[Antioxidant activity of hydroxy derivatives of coumarin].

The inhibition efficiency (antioxidant activity) of hydroxy derivatives of coumarin, such as esculetin, dicumarol, and fraxetin, was studied in the methemalbumin-H2O2-tetramethylbenzidine (TMB) pseudoperoxidase system at 20 degrees C in a buffered physiological solution (pH 7.4) containing 6% DMF and 0.25% DMSO. The inhibitor's efficiency was quantitatively characterized by the inhibition constants (K(i), microM) and the inhibition degree (%). The K(i) values for esculetin, dicumarol, and fraxetin were 9.5, 15, and 26 microM, respectively. Esculetin and fraxetin inhibited pseudoperoxidase oxidation of TMB in a noncompetitive manner; dicumarol, in a mixed manner. The inhibiting activity ofesculetin in peroxidase-catalyzed TMB oxidation at pH 6.4 is characterized by a K(i) value equal to 1.15 microM, and the inhibition process is competitive. Esculetin was found to be the most effective antioxidant of plant origin among all derivatives previously studied in model biochemical systems.

[Antioxidant activity of oxygen-containing aromatic compounds].

Inhibition efficiency (antioxidant activity) of 26 oxygen-containing aromatic compounds was studied in methemalbumin-H202-o-phenylenediamine (PDA) or tetramethylbenzidine (TMB) pseudoperoxidase system at 20 degrees C in buffered physiological solution (pH 7.4) containing 6% DM F and 0.25% DMSO. The inhibitor's efficiency was quantitatively characterized by the inhibition constants (Ki, microM) or the inhibition degree (%). Ki values varied in the range of4 to 500 microM and were influenced by a substrate, the structure of an inhibitor, hydroxyl groups, electron-donating substituents in aromatic ring, and steric hindrances. The type of inhibition at cooxidation of eight pairs was noncompetitive, and that of five pairs was mixed and determined by the substrate nature and the inhibitor structure. Lignin phenolic compounds ofguaiacyl and syringal series exhibited high antioxidant activity (Ki in the range of 10-300 microM), and their efficiency decreased in the following order: caffeic acid > synapaldehyde > syringic acid > coniferyl aldehyde > para-hydroxycou maric acid.

[Highly effective test-system for determination of the total antioxidant activity of human blood serum].

A highly effective test-system for quantitative characterization of the total antioxidant activity (TAA) of human blood serum (HBS), including methemalbumin (MetHa) as biocatalyst, H2O2 as the oxidant, o-phenylenediamine (PDA) as the acceptor of radicals and 2,2,5,7,8-pentamethylchroman-6-ol (PMC) as the inhibitor-calibrator, has been developed and proved under the laboratory environments. The test-system has been optimized for the concentrations of all the components, the reaction conditions and the PDA consumption monitoring at 37 degrees C in the medium of buffered physiological solution, pH 7.4 containing 5% DMFA and 0.5% DMSO. Under strongly standardized conditions by a comparison of the inhibiting action of the HBS and the calibrator PMC at 37 degrees C, the quantitative parameters of the HBS TAA were determined in microg of PMC, equivalent to 1 mg of HBS, or as a reverse value in mg of HBS, equivalent to the action of 1 microg of PMC. The values of the HBS TAA significantly varied for the group of healthy individuals and essentially decreased for the group of patients with thyroid gland pathologies. This underlies necessity of antioxidant therapy in these patients.

[Ultrasonic inactivation of Aspergillus niger glucose oxidase in aqueous solutions].

The inactivation of Aspergillus niger glucose oxidase (GO) was studied in 0.02 M phosphate-citrate buffer (PCB) at various pH, temperatures of 37-59 degrees C, and sonication with low frequency (27 kHz, LF-US) and high frequency (2.64 MHz, HF-US) ultrasound. The GO inactivation was characterized by the effective first-order inactivation rate constants k(in), k(in)*, and k(in)(us), reflecting the total, thermal, and ultrasonic inactivation components. The constants strongly depended on the pH and temperature of solution, GO concentration, and the presence of acceptors of the free radicals HO* -DMF, DMSO, ethanol, butanol, octanol, and mannitol, confirming that the active radicals formed in the ultrasonic cavitation field played an important role in the GO inactivation. The activation energy in the loss of GO catalytic activity considerably decreased when the enzyme solution was treated with LF-US or HF-US. The dissociative scheme of GO inactivation is discussed. Mannitol can be used for protection of GO from inactivation with LF-US or HF-US in the food industry and immunobiotechnology.

[Substituted 1,5,6,7-tetrahydro-4H-benzimidazol-4-ones as inhibitors of urease].

A comparative kinetic study of the inhibition of urea hydrolysis by 9 substituted 1,5,6,7-tetrahydro-4H-benzimidazol-4-ones (BI I-IX) has been carried out. The inhibition had reversibl competitive character; the inhibition constants K(i), varied from 29 up to 754 microM in dependence of the structure of BI I-IX. Three BI I-III, having the K(i) values from 29 to 82 microM, may be used as the potential therapeutic agents for gastroenterology for treatment of stomach and duodenal ulcers.

[Effect of herbicide tralkoxydim and 2-acylcyclohexane-1,3-diones on peroxidase activity].

Effect of 2-acylcyclohexane-1,3-dione derivatives (tralkoxydim and its diketone precursors) on peroxidase-catalyzed oxidation of 3,3',5,5'-tetramethylbenzidine (TMB), o-phenylenediamine (PDA), and the phenol-4-aminoantipyrine (4-AAP) couple has been studied. This effect varies from horseradish peroxidase (HRP) inactivation to activation in the reactions of peroxidation ofTMB, PDA, and, to a lesser extent, the phenol-4-AAP couple. The diketone-mediated HRP activation depends strongly on pH, presence of dimethylformamide, the structures of tralkoxydim and other diketones, and the substrate nature. The type of activation in the course of peroxidation with the presence of tralkoxydim can be noncompetitive (PDA and TMB) or mixed (TMB) depending on conditions. The maximal level of the HRP activation mediated by diketones depends on their structure. It can reach 4000% of the initial HRP-catalyzed peroxidation rate for TMB and ca. 1000% for PDA. A test system is proposed for quantitative tralkoxydim assay at millimolar concentration. It includes HRP and TMB as the substrate with spectrometrical monitoring of the TMB peroxidation product at 655 nm.

[Substituted aminophenols and flavonoids as potential components for test-systems of total antioxidant activity].

A comparative kinetic study of ortho-phenylenediamine (PDA) oxidation in the "pseudoperoxidase" system Methemalbumin-H2O2 in the presence of 2-amino-4-tret-butylphenol (ATBP), 2-amino-4,6-di-tret-butylphenol (ADTBP) and its four N-acyl derivates, as well as flavonoids (quercetin, morin, silibin, hesperidin and naringin) has been carried out under standart conditions at 20 degrees C in phosphate buffered saline, pH 7.4, containing 5.25% ethanol and DMFA. ATBP, ADTBP and two its N-acyl-derivatives as well as all five flavonoids inhibited with different efficiency the PDA oxidation characterized in terms of the inhibition constants, K(i), M, or the percent of inhibition degree at the maximal taking concentrations of these inhibitors. Most effective antioxidants were quercetin (K(i) = 7.7x10(-5) M) and ATBP (K(i) = 1.26x10(-4) M). Using these characteristics and other necessary criteria, the pairs PDA-quercetin and PDA-ATBP were proposed for a practical application in the test-systems for total antioxidant activity of biological objects.

[Initiation and inhibition of free-radical processes in biochemical peroxidase systems: a review].

The role of complexes containing oxygen or peroxide in monooxygenase systems and models thereof, as well as in peroxidase- and quasi-peroxidase-catalyzed processes, has been reviewed. Pathways of conversion of these intermediate complexes involving single-electron (radical) and two-electron (heterolytic) mechanisms are dealt with. Coupled peroxidase-catalyzed oxidation of aromatic amines and phenols is analyzed; inhibition and activation of peroxidase-catalyzed reactions are characterized quantitatively. Oxidation of chromogenic substrates (ABTS, OPD, and TMB) in the presence of phenolic inhibitors or polydisulfides of substituted phenols is characterized by inhibition constants (Ki, micromol). Activation of peroxidase-catalyzed oxidation of the same substrates is characterized by the degree (coefficient) of activation (alpha, M(-1)), which was determined for 2-aminothiazole, melamine, tetrazole, and its 5-substituted derivatives. Examples of applied use of peroxidase-catalyzed enzyme and model systems are given (oxidation of organic compounds, chemical analysis, enzyme immunoassay, tests for antioxidant activity of biological fluids).

[Flavonoids: efficient protectors of glucose-6-phosphate dehydrogenase from ultrasonic cavitation-induced inactivation].

Seven structurally diverse flavonoids have been shown to decrease glucose-6-phosphate dehydrogenase (G6PDH) inactivation in 0.1 M phosphate buffer (pH 7.4), induced by exposure to a high temperature (44 degrees C), or by a low-frequency ultrasound (27 kHz, 60 Wt/cm2). The activity of the compounds was assessed by their ability to change effective first-order rate constants characterizing the total (thermal and ultrasonic), thermal, and ultrasonic inactivation of 2.5 nM G6PDH (k(in), k(in)* [Russian characters: see text] kin(us), respectively). The value dependences of these constants on flavonoid concentrations (0.01-50 microM) were obtained. Rank order of potency exhibited by the compounds in protecting G6PDH appeared as follows: hesperidin > morin > silibin > naringin = quercetin > kampferol >> astragalin. The data obtained confirm the crucial role of free radicals formed in the field of ultrasonic cavitation (HO* and O2*-) in G6PDH inactivation in solutions.

[Stabilization of glucoso-6-phosphate dehydrogenase by its substrate and cofactor in an ultrasonic field].

The inactivation kinetics of glucoso-6-phosphate dehydrogenase (GPDH) and its complexes with glucoso-6-phosphate and NADP+ was characterized in aqueous solutions at 36-47 degrees C under treatment with low frequency (27 kHz, 60 W/cm2) and high frequency ultrasound (880 kHz, 1 W/cm2). To this end, we measured three effective first-order inactivation rate constants: thermal k(in)* , total (thermal and ultrasonic) kin, and ultrasonic kin (US). The values of the constants were found to be higher for the free enzyme than for its complexes GP-DH-GP and GPDH-NADP+ at all temperatures, which confirms the enzyme stabilization by its substrate and cofactor under both thermal and ultrasonic inactivation. Effective values of the activation energies (Ea) were determined and the preexponential factors of the rate constants and thermodynamic activation parameters of inactivation processes (deltaH*, deltaS*, and deltaG*) were calculated from the temperature dependences of the inactivation rate constants of GPDH and its complexes. The sonication of aqueous solutions of free GPDH and its complexes was accompanied by a reduction of Ea and deltaH* values in comparison with the corresponding values for thermal inactivation. The Ea, deltaH*, and deltaS* inactivation values for GPDH are lower than the corresponding values for its complexes. A linear dependence between the growth of the deltaH* and deltaS* values was observed for all the inactivation processes for free GPDH and its complexes.

[Flavonoids as effective protectors of urease from ultrasonic inactivation in solutions].

Inactivation of soybean urease in aqueous solution at pH 5.4, 36 degrees C, and high-frequency sonication (2.64 MHz, 1.0 W/cm2) is substantially reduced in the presence of seven structurally different flavonoids. A comparative kinetic study of the effect of these flavonoids on the effective first-order rate constants that characterize the total (thermal and ultrasonic) inactivation k(i), thermal inactivation k(i)*, and ultrasonic inactivation k(i)(US) of 25 nM enzyme solution was carried out. The dependences of the three inactivation rate constants of the urease on the concentrations of flavonoids within the range from 10(-11) to 10(-4) M were obtained. The following order of the efficiency of the flavonoids used in respect of the urease protection from ultrasonic inactivation was found: astragalin > silybin > naringin > hesperidin > quercetin > kaempferol > morin. The results confirm a significant role in the inactivation of the urease of HO* and HO2*, free radicals, which are formed in the ultrasonic cavitation field.

[Ultrasonic and thermal inactivation of catalases from the bovine liver, the methylotrophic yeast Pichia pastoris, and the fungus Penicillium piceum].

The kinetics of inactivation of catalases from bovine liver (CAT), the fungus Penicillium piceum (CAT1), and the methylotrophic yeast Pichia pastoris (CAT2) was studied in phosphate buffer (pH 5.5 or 7.4) at 45 and 50 degrees C or under the conditions of exposure to low-frequency ultrasound (LFUS; 27 kHz, 60 W/cm2). The processes were characterized by effective first-order rate constants (s(-1)): kin (total inactivation), k*in in (thermal inactivation), and k*in (us) (ultrasonic inactivation). The values of kin and k*in increased in the following order: CAT1 < CAT < CAT2. CD spectra of the enzyme solutions were recorded in the course of inactivation by high temperatures (45 and 50 degrees C) and LFUS, and the ratios of secondary structures were calculated. Processes of thermal and ultrasonic inactivation of catalases were associated with a decrease in the content of alpha helices and an increase in that of antiparallel beta structures and irregular regions (CAT1 < CAT < CAT2). We conclude that the enzymes exhibit the following rank order of resistance: CAT1 > CAT >CAT2. Judging from the characteristics of CAT1, it appears to be an optimum component for antioxidant enzyme complexes.

[Inhibition of peroxidase-catalyzed oxidation of chromogenic substrates by propyl gallate and its polydisulfide].

Peroxidase-catalyzed oxidation of 2,2'-azino-di-(3-ethyl-2,3-dihydrobenzthiazoline-6-sulfonate) (ABTS) was competitively inhibited by propyl gallate (PG) and its polydisulfide (PGPDS) at 20 degrees C in 0.015 M phosphate-citrate buffer (pH 6.0). Under these conditions, the values of the inhibition constant (Ki) were equal to 62 and 5.6 microM, respectively, for PG and PGPDS. The stoichiometric inhibition factor (f; the number of radicals extinguished per molecule of an inhibitor) equaled 2.0 and 14.7, respectively, for PG and PGPDS. Peroxidase-catalyzed oxidation of o-phenylenediamine was barely affected by PG or PGPDS. PGPDS may be used as a stop-reagent of peroxidase-catalyzed ABTS oxidation, whereas PG may serve as a calibrating inhibitor in test systems for measurement of total antioxidant activity (in human biological fluids, natural preparations, juices, wines, and other objects).

[Inhibition of soybean urease by polycarbonyl compounds].

Competitive inhibition of soybean urease was studied at 36 degrees C in aqueous solution (pH 4.95) in the presence of polycarbonyl compounds (PCCs): oxalyldihydrazide (ODH), its polydisulfide (poly(DSODH)), three cyclic beta-triketones (CTKs), and seven cyclic PCC species of differing structure. The inhibition constants of ureolysis (Ki) varied in the range 8.5-3800 microM depending on the structure of organic chelators for the nickel atom in urease. It was shown that pH variation within the range from 3.85 to 7.40 exerted a strong effect on the values of Ki] of three CTKs and hydroxyurea, which was used as a reference: pH dependences of lgK(i) were linear in all cases and displayed a break at pH 6.0-6.5. The most effective inhibitor of ureolysis was poly(DSODH), which contained approximately 28 carbonyl groups in the polymer molecule. The role of such factors as the number of carbonyl groups per PCC molecule, mutual arrangement, and reaction medium pH in the efficiency of the process of urease inhibition is discussed.

[Characterization of peroxidase-catalyzed oxidation of chromogenic substrates by tetrazole and its 5-substituted derivatives].

Peroxidase-catalyzed oxidation of 2,2-azino-di(3-ethyl-benzthiazolydine-6-sulfonic acid) (ABTS) and 3,3',5,5'-tetramethylbenzidine (TMB) is activated by tetrazole and its 5-substituted derivatives--5-amino-(AmT), 5-methyl- (MeT), 5-phenyl- (PhT), and 5-CF3- (CF3-T) tetrazoles. In phosphate-citrate or phosphate buffer (pH 6.4 or 7.2; 20 degrees C), the activating effect of tetrazoles on TMB and ABTS oxidation decreased in the series AmT > MeT > T > PhT > CF3-T and T > AmT > MeT > PhT, respectively. The (coefficient) degree of activation (alpha), expressed in M(-1), determined for both substrates and all activators, depended on substrate type, buffer nature, and pH (it increased as pH increased from 6.4 to 7.2). For TMB oxidation, good correlation between lgalpha and the Hammet constants sigma(meta) for m-substituents in the benzene series NH2, CH3, C6H5, and CF3 was found. It is suggested that AmT, MeT, and T can be used as activators of peroxidase-catalyzed oxidation of TMB and ABTS, as well as in designing peroxidase-based biosensors.

[Inhibition of the peroxidase-catalyzed oxidation of chromogenic substrates by alkyl-substituted diphenols]. / Ingibirovanie peroksidaznogo okisleniia khromogennykh substratov alkilzameshchennymi difenolami.

A comparative kinetic study of the peroxidase oxidation of three chromogenic substrates--2,2'-azino-bis(3-ethyl-2,3-dihydrobenzothiazoline-6-sulfonic acid), o-phenylenediamine (PDA), and 3,3',5,5'-tetramethylbenzidine--inhibited by trimethylhydroquinone and six tert-butylated pyrocatechols (InH) was carried out at 20 degrees C in 0.015 M phosphate-citrate buffer (pH 6.0) containing organic cosolvents (0-10% ethanol or DMF). The inhibitors were quantitatively characterized by the inhibition constants (Ki), the duration of the lag period in the oxidation product formation (delta tau), and the stoichiometric coefficient of inhibition that specifies the number of radicals terminated by one InH molecule (f). The inhibition could be competitive, noncompetitive, mixed, or uncompetitive, which depended on the nature and structure of the chromogenic substrate-diatomic phenol pair. Various substrate-diatomic phenol pairs exhibited Ki values within the range of 11-240 microM and f values from 0.7 to 2.6. The absence of a lag period was characteristic of oxidation of the substituted o-phenylenediamine-substituted pyrocatechol. The total kinetic parameters and properties of the components allowed us to suggest six chromogenic substrate-substituted diatomic phenol pairs for use in test systems for the determination of antioxidant activity in human body fluids, natural biological preparations, and food. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 5; see also http: // www.maik.ru.

[Inhibition of urease by cyclic beta-triketones and fluoride ions]. / Ingibirovanie ureazy tsiklicheskimi beta-triketonami i ftorid-ionom.

Competitive inhibition of soybean urease by 11 cyclic beta-triketones was studied in aqueous solutions at pH 7.4 and 36 degrees C. This process was characterized quantitatively by the inhibition constant (Ki), which showed a strong dependence on the structure of organic chelating agents (nickel atoms in urease) and varied from 58.4 to 847 microM. Under similar conditions, the substrate analogue (hydroxyurea) acted as a weak urease inhibitor (Ki = 6.47 mM). At 20 degrees C, competitive inhibition of urease with the ligand of nickel atoms (fluoride anion) was pH-dependent. At pH 3.85-6.45, the value of Ki for the process ranged from 36.5 to 4060 microM. Three nontoxic cyclic beta-triketones with Ki values of 58.4, 71.4, and 88.0 microM (36 degrees C) were the most potent inhibitors of urease. Their efficacy was determined by the presence of three >C=O- groups in the molecule and minimum steric hindrances to binding with metal sites in soybean urease.

[The effect of tetrazole and its amino derivatives on the kinetics of peroxidase oxidation of chromogenic substrates]. / Vliianie tetrazola i ego aminoproizvodnykh na kinetiku peroksidaznogo okisleniia khromogennykh substratov.

The peroxidase-catalyzed oxidation of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine (PDA), and 3,3',5,5'-tetramethylbenzidine (TMB) was found to be activated by tetrazole and 5-aminotetrazole (AT) and weakly inhibited by 1,5-diaminotetrazole. The activating action of tetrazole and AT on the PDA and TMB oxidation was clearly discompetitive and that on ABTS was non-competitive. The coefficients (degrees) of activation alpha were determined for three substrates and two activators; they depended on the substrate type and the buffer nature and increased along with the pH growth from 6.4 to 7.2. For AT and tetrazole, the maximal alpha values were 4140 and 800 M(-1), respectively, upon the PDA oxidation and 3570 and 540 M(-1), respectively, upon the TMB oxidation. Lower alpha values (145 and 58 M(-1) for tetrazole and AT, respectively) were characteristic of the peroxidase oxidation of ABTS. The activation of peroxidase oxidation of the substrates by tetrazole and AT at pH > or = 5.4 was explained by the nucleophilic nature of the activators interacting with the amino acid residues in the peroxidase active site according to the mechanism of acid-base catalysis. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 3; see also http://www.maik.ru.

[Inactivation of glucose-6-phosphate dehydrogenase in solution by low- and high-frequency ultrasound]. / Inaktivatsiia gliukozo-6-fosfat-degidrogenazy v rastvorakh nizko- i vesokochastotnym ul'trazvukom.

Kinetics of inactivation of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) in 0.1 M phosphate buffer (pH 7.4) within temperature range from 36 to 50 degrees C was studied comparatively under conditions of exposure of enzyme solution to low-frequency (LF, 27 kHz, 60 W/cm2) or high-frequency (HF, 880 kHz, 1.0 W/cm2) ultrasound (USD). Inactivation of G6PDH was characterized by effective first-order rate constants: (k(in)) total (summarized) inactivation; (k(in)*) thermal inactivation; and (k(in)(usd)) ultrasonic inactivation. Dilution of enzyme solution from 20 to 3 nM was accompanied by a significant increase in the values of the three rate constants. The following inequality was valid in all cases: k(in) > k(in)*. The rate constants increased upon increasing the temperature. The Arrhenius plots of the temperature dependencies of k(in) and k(in) (usd) have a salient point at 44 degrees C. The activation energy (Eact) of the total inactivation of G6PDH was higher than Eact for the process of ultrasonic inactivation of this enzyme. The two values were found to depend on USD frequency: Eact in case of inactivation with low-frequency ultrasound (LF-USD) was higher than in case of inactivation with high-frequency ultrasound (HF-USD). The rate of the ultrasonic induced inactivation of this enzyme substantially decreased in the presence of low concentrations of traps of radicals HO. (dimethylformamide, ethanol, and mannitol). This fact supports the conclusion that free radicals are involved in the mechanism of the G6PDH inactivation in solutions exposed to LF-USD and HF-USD. Ethanol was an effective protector of G6PDH inactivation in enzyme solutions exposed to USD.

[Inhibition of peroxidase oxidation of aromatic amines by substituted phenols]. / Ingibirovanie peroksidaznogo okisleniia aromaticheskikh aminov zameshchennymi fenolami.

Peroxidase-catalyzed oxidation of o-phenylene diamine (OPD) was competitively inhibited by trimethylhydroquinone (TMHQ), 4-tert-butylpyrocatechol (In5), and 4,6-di-tert-butyl-3-sulfanyl-1,2-dihydroxybenzene (In6). In6 was the most efficient inhibitor (Ki = 11 microM at 20 degrees C in 0.015 M phosphate-citrate buffer, pH 6.0). The effects of In5 and In6 were not preceded by periods of induction of OPD oxidation products (contrary to TMHQ). Peroxidase-catalyzed oxidation of tetramethylbenzidine (TMB) was non-competitively inhibited by In6 and 3-(2-hydroxyethylthio)-4,6-di-tert-butylpyrocatechol (In4), whereas o-aminophenol (OAP) acted as a mixed-type inhibitor. The effects of all three inhibitors were preceded by an induction period, during which TMB oxidation products were formed. Again, In6 was the most efficient inhibitor (Ki = 16 microM at 20 degrees C in 0.015 M phosphate-citrate buffer supplemented with 5% ethanol, pH 6.0). Judging by the characteristics of the inhibitors, taken in aggregate, it is advisable to use the pairs OPD-In5 and OPD-In6 in systems for testing the total antioxidant activity of biological fluids of humans.