Enzymatic method for the determination of phenols with the use of peanut peroxidase

Cationic peanut peroxidase was used for the first time for the determination of phenols at a level of 0.5–10μM. The examined phenols were found to be inhibitors or second substrates of peanut peroxidase in the indicator reaction of the oxidation ofo-dianisidine by hydrogen peroxide. The effect of phenols on the rate of the indicator reaction depends on their redox properties. The data on the effects of phenols on the catalytic activities of peroxidases isolated from different sources (peanut, horseradish roots,Medicago sativa alfalfa cells, and the xylotrophic fungusPhellinius igniarius) were compared     

Investigation of the electro-oxidation and oxidation of catechol in the presence of sulfanilic acid

The electrochemical oxidation of catechol (1) in the presence of sulfanilic acid (2) was investigated. Some electrochemical (EC) techniques such as cyclic voltammetry and controlledpotential coulometry were used. The oxidation reaction of catechol (1) with periodate in the presence of sulfanilic acid (2) was also investigated spectrophotometrically. The results indicate that the o-quinone derived from catechol participate in Michael addition reaction with sulfanilic acid (2). In addition, according to the ECE mechanism, the observed homogeneous rate constant (k _obs) for the reaction ofo-quinone derived from catechol (1) with sulfanilic acid (2) has been estimated by digital simulation of cyclic voltammograms.     

Enzyme repurposing of a hydrolase as an emergent peroxidase upon metal binding

As an alternative to Darwinian evolution relying on catalytic promiscuity, a protein may acquire auxiliary function upon metal binding, thus providing it with a novel catalytic machinery. Here we show that addition of cupric ions to a 6-phosphogluconolactonase 6-PGLac bearing a putative metal binding site leads to the emergence of peroxidase activity (k_cat 7.8 × 10^–2 s^–1, K_M 1.1 × 10^–5 M). Both X-ray crystallographic and EPR data of the copper-loaded enzyme Cu·6-PGLac reveal a bis-histidine coordination site, located within a shallow binding pocket capable of accommodating the o-dianisidine substrate.     

Mechanism and kinetics of the vanadium-catalyzed o-dianisidine—t-butyl hydroperoxide reaction in non-aqueous media

The vanadium-catalyzed oxidation of o-dianisidine by t-butyl hydroperoxide is studied for the determination of trace vanadium in non-aqueous media. In acetonitrile, vanadium acts as a one-eletron mediator between the reductant and oxidant. Mechanistic studies, based on e.p.r. spectrometry and visible spectrophotometry, show that the hydroperoxide oxidizes vanadium(IV) via a radical mecahnism. The kinetics of V(IV) oxidation are investigated and a rate equation is proposed. Oxidation of o-dianisidine by the vanadium(V) formed in a one-electron step with formation of the o-dianisidine radical completes the catalytic cycle, as shown by spectroelectrochemical measurements. The radical reacts further with excess of t-butyl hydroperoxide to form the diimine, which may be used for sensitive spectrophotometric monitoring of the indicator reaction at 450 nm.     

Enzymatic determination of some pharmaceuticals in direct and reversed sodium dodecyl sulfate micelles

The properties of horseradish peroxidase in sodium dodecyl sulfate (DDS) reversed micelles in benzene-pentanol-water solutions are studied. The potential of the analytical application of direct and reversed DDS micelles is demonstrated using newly developed methods for the determination of peroxidase substrates (hydrogen peroxide and cystein), inhibitor (sulfanylamide), and activator (imidazole) via the oxidation of o-dianisidine (o-D) with hydrogen peroxide.     

Superactivity of peroxidase solubilized in reversed micellar systems

Vaccinium mirtyllus peroxidase solubilized in reversed micelles was used for the oxidation of guaiacol. Some relevant parameters for the enzymatic activity, such as pH,w _o (molar ratio water/surfactant), surfactant type and concentration, and cosurfactant concentration, were investigated. The peroxidase showed higher activities in reversed micelles than in aqueous solution. The stability of the peroxidase in reversed micelles was also studied, namely, the effect ofw _o and temperature on enzyme deactivation. The peroxidase displayed higher stabilities in CTAB/hexanol in isooctane reversed micelles, with halflife times higher than 500 h.     

Stabilizing and suicide-peroxide protecting effect of Ni2+ on horseradish peroxidase

The present research discusses the structure stabilizing and protecting effects of Ni²⁺ against suicide-peroxide inactivation of horseradish peroxidase (HRP). Suicide inactivation of HRP by hydrogen peroxide (3 mM) was monitored by measuring change in the absorbance of the colored product (tetraguaiacol) of the catalytic reaction cycle at 470 nm. Progress curves of the catalytic reaction cycle were obtained at 27 °C, phosphate buffer (5 mM), pH 7.0. The corresponding kinetic parameters (e.g., initial enzyme activity (α_o) and the apparent rate constant (k_i) of suicide inactivation of HRP by peroxide) were evaluated using a kinetic equation derived in this study. Comparative activatory and inhibitory effects of Ni²⁺ on the kinetics of suicide-peroxide inactivation of HRP are discussed.     

Phenol oxidases production and wood degradation by a thermophilic fungusThermoascus aurantiacus

The ability of a Brazilian strain ofThermoascus aurantiacus, a thermophilic fungus, to produce extracellular phenol oxidases and to degradeEucalyptus grandis sawdust was studied.T. aurantiacus was capable of good growth in liquid culture containing 1.5% (w/v) of various lignocellulosic substrates (sugar cane bagasse, rice hulls, and chips and sawdust ofE. grandis) plus 5 mg/mL of glucose. When lignocellulosic substrates were used, enzymes involved in cellulose and hemicellulose metabolism were stimulated inT. aurantiacus. It was also found that these substrates have an inductive effect on phenol oxidase production. The most effective inducer of phenol oxidase activity wasE. grandis sawdust, which led to the production of 0.80 U/mL (o-dianisidine oxidation) on day 12. Low phenol oxidase activity was observed at cultures when only glucose was used. Cultures ofT. aurantiacus also exhibited cellobiose-quinone oxidoreductase activity when lignocellulosic materials were used as substrate. However, under our experimental conditions, lignin peroxidase activity was not detected.E. grandis sawdust supplemented with 5 mg/mL of glucose suffered a total weight loss of 6.7% accompanied by 15% lignin loss and 64.4% extractive loss after 21 d incubation withT. aurantiacus.     

Features of the kinetics of the liquid-phase oxidation of cyclohexanol

The kinetics of oxygen uptake in the cumyl peroxide-initiated oxidation of cyclohexanol (373 K, o-dichlorobenzene) is studied. The parameters of the oxidizability of k _p (2k _t )^?0.5 (which depend on [RH]) and the rate constants of the bi- and trimolecular reactions of chain initiation (k ₀ = 1.25 × 10^?8 L/(mol s) and k′₀ = 2.5 × 10^?9 L²/(mol² s), respectively) are determined by solving the inverse kinetic problem. It is demonstrated that the quadratic-law recombination of peroxyl radicals during cyclohexanol oxidation also occurs without chain termination. The recombination rates of peroxyl radicals with and without chain termination (k′/k _t ) are found to grow with increasing [RH], reaching their maxima at [RH] = 1.0 mol/L, and to diminish subsequently. We conclude that this can be attributed to changes in the ratio between the propagating peroxyl radicals (hydroperoxyl and 1-hydroxycyclohexylperoxyl) in the reaction medium.     

The oxidation of benzidine, o,o′-tolidine and o,o′-dianisidine by manganese dioxide

The oxidation of benzidine, o,o′-tolidine, and o,o′-dianisidine by manganese dioxide was studied in an acidic medium. It has been shown that the studied substances are quantitatively oxidized by the reagent to the corresponding quinonediimines and that this reaction can be utilized for the indirect titrimetric determination of benzidine and o,o′-tolidine, based either on ferrometric titration of unconsumed manganese dioxide or on ascorbinometric titration of the quinonediimine formed.     

The catalytic effect of catiohic amino micelles on the hydrolysis of substituted phenyl esters

The catalytic effects of two aminocationic micelles on the hydrolysis of substituted phenyldecanoate esters and a positively charched benzoate ester (CPNBA) were determined. The micellaric catalysts were of the general structure [CH₃(CH₂)₃N(CH₃)₂(CH₂)_nNH₂]Br where n=2 (micelle 1); n=3 (micelle 2). The kinetics followed the expression: k_obs =k_o+k_cat x K_a/(K_a+H⁺)+k^o_OH[OH^-]. From the comparison of the kc OH rates with specific base catalysis rates deduced from reactions in non catalytic micelles, it was concluded that the kc OH term, is compatible mainly with an aminolysis reaction catalyzed by hydorxide ion. The Hammett and Bronsted correlations (p=2.8; β=1.0), in addition to the very small deuterium isotope effect, suggested that kcat corresponded with a nucleophilic mechanism. The Bronsted plot of log k_cat vs pKa of the phenolate leaving groups in micelles 1 and 2 showed a biphasic behaviour. The break in the curve occured at pK_o=5.89 and pK_o=6.78 respectively. The partition ratio k^±/k_-a of the zwiterionic tetrahedral intermediate was derived from the experimental data and produced the following correlation: log k^±/k_-a=-0.92pK_o+0.43pK_N+2.466. The ester CPNBA exhibited a deuterium isotope effect of 2.1. From product analysis it was concluded that the reaction proceeds via a general base catalysis of aminolysis.     

Product distribution in preparative scale electrolysis: Part VI. Competition between dimerization and first-order deactivation

The dependence of product distribution from the various experimental parameters (rates, concentration diffusion layer thickness, current density) is analyzed for a reaction scheme involving the competition between dimerization and first-order deactivation of the intermediate resulting from the first electron uptake (or removal). Two cases are considered, corresponding to the product of the first-order deactivation being either electroinactive or undergoing a further electron transfer at the electrode or in the solution. It is shown that the dimer yield is not very sensitive to the further fate of the deactivation product. It is in all cases a function of the competition parameter σ=k₁k₂^?3/2D^1/2 c⁰ δ^?1 in potentiostatic conditions and σ^Δ=k₁k₂^?3/2D^?1/2(i⁰/FS) in galvanostatic conditions (k₁, k₂ rate constants of the dimerization and deactivation reactions respectively; D diffusion coefficient; c⁰ concentration of substrate; δ diffusion layer thickness; (i⁰/S) current density). The reduction of CO₂ in DMF with competitive formation of oxalate and formate is taken as an example illustrating the theoretical analysis.     

Determination of Mercury at the Picogram per Milliliter Level Using Immobilized Horseradish Peroxidase

Abstract

New test method and test device for the mercury (II) determination at the pg/mL level were developed based on the mercury inhibitory action on horseradish peroxidase immobilized on solid supports – in the cells of the polystyrene plate and on the chromatographic paper. The reactions of o-dianisidine, 3,3′,5,5′-tetramethylbenzidine and o-phenylenediamine oxidation by hydrogen peroxide were used as the indicator reactions. The mercury inhibitory effect increased in the presence of thiourea. Under the elucidated optimal conditions the calibration curves for the mercury determination showed a linear relationship between the peroxidase inhibition degree and the mercury concentration in the range of 0,1–1000 pg/mL. The mercury detection limits were 0,1–10 pg/mL in dependence on the concrete indicator reaction. The analysis completed in 15 min. The proposed test device was applied to the mercury determination in underground waters of Moscow region. The mercury content obtained was coincident with that obtained by atomic-fluorescent method with cold vapour.     

Effectiveness of the Accessory Yellow Fluorescent Protein in the Bacterial Luciferase Reaction Correlates with the Lifetime of the Peroxyhemiacetal Intermediate: The Stereochemistry of the Reaction

In the in vitro reaction of Vibrio fischeri Y-1 luciferase, the dependence of the initial luminescence intensity (I_o) and its rate of decay (k_d) on the chain-length of the aliphatic aldehyde are greatly altered by the presence of yellow fluorescent protein (YFP), which functions as an accessory emitter. Whereas with no YFP both k_d and I_o are maximum for chain lengths ≥ 12, the fraction of the light emitted from the accessory chromophore, measured as the ratio of yellow to blue light (Y/B), is greater with shorter chain-length aldehydes. Thus, aldehydes that are least efficient in the absence of YFP are more efficient for causing yellow emission in its presence. These results are interpreted on the basis of the expected lifetimes of the peroxyhem-iacetals with which YFP interacts: high values of k_d reflect short peroxyhemiacetal lifetimes, hence less chance of interaction with YFP. The critical dependence on aldehyde chain-length underlines the importance of stereochemical factors in the bacterial reaction, which are discussed here in the framework of a chemically initiated electron exchange luminescence model.     

Kinetics of inhibition of peroxidase activity of myeloperoxidase by quercetin

The inhibition of myeloperoxidase (MPO), isolated from human neutrophils, by quercetin was investigated by following peroxidase activity of the enzyme using o-dianisidine as the substrate. The inhibition parameters (IC₅₀) were obtained by graphical analysis of the inhibition curves. A reaction mechanism, which involved the enzyme inhibition by quercetin and H₂O₂ in excess, was proposed. The rate and equilibrium constants for the proposed reaction path were calculated from experimental data. Kinetic analysis in noninhibiting H₂O₂ concentration range in the absence and the presence of quercetin revealed that the reaction mechanism underwent Michaelis–Menten kinetics. K and V values indicated that quercetin was a mixed inhibitor of MPO activity. The initial reaction rates were recalculated using the obtained results. Calculated curves fitted the experimental results within the range of experimental error. © 2008 Wiley Periodicals, Inc. Int J Chem Kinet 40: 384–394, 2008     

The use of redox reactions in the analysis of dyes and dye industry intermediates: IV. Oxidation of benzidine,o-tolidine,and o-dianisidine with chloramine T and N-bromosuccinimide

The oxidation of benzidine, o-tolidine, and o-dianisidine with chloramine T and N-bromosuccinimide was studied, the stoichiometry was established, and a mechanism was proposed. An extraction-photometric determination of these diamines was developed, based on the reaction with excess chloramine T or N-bromosuccinimide and the measurement of the absorbance of the N,N′-dihalogeno-diphenoquinonediimines formed, after their extraction into chloroform.     

Palladium-catalyzed oxidation of lower aliphatic alcohols with oxygen in the presence of aromatic nitriles in aqueous medium

The kinetics of oxidation of lower aliphatic alcohols (C₁–C₄) to the corresponding carbonyl compounds with oxygen in the presence of palladium(II) tetraaqua complex and aromatic nitriles (benzonitrile, phenylacetonitrile, and o-tolunitrile) in aqueous medium (c = 0.01 M) at 65°C under atmospheric pressure were studied. A probable reaction mechanism and kinetic equation were proposed. Aromatic nitriles were found to stabilize decomposition of low-valence palladium species, ensuring activation of molecular oxygen and subsequent oxidation of alcohols.     

Photoreduction ofortho-benzoquinones in the presence ofpara-substitutedN,N-dimethylanilines

Photoreduction ofo-benzoquinones irradiated at the wavelengths λ_max ≈ 400 and 600 nm corresponding to the S(π → π*) and S(n → π*) electron transitions in the >C=0 groups, respectively, in the presence ofN,N-dimethylaniline and its derivatives was studied. The apparent rate constants of the photoreduction (k _H) ofo-quinones are determined by the free energy of electron transfer from the amine molecule to a photoexcitedo-quinone molecule (ΔG _e.t). The ΔG _e.t. values are calculated as the sums of the energies of the 0→0 transitions of the lowest triplet excited state ofo-quinones, the reduction energies ofo-quinones, and the oxidation energies of amines (the last two terms are numerically equal to the corresponding redox potentials). The maximum rate of photoreduction was found for ΔG _e.t≈0. The reaction mechanism is proposed, in which the reversible formation of a triplet exiplex is the rate-determining stage and hydrogen transfer proceeds in parallel with electron transfer within the exiplex. Published inIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 9, pp. 1515–1521, September, 2000.     

Kinetics and mechanism of the doping-addition of Arscl to olefins

A lithium percholate effect on the rate and product ratio of 2,4-dinitrobenzenesulfenyl chloride doping-addition to 1-methylcyclopropene (5) and tetrafluorobenzobarrelene (10) in acetic acid has been examined. The rates of both doping-addition reactions follow the normal salt effect dependence. However, the yields of doping-addition products is much higher than expected from the term k_o.b.[LiClO₄] at low concentrations of the salt added and, hence, the catalytic influence of LiClO₄ on product formation is much more pronounced than expected from normal salt effect dependence. The mechanism of doping-addition is analyzed in detail.