The oxidation of oxytocin and vasopressin by peroxidase/H2O2 system

Summary Oxytocin and vasopressin are oxidized by horseradish peroxidase and by lactoperoxidase, in the presence of hydrogen peroxide. Spectrophotometric measurements are indicative of the formation of dityrosine. Kinetic parameters indicate that the affinity of horseradish peroxidase is slightly higher for oxytocin with respect to vasopressin and that the two hormones are better substrates for both peroxidases than free tyrosine.     

Hemin/nitrite/H2O2 induces brain homogenate oxidation and nitration: effects of some flavonoids

Oxidative injury has been implicated in the pathogenesis of numerous neurodegenerative diseases. Recently, it has been found that with the existence of hydrogen peroxide and nitrite, hemin catalyzes protein nitration. We hypothesize under certain pathological conditions, hemin catalyzed protein nitration may happen in the brain. In this paper, the effects of three flavonoids, i.e. quercetin, catachin and baicalein on hemin/nitrite/H2O2 induced brain homogenate oxidation and nitration were studied. The results showed that hemin/nitrite/H2O2 system could effectively induce brain homogenate protein oxidation and nitration. Quercetin, catachin and baicalein dose-dependently inhibited hemin/nitrite/H2O2 system-induced protein nitration in a dose-dependent manner, the inhibition of protein nitration was in the order of quercetin>catachin>baicalein. These compounds also inhibited hemin/H2O2 system-induced lipid peroxidation, the inhibition order was baicalein >quercetin>catachin. However, these flavonoids showed marginal effect on hemin/nitrite/H2O2 system caused protein oxidation and thiol oxidation. The inhibition activities of flavonoids on hemin/nitrite/H2O2 system-induced protein nitration may closely relate to their radical scavenging activities, since the inhibition order of protein nitration is the same as the radical scavenging order. These results indicate hemin/nitrite/H2O2 system induces different types of oxidative assault on bio-molecules. Flavonoids could act as antioxidants inhibiting ROS and RNS caused brain damage.     

A novel and efficient polymerization of lignosulfonates by horseradish peroxidase/H2O2 incubation

Lignosulfonates(LSs), by-products from chemical pulping processes, are low-value products with limited dispersion properties. The ability of commercially available horseradish peroxidase (HRP) to polymerize LS macromolecules and improve the dispersion properties of LSs was investigated. The polymerization of LSs proceeded efficiently under mild reaction conditions in an aqueous solution with HRP/H₂O₂. Gel permeation chromatography showed a significant increase in weight-average molecular weight (M _w ) of sulfonated kraft lignin and sodium lignosulfonate (NaLS) by 8.5-fold and 4.7-fold, respectively. The mechanism of polymerization was investigated by elemental analysis, surface charge measurement, headspace gas chromatography, infrared spectroscopy (IR), and hydrogen nuclear magnetic resonance spectrometry (¹H-NMR). The functional group measurements indicated that HRP incubation did not reduce the sulfonic group content. However, it decreased the phenolic and methoxyl group contents. As the phenolic group content decreased, M _w increased as a power function. The polymerization was proposed to involve the random coupling of phenoxy radical intermediates. The radicals coupled with each other to form different inter-unit linkages, most of which were the β-O-4’ type, as the ¹H-NMR spectra indicated. Moreover, the HRP/H₂O₂ incubation induced a significant improvement in the adsorption and dispersion properties of LSs. Therefore, the HRP/H₂O₂ incubation is a promising approach for industrial applications of LSs.     

A novel oxidation of the carcinogen N-hydroxy-N-2-fluorenylacetamide catalyzed by peroxidase/H2O2/Br-

N-Hydroxy-N-2-fluorenylacetamide, a proximate carcinogenic metabolite of N-2-fluorenylacetamide, is oxidized largely to 2-nitrosofluorene by lactoperoxidase or extract of peroxidative activity of rat uterus in an H2O2- and Br- -dependent reaction. Evidence is presented that the oxidizing species includes OBr- (HOBr). This novel oxidation may be involved in carcinogenesis by N-arylhydroxamic acids.     

Singlet oxygen formation by a peroxidase, H2O2 and halide system.

Evidence for singlet oxygen formation has been obtained for the lactoperoxidase, H2O2 and bromide system by monitoring 2,3-diphenylfuran and diphenylisobenzofuran oxidation, O2 evolution, and chemiluminescence. This could provide an explanation for the cytotoxic and microbicidal activity of peroxidases and polymorphonuclear leukocytes. Evidence for singlet oxygen formation included the following. (a) Chemiluminescence accompanying the enzymic reaction was doubled in a deuterated buffer and inhibited by singlet oxygen traps. (b) The singlet oxygen traps, diphenylfuran and diphenylisobenzofuran, were oxidized to their known singlet oxygen oxidation products in the presence of lactoperoxidase, hydrogen peroxide and bromide. (c) The rate of oxidation of diphenylfuran and diphenylisobenzofuran was inhibited when monitored in the presence of known singlet oxygen traps or quenchers. (d) Oxygen evolution from the enzymic reaction was inhibited by singlet oxygen traps but not by singlet oxygen quenchers. (e) The traps or quenchers which were effective inhibitors in the experiments above did not inhibit peroxidase activity, were not competitive peroxidase substrates and did not react with the hypobromite intermediate since they did not inhibit hydrogen peroxide consumption by the enzyme. Using these criteria, various biological molecules were tested for their reactivity with singlet oxygen. Furthermore, by studying their effect on oxygen release by the enzymic reaction, it could be ascertained whether they were acting as singlet oxygen traps or quenchers.     

H2O2 and (.)NO scavenging by Mycobacterium leprae truncated hemoglobin O

Kinetics of ferric Mycobacterium leprae truncated hemoglobin O (trHbOFe(III)) oxidation by H₂O₂ and of trHbOFe(IV)O reduction by NO and NO₂⁻ are reported. The value of the second-order rate constant for H₂O₂-mediated oxidation of trHbOFe(III) is 2.4 × 10³ M⁻¹ s⁻¹. The value of the second-order rate constant for NO-mediated reduction of trHbOFe(IV)O is 7.8 × 10⁶ M⁻¹ s⁻¹. The value of the first-order rate constant for trHbOFe(III)ONO decay to the resting form trHbOFe(III) is 2.1 × 10¹ s⁻¹. The value of the second-order rate constant for NO₂⁻-mediated reduction of trHbOFe(IV)O is 3.1 × 10³ M⁻¹ s⁻¹. As a whole, trHbOFe(IV)O, generated upon reaction with H₂O₂, catalyzes NO reduction to NO₂⁻. In turn, NO and NO₂⁻ act as antioxidants of trHbOFe(IV)O, which could be responsible for the oxidative damage of the mycobacterium. Therefore, Mycobacterium leprae trHbO could be involved in both H₂O₂ and NO scavenging, protecting from nitrosative and oxidative stress, and sustaining mycobacterial respiration.     

Inhibitory and enhancing effects of NO on H2O2 toxicity: Dependence on the concentrations of NO and H2O2

Nitric oxide (NO) has been shown to both enhance hydrogen peroxide (H₂O₂) toxicity and protect cells against H₂O₂ toxicity. In order to resolve this apparent contradiction, we here studied the effects of NO on H₂O₂ toxicity in cultured liver endothelial cells over a wide range of NO and H₂O₂ concentrations. NO was generated by spermine NONOate (SpNO, 0.001–1 mM), H₂O₂ was generated continuously by glucose/glucose oxidase (GOD, 20–300 U/l), or added as a bolus (200 μM). SpNO concentrations between 0.01 and 0.1 mM provided protection against H₂O₂-induced cell death. SpNO concentrations >0.1 mM were injurious with low H₂O₂ concentrations, but protective at high H₂O₂ concentrations. Protection appeared to be mainly due to inhibition of lipid peroxidation, for which SpNO concentrations as low as 0.01 mM were sufficient. SpNO in high concentration (1 mM) consistently raised H₂O₂ steady-state levels in line with inhibition of H₂O₂ degradation. Thus, the overall effect of NO on H₂O₂ toxicity can be switched within the same cellular model, with protection being predominant at low NO and high H₂O₂ levels and enhancement being predominant with high NO and low H₂O₂ levels.     

Mechanisms of acetylcholine-dependent production of H2O2 and NO2- by stromal cells of endometrium

The metabolism of NO(NO2-) and H2O2(O2-) by stroma cells of pig endometrium is NAD(P)H and glutathione-dependent process. The efficiency of biosynthesis and utilization of these metabolites appreciably depends on the state of SH-groups of the conforming ferment systems. And the reversible oxidation of SH-groups (maybe by the reaction products) results in the drop of biosynthesis rate. The NO and H2O2 metabolism is also defined by the state of oxidative metabolism of arachidonic acid (depending on salicylate), and also intensity of a course of redox-processes on plasmalemma (is regulated by cytochrome c). The NO2- biosynthesis by stroma cells is strongly inhibited by the agents, which super produce H2O2(O2-) (salicylate and cytochrome c). The NO(NO2-) and H2O2(O2-) metabolism at stimulation by acetylcholine is of cyclic character, and the infringement of any link during biosynthesis or utilization of these compositions results in losses of cyclicity. In contrast to this the formation of nitrosoglutathione with time achieves the saturation, which reflects its buffer and depositing with respect to NO function and permits to consider formation of the latter as one of mechanisms of effective utilization of NO(NO2-)--by the stroma cells.     

Spin trapping of the azidyl radical in azide/catalase/H2O2 and various azide/peroxidase/H2O2 peroxidizing systems

The azidyl radical is formed during the oxidation of sodium azide by the catalase/hydrogen peroxide system, as detected by the ESR spin-trapping technique. The oxidation of azide by horseradish peroxidase, chloroperoxidase, lactoperoxidase, and myeloperoxidase also forms azidyl radical. It is suggested that the evolution of nitrogen gas and nitrogen oxides reported in the azide/catalase/hydrogen peroxide system results from reactions of the azidyl radical. The azide/horseradish peroxidase/hydrogen peroxide system consumes oxygen, and this oxygen uptake is inhibited by the spin trap 5,5-dimethyl-1-pyrroline-N-oxide, presumably due to the competition with oxygen for the azidyl radical. Although azide is used routinely as an inhibitor of myeloperoxidase and catalase, some consideration should be given to the biochemical consequences of the formation of the highly reactive azidyl radical by the peroxidase activity of these enzymes.     

Scavenging of H2O2 and production of oxygen by horseradish peroxidase

Peroxidases catalyze many reactions, the most common being the utilization of H2O2 to oxidize numerous substrates (peroxidative mode). Peroxidases have also been proposed to produce H2O2 via utilization of NAD(P)H, thus providing oxidant either for the first step of lignification or for the "oxidative burst" associated with plant-pathogen interactions. The current study with horseradish peroxidase characterizes a third type of peroxidase activity that mimics the action of catalase; molecular oxygen is produced at the expense of H2O2 in the absence of other reactants. The oxygen production and H2O2-scavenging activities had temperature coefficients, Q10, of nearly 3 and 2, which is consistent with enzymatic reactions. Both activities were inhibited by autoclaving the enzyme and both activities had fairly broad pH optima in the neutral-to-alkaline region. The apparent Km values for the oxygen production and H2O2-scavenging reactions were near 1.0 mM H2O2. Irreversible inactivation of horseradish peroxidase by exposure to high concentrations of H2O2 coincided with the formation of an absorbance peak at 670 nm. Addition of superoxide dismutase (SOD) to reaction mixtures accelerated the reaction, suggesting that superoxide intermediates were involved. It appears that horseradish peroxidase is capable of using H2O2 both as an oxidant and as a reductant. A model is proposed and the relevance of the mechanism in plant-bacterial systems is discussed.     

Competing peroxidase and oxidase reactions in scopoletin-dependent H2O2-initiated oxidation of NADH by horseradish peroxidase

Addition of NADH inhibited the peroxidative loss of scopoletin in presence of horseradish and H₂O₂ and decreased the ratio of scopoletin (consumed):H₂O₂ (added). Concomitantly NADH was oxidized and oxygen was consumed with a stoichiometry of NADH:O₂ of 2:1. On step-wise addition of a small concentration of H₂O₂ a high rate of NADH oxidation was obtained for a progressively decreasing time period followed by termination of the reaction with NADH:H₂O₂ ratio decreasing from about 40 to 10. The rate of NADH oxidation increased linearly with increase in scopoletin concentration. Other phenolic compounds including p-coumarate also supported this reaction to a variable degree. A 418-nm absorbing compound accumulated during oxidation of NADH. The effectiveness of a small concentration of H₂O₂ in supporting NADH oxidation increased in presence of SOD and decreased in presence of cytochrome c, but the reaction terminated even in their presence. The results indicate that the peroxidase is not continuously generating H₂O₂ during scopoletin-mediated NADH oxidation and that both peroxidase and oxidase reactions occur simultaneously competing for an active form of the enzyme.     

Heterotrophic nitrification by Alcaligenes faecalis: NO2-, NO3-, N2O, and NO production in exponentially growing cultures

Heterotrophic nitrification by Alcaligenes faecalis DSM 30030 was not restricted to media containing organic forms of nitrogen. In both peptone-meat extract and defined media with ammonium and citrate as the sole nitrogen and carbon sources, respectively, NO2-, NO3-, NO, and N2O were produced under aerobic growth conditions. Heterotrophic nitrification was not attributable to old or dying cell populations. Production of NO2-, NO3-, NO, and N2O was detectable shortly after cultures started growth and proceeded exponentially during the logarithmic growth phase. NO2- and NO3- production rates were higher for cultures inoculated in media with pH values below 7 than for those in media at alkaline pH. Neither assimilatory nor dissimilatory nitrate or nitrite reductase activities were detectable in aerobic cultures.     

Oxidation of cyanide to the cyanyl radical by peroxidase/H2O2 systems as determined by spin trapping

The cyanyl radical was formed during the oxidation of potassium or sodium cyanide by horseradish peroxidase, lactoperoxidase, chloroperoxidase, NADH peroxidase, or methemoglobin in the presence of hydrogen peroxide. The spin adducts of the cyanyl radical with 5,5-dimethyl-1-pyrroline-N-oxide and N-tert-butyl-alpha-phenylnitrone were quite stable at neutral pH. The identity of these spin adducts could be demonstrated using 13C-labeled cyanide and by comparison with the spin adducts of the formamide radical, a hydrolysis product of the cyanyl radical adduct. The enzymatic conversion of cyanide to cyanyl radical by peroxidases should be considered in addition to its well-known role as a metal ligand. Furthermore, since cyanide is used routinely as an inhibitor of peroxidases, some consideration should be given to the biochemical consequences of this formation of the cyanyl radical by the catalytic activity of these enzymes.     

Heterotrophic nitrification by Alcaligenes faecalis: NO2-, NO3-, N2O, and NO production in exponentially growing cultures.

Heterotrophic nitrification by Alcaligenes faecalis DSM 30030 was not restricted to media containing organic forms of nitrogen. In both peptone-meat extract and defined media with ammonium and citrate as the sole nitrogen and carbon sources, respectively, NO2-, NO3-, NO, and N2O were produced under aerobic growth conditions. Heterotrophic nitrification was not attributable to old or dying cell populations. Production of NO2-, NO3-, NO, and N2O was detectable shortly after cultures started growth and proceeded exponentially during the logarithmic growth phase. NO2- and NO3- production rates were higher for cultures inoculated in media with pH values below 7 than for those in media at alkaline pH. Neither assimilatory nor dissimilatory nitrate or nitrite reductase activities were detectable in aerobic cultures.     

Engineering of the H2O2-binding pocket region of a recombinant manganese peroxidase to be resistant to H2O2.

The manganese peroxidase produced by Phanerochaete chrysosporium, which catalyzes the oxidation of Mn(2+) to Mn(3+), is easily inactivated by the hydrogen peroxide (H2O2) presented in the reaction. We attempted to increase H2O2 resistance by the conformational stabilization around the H2O2-binding pocket. Based on its structural model, engineering of oxidizable Met273 located near the pocket to a non-oxidizable Leu showed a great improvement. Furthermore, after treatment at 1 mM H2O2 where the wild-type is completely inactivated, full activity can be retained by engineering the Asn81, which might have conformational changes due to the environment of the pocket, to a non-bulky and non-oxidizable Ser.     

Role of extra- and intracellular Ca2+ in changes of NO2- and H2O2 production by endometrium

The work is aimed to find out the role of extra- and intracellular Ca2+ in cyclic mechanisms of NO2- and H2O2 metabolism in the stroma cells of endometrium activated by acetylcholine. High activity of Mg2+, Ca(2+)-ATP-ase is characteristic of stroma cells of the endometrium. This activity is tested in the presence of 0.01% of the Triton X-100 (36 +/- +/- 2 mumole Pi/mg of protein for 1 hour). The acetylcholinesterase activity in these systems is equal to 9.8 +/- 0.2 mumole thiocholinebromide/mg of protein for 1 hour. Acetylcholine (10 microM) elevated essentially the concentration of cytosolic Ca2+ in them. It was established, that in the control the suspension of stroma cells produced 1 nmol/NO2-/10(6) of cells, this value being constant for the experimental period of time in the range of 5-60 s. The activation of cells (1 microM acetylcholine + 10 microM Ca2+) results in the appearance of cyclicity (maxima on 5, 15 and 60 s) and 5-fold intensification of NO2- production. The rise of extracellular concentration of Ca2+ up to 0.1-1--10 mM results in essential change of the character of the time dependence of NO2- metabolism and only in inappreciable intensification of the response amplitude. Such a pattern is observed for H2O2: 0.77 mumol H2O2/10(6) of cells in the control, at 10 microM Ca2+ maxima of production on the 5 and 30 s, change of the form of the time response, instead of the amplitude under the increase of concentration of extracellular Ca2+. Preincubation of cells with modifiers endoplasmic reticulum ryanodine, caffeine (1 mM) and heparine (10 mM) results in essential drop of NO2- production and infringement of cyclicity, the effect of ryanodine being more expressed on 5 s, than on 15 s, and heparine--also on 5 s, and on 15 s. Preincubation of cells with methylene blue (10 mM), which inhibits guanilate cyclase, result in considerable intensification of NO2- and H2O2 formation and cyclicity losses. Dynamics of NO2- formation (is controversy) reciprocated with cGMP, whereas nitrosoglutathione production and NO3- tends to saturation in the course of time. Thus, acetylcholine-dependent variations of NO2- and H2O2 concentrations in the suspension of stroma cells depend on the state of extra- and intracellular Ca(2+)-stores, are controlled by cGMP. It may be assumed, that the NO2- production minima are caused by its transfer in NO3- and its binding with glutathione.     

Metabolism of [17-2H]pregnenolone into 5-[17 beta-2H, 17 alpha-18O]androstene-3 beta, 17 alpha-diol and other products by incubation with the microsomal fraction of boar testis under 18O2 atmosphere.

[17-2H]Pregnenolone was incubated with the microsomal fraction of boar testis under an 18O2 atmosphere. The metabolites were analyzed by gas chromatography-mass spectrometry, and the following six metabolites labeled with 2H or 18O (or both) were identified: 17 alpha-[17-18O]hydroxypregnenolone, [17-18O]dehydroepiandrosterone, 5-[17-18O]androstene-3 beta, 17 beta-diol, 16 alpha-[16-18O]hydroxy[17-2H]pregnenolone, 5-[17 beta-2H, 17-18O]androstene-3 beta,17 alpha-diol, and 5,16-[17-2H]androstadien-3 beta-ol. The time course of the formation of these metabolites from pregnenolone was also studied using 14C-labeled substrate. The results obtained from these experiments suggest that the first three metabolites were synthesized by a well-documented pathway--pregnenolone yields 17 alpha-hydroxypregnenolone yields dehydroepiandrosterone yields 5-androstene-3 beta,17 beta-diol--, and that 16 alpha-hydroxypregnenolone, 5-androstene-3 beta,17 alpha-diol and 5,16-androstadien-3 beta-ol were synthesized from [17-2H]pregnenolone with retention of 17-2H.     

The mechanism of oleic acid nitration by *NO(2)

Fatty acid nitration is a recently discovered process that generates biologically active nitro lipids; however, its mechanism has not been fully characterized. For example, some structural details such as vinyl and allyl isomers of the nitro fatty acids have not been established. To characterize lipids that originated from a biomimetic reaction of *NO(2) with oleic acid, we synthesized several isomers of nitro oleic acids and studied their chromatography and mass spectra by various techniques of mass spectrometry. LC/MS analysis performed on a high resolution micro column detected molecular carboxylic anions of various oleic acid nitro isomers (NO(2)OA). Esterification of NO(2)OA with pentafluorobenzyl bromide and diisopropylethylamine as a catalyst produced a unique isoxazole ester derivative exclusively from allyl NO(2)OA isomers via dehydration of the nitro group at ambient temperatures. This new analytical procedure revealed that *NO(2) generated two vinyl and two allyl isomers of NO(2)OA. The vinyl isomers showed high regioselectivity with the 1.8:1 preference for the 10-NO(2)OA isomer that was absent among allylic isomers. The nitration also generated elaidic acid via cis-trans isomerization and diatereoisomers of vicinal nitro hydroxy, nitro keto and alpha-nitro epoxy stearic acids with high stereo and regioselectivity. Nitration of small unilamelar phospholipid vesicles resulted in several phospholipids containing nitro lipids and elaidic acid amenable to hydrolysis by phospholipase A(2).     

NADPH oxidation catalyzed by the peroxidase/H2O2 system. Iodide-mediated oxidation of NADPH to iodinated NADP

Oxidation of NADPH catalyzed by the peroxidase/H2O2 system is known to require the presence of mediating molecules. Using either lactoperoxidase or horseradish peroxidase, we demonstrated that in the peroxidase/H2O2 system, NADPH oxidation was mediated by iodide. The oxidation product was the iodinated NADP. This product was shown to possess spectral characteristics different from those of NADP+ and NADPH, since for iodinated NADP, increased absorbance was observed in the 280-nm region and was directly proportional to the rate of iodination. It is suggested that oxidation and iodination of NADPH proceed via a single reaction between the intermediary iodide oxidation species and NADPH. Experiments with different molecules of NADPH analogues indicated that iodination occurred in the nicotinamide part of the NADPH molecule.