[Flavonoid oxidation kinetics in aqueous and aqueous organic media in the presence of peroxidase, tyrosynase, and hemoglobin].

The kinetics of oxidation reactions of flavonoids, quercetin, dihydroquercetin, and epicatechin has been studied in the presence of biocatalysts of different natures: horseradish peroxidase, mushroom tyrosinase, and hemoglobin from bull blood. Comparison of the kinetic parameters of the oxidation reaction showed that peroxidase appeared to be the most effective biocatalyst in these processes. The specificity of the enzyme for quercetin increased with increasing the polarity of the solvent in a series of ethanol­acetonitrile­dimethyl sulfoxide.

A solid-phase fluorescent biosensor for the determination of phenolic compounds and peroxides in samples with complex matrices.

A solid-phase fluorescent biosensor for the determination of phenolic compounds (simple substituted phenols and catecholamines) and peroxides has been developed. The biosensor has a simple construction and the analytical signal is measured directly in a biosensitive layer {peroxidase-chitosan} on the sensor surface. This approach allowed analyzing samples with complex matrices (including water-insoluble samples and nontransparent solutions) without their preliminary pretreatment. Two novel fluorescent indicator reactions for the determination of the above-mentioned analytes in wide concentration ranges (from nmol l(-1) to mm l(-1)) which provided an analytical signal registration on a solid phase were proposed. The developed sensor was applied successfully for the analysis of urine, cosmetics, pharmaceuticals preparations, etc.

Properties and analytical applications of the self-assembled complex {peroxidase-chitosan}.

A novel promising approach to the improvement of analytical properties of horseradish peroxidase based on its inclusion into self-assembled structures of chitosan is discussed. It is shown that the reasonable choice of a polyelectrolyte, a detailed investigation of its interaction with the enzyme and the conditions of the {peroxidase-polyelectrolyte} complex formation allow for stabilizing the biocatalyst in aqueous and aqueous-organic media without a substantial loss in its activity and developing corresponding analytical procedures and biosensors. The latter provides highly selective determination of a number of organic compounds and sensitive determination of heavy metal ions that becomes possible due to the specific interactions of the analytes with the polymer matrix. Besides, the application of the proposed analytical systems and biosensors provides the expansion of the range of the compounds, and poorly water soluble and slowly oxidized substrates of peroxidase as well, which could be determined and real samples which could be analyzed by enzymatic methods. Analytical performance of the developed spectrophotometric indicator procedures and biosensors based on the self-assembled complex {peroxidase-chitosan} is demonstrated in the determination of metal ions (Hg(II), Cd(II), and Pb(II)), phenothiazines (promazine, chloropromazine, and trifluoroperazine), phenolic compounds (phenol, hydroquinone, catechol, pyrogallol, quercetin, rutin, and esculetin), organic peroxides (tert-butyl peroxide, 2-butanone peroxide, and benzoyl peroxide) in various samples, including water-insoluble matrices.

[Catalytic activity and the stability of horseradish peroxidase increase as a result of its incorporation into a polyelectrolyte complex with chitosan].

The incorporation of horseradish peroxidase into polyelectrolyte complexes with chitosans of different molecular weights (MW 5-150 kDa) yielded highly active and stable enzyme preparations. As a result of the selection of optimal conditions for the formation of peroxidase-chitosan complexes, it was found that 0.1% chitosan with a MW of 10 kDa had the strongest activatory effect on peroxidase (activation degree, > 70%) in the reaction of o-dianisidine oxidation by hydrogen peroxide. The complex formed by 0.001% chitosan with a molecular weight of 150 kDa was most stable: when immobilized on foamed polyurethane, it retained at least 50% of the initial activity for 550 days. The highest catalytic activity was exhibited in a 0.05 M phthalate buffer (pH 5.9-6.2) by the complex containing 0.006-0.009% chitosan in the indicator reaction. The activatory effect of the polysaccharide on the enzyme was determined by its influence on the binding and conversion of the reducting substrate peroxidase.

[Mechanisms of peroxidase oxidation of o-dianisidine, 3,3',5,5'-tetramethylbenzidine, and o-phenylenediamine in the presence of sodium dodecyl sulfate].

Peroxidase oxidation of o-dianisidine, 3,3',5,5'-tetramethylbenzidine, and o-phenylenediamine in the presence of sodium dodecyl sulfate (SDS), an anionic surfactant, was spectrophotometrically studied. It was found that 0.1-100 mM SDS concentrations stabilize intermediates formed in the peroxidase oxidation of these substrates. The cause of the stabilization is an electrostatic interaction between positively charged intermediates and negatively charged surfactant.

Enzymatic determination of phenols using peanut peroxidase.

The influence of phenol and its derivatives on the kinetics of oxidation of aryldiamines (indicator-substrates) catalyzed by novel plant peroxidase-cationic peanut peroxidase-was studied. The character of influence of phenols on the kinetics of enzymatic oxidation of benzidine, o-dianisidine, and 3,3',5,5'-tetramethylbenzidine (TMB) with hydrogen peroxide was found to depend on a correlation between redox properties of phenols and the indicator-substrate of peroxidase. Thus, the catalytic activity of peanut peroxidase is inhibited by phenols with redox potentials higher than that of aryldiamines mentioned above, whereas phenols with potentials below those of aryldiamines, play the role of second substrates of the enzyme. The enzymatic procedures for the determination of numerous phenols on the level of their concentrations 0.05-80 muM were developed using the reactions of benzidine, o-dianisidine, and TMB oxidation. Different analytical signals-the indicator reaction rate and the induction period duration-were used for the determination of phenols, belonging to various groups-the inhibitors and second substrates of the enzyme, respectively.

Inhibition of peroxidase, trypsin, and alpha-chymotrypsin by platinum (II) and platinum (IV) complexes.

Effects of various complexes of platinum (II) and platinum (IV) on activities of trypsin, alpha-chymotrypsin, and peroxidase were compared. The platinum (II) complexes were found to inhibit these enzymes, though with variable efficiency. The platinum (IV) complexes at concentrations < or = 0.2 mM efficiently inhibited peroxidase but had no effect on the proteases. An enzymatic assay was developed to measure the most effective peroxidase inhibitor (cisplatin) at concentrations of 5-50 microM in the presence of fivefold excess of its isomer (transplatin).

Assay of enzyme effectors.

The effects of various classes of organic compounds and of metal ions on the catalytic activity of horseradish peroxidase in hydrogen peroxide-catalysed o-dianisidine oxidation and, on the activity of alkaline phosphatase in p-nitrophenyl phosphate hydrolysis have been studied. Enzymic methods have been developed for determination of sulphur compounds at 10(-5)-10(-4)M, nitrogen compounds at 2 x 10(-7)-3 x 10(-5)M mercury at 3 x 10(-7) mu/ml and lead at 6 x 10(-4) mu/ml concentration.

Bioluminescent assay of creatine kinase activity using immobilized firefly extract.

A bioluminescent method has been developed for creatine kinase (CK) assay using immobilized firefly extract containing the bioluminescent coimmobilized system: adenylate kinase + luciferase. ADP for the reaction with CK was produced from the initial mixture of AMP and ATP. The ATP formed in the reaction with CK was quantified using firefly luciferase. The lowest detection limit for CK activity was 0.5 +/- 0.2 U/liter in the sample. A linear range of the determined CK activities was 0.5-1000 U/liter. The correlation coefficient between the bioluminescent and spectrophotometric methods was 0.981 (n = 40). The use of immobilized firefly extract for analysis has been shown to be advantageous compared to soluble enzyme.

[Inhibition and inactivation of horseradish peroxidase by thiourea]. / Ingibirovanie i inaktivatsiia peroksidazy iz khrena tiomochevinoi.

The kinetics of horseradish peroxidase (EC 1.11.1.7)-catalyzed oxidation of o-dianisidine by hydrogen peroxide in the presence of thiourea were studied. At the first, fast step of this process thiourea acts as a competitive reversible inhibitor with respect to o-dianisidine (Ki = 0.22 mM). The formation of a thiourea-peroxidase complex was determined by the increase in the absorbance at A495 and A638 of the enzyme. The dissociation constant for the peroxidase-thiourea complex is equal to 2.0-2.7 mM. Thiourea is not a specific substrate of peroxidase during the oxidation reaction by H2O2, but is an oxidase substrate (although not a very active one) of peroxidase. The irreversible inactivation of the enzyme during its incubation with thiourea was studied. The first-order inactivation rate constant (kin) was shown to increase with a fall in the enzyme concentration. The curve of the dependence of kin on the initial concentration of thiourea shows a maximum at 5-7 mM. The enzyme inactivation is due to its modification by intermediate free radical products of thiourea oxidation. The inhibitors of the free radical reactions (o-dianisidine) protect the enzyme against inactivation. The degree of inactivation depends on concentrations and ratio of thiourea and peroxidase. A possible mechanism of peroxidase interaction with thiourea is discussed.