 to 5.4. TBARS production increased when homogenates were incubated in the pH (5.4-6.8) medium both in the absence and presence of Fe(II). These data indicate that lipid peroxidation processes, mediated by iron, are enhanced with decreasing pH. The iron mobilization may come from reserves where it is weakly bound. Diphenyl ditelluride significantly protected TBARS production at all studied pH values in a concentration dependent manner in brain homogenate. This study provides in vitro evidence for acidosis induced oxidative stress and anti-oxidant action of diphenyl ditelluride.     

Active site structure and catalytic mechanisms of human peroxidases

Myeloperoxidase (MPO), eosinophil peroxidase, lactoperoxidase, and thyroid peroxidase are heme-containing oxidoreductases (EC 1.7.1.11), which bind ligands and/or undergo a series of redox reactions. Though sharing functional and structural homology, reflecting their phylogenetic origin, differences are observed regarding their spectral features, substrate specificities, redox properties, and kinetics of interconversion of the relevant redox intermediates ferric and ferrous peroxidase, compound I, compound II, and compound III. Depending on substrate availability, these heme enzymes path through the halogenation cycle and/or the peroxidase cycle and/or act as poor (pseudo-)catalases. Based on the published crystal structures of free MPO and its complexes with cyanide, bromide and thiocyanate as well as on sequence analysis and modeling, we critically discuss structure-function relationships. This analysis highlights similarities and distinguishing features within the mammalian peroxidases and intents to provide the molecular and enzymatic basis to understand the prominent role of these heme enzymes in host defense against infection, hormone biosynthesis, and pathogenesis.     

Evidence for the formation of Michael adducts from reactions of (E,E)-muconaldehyde with glutathione and other thiols

Glutathione induces the rapid isomerization of (Z,Z)-muconaldehyde to (E,E)-muconaldehyde via (E,Z)-muconaldehyde, probably via reversible Michael addition of the thiol to one of the enal moieties of the muconaldehyde. Reactions of (E,E)-muconaldehyde with glutathione (in the presence and absence of equine glutathione S-transferase), phenylmethanethiol, N-acetyl-l-cysteine, and N-acetyl-l-cysteine methyl ester were investigated using mass spectrometric techniques. In each case, evidence was obtained for the formation of Michael adducts, e.g., reaction between (E,E)-muconaldehyde and glutathione gave 4-glutathionyl-hex-2-enedial and 3,4-bis-glutathionyl-hexanedial. These experiments suggest that (Z,Z)-muconaldehyde, a putative metabolite of benzene, could lead to the long established urinary metabolite of benzene, (E,E)-muconic acid, via glutathione-mediated isomerization to (E,E)-muconaldehyde.     

Reactivity of selenium-containing compounds with myeloperoxidase-derived chlorinating oxidants: Second-order rate constants and implications for biological damage

Hypochlorous acid (HOCl) and N-chloramines are produced by myeloperoxidase (MPO) as part of the immune response to destroy invading pathogens. However, MPO also plays a detrimental role in inflammatory pathologies, including atherosclerosis, as inappropriate production of oxidants, including HOCl and N-chloramines, causes damage to host tissue. Low molecular mass thiol compounds, including glutathione (GSH) and methionine (Met), have demonstrated efficacy in scavenging MPO-derived oxidants, which prevents oxidative damage in vitro and ex vivo. Selenium species typically have greater reactivity toward oxidants compared to the analogous sulfur compounds, and are known to be efficient scavengers of HOCl and other hypohalous acids produced by MPO. In this study, we examined the efficacy of a number of sulfur and selenium compounds to scavenge a range of biologically relevant N-chloramines and oxidants produced by both isolated MPO and activated neutrophils and characterized the resulting selenium-derived oxidation products in each case. A dose-dependent decrease in the concentration of each N-chloramine was observed on addition of the sulfur compounds (cysteine, methionine) and selenium compounds (selenomethionine, methylselenocysteine, 1,4-anhydro-4-seleno-L-talitol, 1,5-anhydro-5-selenogulitol) studied. In general, selenomethionine was the most reactive with N-chloramines (k₂ 0.8–3.4×10³ M^–1 s^–1) with 1,5-anhydro-5-selenogulitol and 1,4-anhydro-4-seleno-L-talitol (k₂ 1.1–6.8×10² M^–1 s^–1) showing lower reactivity. This resulted in the formation of the respective selenoxides as the primary oxidation products. The selenium compounds demonstrated greater ability to remove protein N-chloramines compared to the analogous sulfur compounds. These reactions may have implications for preventing cellular damage in vivo, particularly under chronic inflammatory conditions.     

Origin, structure, and biological activities of peroxidases in human saliva

Human whole saliva contains two peroxidases, salivary peroxidase (hSPO) and myeloperoxidase (hMPO), which are part of the innate host defence in oral cavity. Both hSPO as well as human milk lactoperoxidase (hLPO) are coded by the same gene, but to what extent the different producing glands, salivary and mammary glands, affect the final conformation of the enzymes is not known. In human saliva the major function of hSPO and hMPO is to catalyze the oxidation of thiocyanate (SCN(-)) in the presence of hydrogen peroxide (H(2)O(2)) resulting in end products of wide antimicrobial potential. In addition cytotoxic H(2)O(2) is degraded. Similar peroxidation reactions inactivate some mutagenic and carcinogenic compounds, which suggests another protective mechanism of peroxidases in human saliva. Although being target of an active antimicrobial research, the structure-function relationships of hSPO are poorly known. However, recently published method for recombinant hSPO production offers new tools for those investigations.     

Heme-protein covalent bonds in peroxidases and resistance to heme modification during halide oxidation

Plant peroxidases, as typified by horseradish peroxidase (HRP), primarily catalyze the one-electron oxidation of phenols and other low oxidation potential substrates. In contrast, the mammalian homologues such as lactoperoxidase (LPO) and myeloperoxidase primarily oxidize halides and pseudohalides to the corresponding hypohalides (e.g., Br(-) to HOBr, Cl(-) to HOCl). A further feature that distinguishes the mammalian from the plant and fungal enzymes is the presence of two or more covalent bonds between the heme and the protein only in the mammalian enzymes. The functional roles of these covalent links in mammalian peroxidases remain uncertain. We have previously reported that HRP can oxidize chloride and bromide ions, but during oxidation of these ions undergoes autocatalytic modification of its heme vinyl groups that virtually inactivates the enzyme. We report here that autocatalytic heme modification during halide oxidation is not unique to HRP but is a general feature of the oxidation of halide ions by fungal and plant peroxidases, as illustrated by studies with Arthromyces ramosus and soybean peroxidases. In contrast, LPO, a prototypical mammalian peroxidase, is protected from heme modification and its heme remains intact during the oxidation of halide ions. These results support the hypothesis that the covalent heme-protein links in the mammalian peroxidases protect the heme from modification during the oxidation of halide ions.     

The peroxidase isozymes of the wheat kernel: tissue and substrate specificity and their chromosomal location

Summary Peroxidase isozymes were studied in the Triticum aestivum L. kernel and in nullisomic-tetrasomic and ditelocentric combinations of Chinese Spring wheat. Analyses were carried out on different parts of dry kernels (embryo plus scutellum and endosperm) using polyacrylamide and starch gel electrophoresis, different electrophoretic buffer systems and various staining methods. The peroxidase isozymes showed a low substrate-specificity and a high tissue-specificity. The embryo plus scutellum and the endosperm always presented different peroxidase patterns. Endosperm peroxidases were associated with chromosome arms 7DS, 4BL and 7AS; whereas the embryo plus scutellum isozymes were related to chromosome arms 3AL, 3BL and 3DS. The different results obtained using various electrophoretic techniques are due to the buffer system used. All staining procedures employed revealed the same peroxidase isozymes.     

The photoprotein pholasin as a luminescence substrate for detection of superoxide anion radicals and myeloperoxidase activity in stimulated neutrophils

Pholasin, the photoprotein of the common piddock Pholas dactylus, emits an intense luminescence upon oxidation. The contribution of superoxide anion radicals and myeloperoxidase (MPO) to Pholasin luminescence in stimulated neutrophils was investigated. Data on Pholasin luminescence were compared with results of superoxide anion radical generation detected by the cytochrome c test as well as with the release of elastase and MPO. In N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulated neutrophils, most of the luminescence is caused by superoxide anion radicals, whereas MPO shows only a small effect as shown by coincubation with superoxide dismutase (SOD) as well as potassium cyanide (KCN), an inhibitor of MPO. However, both, O₂^- and MPO contribute to light emission in fMLP/cytochalasin B and phorbol myristoyl acetate (PMA) stimulated cells. Thus, the kinetics of O₂^- generation and MPO release can be very well detected by Pholasin luminescence in stimulated neutrophils.

Degranulation of azurophilic granules was assessed using an ELISA test kit for released MPO or detection of elastase activity with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide in the supernatant of stimulated cells. Both approaches revealed concurrently similar results concerning the amount and kinetics of enzyme release with data of Pholasin luminescence. Both, cytochrome c measurements and Pholasin luminescence indicate that fMLP/cytochalasin B and PMA stimulated neutrophils produce more O₂^- than fMLP stimulated cells. Thus, Pholasin luminescence can be used to detect, sensitively and specifically, O₂^- production and MPO release from stimulated neutrophils.