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.     

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.     

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.     

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.     

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.     

Interactions between selenium and methylmercury in rat brain

The interaction of selenium with methylmercury was investigated in brain of animals labeled with ⁷⁵SeO₃^2? and CH₃²⁰³Hg⁺. Brains were fractionated into subcellular components and the cytosol was further fractionated by chromatography on Sephadex G-150 and G-200. The main result of these studies was evidence suggesting a shift of ⁷⁵Se from the cytosol to the mitochondrial fraction in brain when CH₃Hg⁺ was given. Concurrent equimolar (10 μmoles/kg) selenite injections increased the uptake of Hg but did not alter ²⁰³Hg distribution in brain. Changing the dose of CH₃Hg⁺ from 1 to 38 μmoles/kg had little effect on Hg uptake (% of dose per g). Gel filtrations on Sephadex G-150 and G-200 revealed that ²⁰³Hg in cytosol followed a pattern more closely related to protein (A₂₈₀) than to ⁷⁵Se, although a considerable portion of both isotopes eluted with proteins in the void volume. Assays of whole brain homogenates revealed a slight reduction in glutathione peroxidase activity in CH₃Hg⁺-treated rats which was not seen when equimolar selenite was injected with the CH₃Hg⁺.     

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.     

Synergism between halide binding and proton transport in a CLC-type exchanger

The Cl-/H+ exchange-transporter CLC-ec1 mediates stoichiometric transmembrane exchange of two Cl- ions for one proton. A conserved tyrosine residue, Y445, coordinates one of the bound Cl- ions visible in the structure of this protein and is located near the intersection of the Cl- and H+ pathways. Mutants of this tyrosine were scrutinized for effects on the coupled transport of Cl- and H+ determined electrophysiologically and on protein structure determined crystallographically. Despite the strong conservation of Y445 in the CLC family, substitution of F or W at this position preserves wild-type transport behavior. Substitution by A, E, or H, however, produces uncoupled proteins with robust Cl- transport but greatly impaired movement of H+. The obligatory 2 Cl-/1 H+ stoichiometry is thus lost in these mutants. The structures of all the mutants are essentially identical to wild-type, but apparent anion occupancy in the Cl- binding region correlates with functional H+ coupling. In particular, as determined by anomalous diffraction in crystals grown in Br-, an electrophysiologically competent Cl- analogue, the well-coupled transporters show strong Br- electron density at the "inner" and "central" Cl- binding sites. However, in the uncoupled mutants, Br- density is absent at the central site, while still present at the inner site. An additional mutant, Y445L, is intermediate in both functional and structural features. This mutant clearly exchanges H+ for Cl-, but at a reduced H+-to-Cl- ratio; likewise, both the central and inner sites are occupied by Br-, but the central site shows lower Br- density than in wild-type (or in Y445F,W). The correlation between proton coupling and central-site occupancy argues that halide binding to the central transport site somehow facilitates movement of H+, a synergism that is not readily understood in terms of alternating-site antiport schemes.     

Synergism between Gibberellin and Auxin in the Growth of Intact Tomato

Gibberellin A_4&7 was more effective than gibberellic acid in increasing shoot elongation when applied to the apex of intact Lycopersicum esculentum seedlings of Tiny Tim, a dwarf cultivar, and Winsall, a tall cultivar. After 14 days, gibberellic acid and gibberellin A_4&7 stimulated growth of the dwarf more than the tall tomato. In tall tomato the application of indole-3-acetic acid alone (6.1 μg/plant) showed an inhibitory growth effect, but when applied with 17.5 μg per plant of gibberellic acid, it had a synergistic effect at 7 days but not at 14 days. When the auxin concentration was reduced to 0.61 μg per plant a synergistic effect was observed on tall plants at 7 and 14 days between indole-3-acetic acid and gibberellic acid. Application of gibberellin A_4&7 with auxin did not give a synergistic response in tall or dwarf tomato.     

Effect of seed soaking with thiols on the antioxidant enzymes and photosystem activities in wheat subjected to water stress

Photosystem 1 and 2 and antioxidant enzyme activities were determined in wheat (Triticum aestivum L. cv. Sonalika) leaves. Seedlings from both control seeds and seeds soaked in solutions like dithiothreitol, thioglycollic acid and thiourea were subjected to water stress induced by polyethylene glycol. Photosystem 1 and 2 activities were less inhibited by water stress due to seed soaking with sulphydryl compounds. The changes in activities of antioxidant enzymes induced by water stress were higher in seedlings from thiol-pretreated seeds than from water-soaked seeds.