ABSTRACT
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.
Subject(s)
Catechin/chemistry , Hemoglobins/chemistry , Horseradish Peroxidase/chemistry , Monophenol Monooxygenase/chemistry , Quercetin/analogs & derivatives , Quercetin/chemistry , Acetonitriles/chemistry , Agaricales/chemistry , Agaricales/enzymology , Animals , Cattle , Dimethyl Sulfoxide/chemistry , Ethanol/chemistry , Fungal Proteins/chemistry , Kinetics , Oxidation-Reduction , Solutions , Solvents/chemistryABSTRACT
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.
Subject(s)
Biosensing Techniques , Catecholamines/urine , Peroxides/analysis , Phenols/urine , Chitosan/chemistry , Cosmetics/chemistry , Fluorescent Dyes , Horseradish Peroxidase/chemistry , Laccase/chemistry , Monophenol Monooxygenase/chemistry , Rhodamines , Spectrometry, FluorescenceABSTRACT
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.
Subject(s)
Biosensing Techniques , Chitosan/chemistry , Horseradish Peroxidase/chemistry , Catalysis , Cosmetics/analysis , Dermatologic Agents/analysis , Dietary Supplements/analysis , Dimethyl Sulfoxide , Ointments/analysis , Peroxides/analysis , Phenols/analysis , Phenothiazines/analysisABSTRACT
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.
Subject(s)
Chitosan/chemistry , Enzymes, Immobilized/chemistry , Horseradish Peroxidase/chemistry , Hydrogen Peroxide/chemistry , Polyurethanes/chemistry , Catalysis , Hydrogen-Ion ConcentrationABSTRACT
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.
