T. N. Shekhovtsova

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.

Ionic liquids in enzymatic catalysis and biochemical methods of analysis: Capabilities and prospects

The first Russian review systematizes and discusses the most important and promising published data on the use of ionic liquids in biocatalysis and, especially, biochemical methods of analysis. Studies on the use of ionic liquids as solvents for enzymes, new reaction media for enzymatic reactions, and components of the biosensitive layers of sensors are analyzed. The physical and chemical properties of ionic liquids used in biocatalysis are discussed. The advantages of ionic liquids over the usual solvents in homogeneous and heterogeneous reactions with the participation of enzymes from various classes are demonstrated, procedures for the coimmobilization of biocatalysts and ionic liquids with cellulose onto polymer supports and electrodes are described, and prospects for the use of enzyme-ionic liquid compositions in biochemical methods of analysis are considered.

Determination of organomercury compounds using immobilized peroxidase

Abstract The effect of organomercury compounds (methyl-, ethyl- and phenylmercury) on the activity of immobilized peroxidase was studied. Peroxidase was immobilized in chitosan films in wells on a polystyrene plate and on chromatography paper. The oxidation reactions of o -dianisidine, o -phenylenediamine and 3,3′, 5,5′-tetramethylbenzidine by H 2 O 2 were used as indicators. The rate of all the indicator reactions was monitored visually by measuring the time of appearance of the colour of the oxidation product. The liberating effect of organomercury compounds on peroxidase immobilized on both the above mentioned supports using o -dianisidine oxidation in the presence of the inhibitor phenylthiourea and the effect of organomercury compounds on the duration of an induction period of 3,3′, 5,5′-tetramethylbenzidine oxidation in the presence of diethyldithiocarbamate catalysed by paper-immobilized peroxidase were used for the development of enzymatic test procedures for the determination of organomercury compounds at concentrations of 0.02–1000 μM. The relative standard deviation ( n = 3) at the lower limits of their analytical concentration ranges in the proposed test procedures using o -dianisidine are 18–23% ( n = 3).

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 o...

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.

Determination of phenols using various peroxidases

Abstract Peroxidases of different origin — horseradish peroxidase isozyme C, alfalfa and peanut cationic peroxidases, tobacco leaves and novel fungal anionic peroxidases – were used to determine phenol and its analogues. Phenol and resorcinol were shown to be the inhibitors of the peroxidase activity towards o—dianisidine for all the enzymes tested, whereas pyrogallol and hydroquinone caused an appearance of a lag—period on a kinetic curve. The duration of a lag—period was proportional to the effector concentration and could be used to determine it. The novel fungal peroxidase from Phellinus igniarius exhibited the highest sensitivity towards phenols and they could be determined at the 10–6 – 10–7 M concentration levels.

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.

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.

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.

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.