The role of the extent of hydration of reversed micelles of surfactant s in the regulation of the peroxidase activity of ferritin and immunocomplexes of cortisol-peroxidase conjugates

The effect of the degree of hydration (W0) of reversed micelles of Aerosol OT (AOT) and its mixture with Triton X-45 in heptane on the peroxidase activity of horse spleen ferritin in the oxidation of various substrates by hydrogen peroxide and organic hydroperoxides and on the activity of solubilized or immobilized immunocomplexes of horseradish peroxidase-cortisol conjugates (HP-COR) was studied. The peroxidase activity versus W0 plot has maxima at W0 8-14 and 19-22, which cannot be attributed to dissociation of immunocomplexes into its components or of ferritin into its subunits. The possibility of the stabilization of the conformers of oligomeric proteins by reversed micelles and the effect of the self-association of micelles on the peroxidase activity of the HP-COR immunocomplexes and ferritin were discussed. A procedure for the isolation of the iron-containing cluster from the ferritin molecule without reduction of the Fe3+ ions was suggested.     

Human thyroid peroxidase: inhibition of iodide ion and 3,3',5,5'-tetramethylbenzidine hydrolysis by phenol antioxidants]

A comparative kinetic study on the poly(gallic acid disulfide) (poly(DSGA)) inhibition of the iodide ion oxidation and on the 2-hydroxy-3,5-di-tert-butyl-N-phenylaniline (butaminophene) inhibition of 3,3',5,5'-tetramethylbenzidine (TMB) oxidation involving human thyroid peroxidase (hTPO) and horseradish peroxidase (HRP) was performed. The inhibition processes were characterized with the inhibition constants Ki and stoichiometric inhibition coefficients f, indicating the number of radical particles perishing on one inhibitor molecule. In the case of poly(DSGA), the Ki values for the I- oxidation were 0.60 and 0.04 microM, and the coefficients f were 13.6 and 16.5 for hTPO and HRP, respectively, which evidences the regeneration and high effectiveness of the polymeric inhibitor. In the case of butaminophene, the Ki values for TMB oxidation were 38 and 46 microM for hTPO and HRP, respectively. The coefficients f were 1.33 and 1.47, respectively, to reveal that butaminophene does not regenerate. The inhibition mechanisms for I- and TMB oxidation involving the two peroxidases are discussed.     

Changes in the antioxidant properties of substituted phenol polydisulfides during interaction with human serum albumin

Gallic acid polydisulfide and poly(2-aminodisulfide-4-nitrophenol) in aqueous solutions were shown to form polycomplexes with human serum albumin. This process was accompanied by considerable changes in the spectrum of protein circular dichroism recorded in distilled water in the far UV range at 20 degrees C. Complex formation between human serum albumin and polydisulfides was followed by a marked decrease in the content of alpha-helices and increase in the count of antiparallel beta-structures in the protein. Stable complexes containing 1.5, 2.8, and 7.7 poly(2-aminodisulfide-4-nitrophenol) molecules per human serum albumin molecule were formed in bicarbonate buffer (pH 9.0). In these complexes, the secondary protein structure underwent changes similar to those in polycomplexes of human serum albumin and polydisulfides. Gallic acid polydisulfide and poly(2-aminodisulfide-4-nitrophenol) inhibited the catalase-induced degradation of 50 mM H2O2. Complexes of human serum albumin and poly(2-aminodisulfide-4-nitrophenol) increased the catalytic activity and operational stability of catalase 1.5 and 4-7-fold, respectively. This was characterized by the effective reaction rate constant (kin, s-1). Our results indicate that complexes of human serum albumin and substituted phenol polydisulfides act as potent protectors and activators of catalase during enzymatic degradation of H2O2 at high concentrations.     

Inhibition of peroxidase oxidation of aromatic amines by substituted phenols

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.     

Inactivation of the human thyroid peroxidase by ultrasound cavitation]

The inactivation kinetics of a human thyroid peroxidase protein fraction upon sonication (ultrasound frequency 27 kHz, power 60 W/cm2) of the enzyme solution in 15 mM phosphate buffer, pH 7.5, was studied. To quantitatively characterize the dependence of the slowest stage of the human thyroid peroxidase inactivation on temperature (36.0-50.4) degrees C, an effective constant of ultrasound inactivation rate Kin(US) was used. From the temperature dependence of Kin(US) at temperatures below 43 degrees C, the activation energy was estimated to be 8.11 kcal/mol. It was shown that the rate of human thyroid peroxidase inactivation strongly depends on the concentration of total protein in solution: the kin(US) value decreases more than sixfold in the protein concentration range from 0.2 to 0.8 mg/ml. It was also shown that poly(2-aminodisulfide-4-nitrophenol), its complexes with human serum albumin as well as the complexes human serum albumin--poly(gallic acid disulfide) substantially inhibit the ultrasound-induced inactivation of the enzyme and can be its effective stabilizers in the ultrasound cavitation field. This confirms the suggestion that active free radicals HO., O2.- and HO2. play a key role in the inactivation of human thyroid peroxidase. A general scheme of the inactivation of human thyroid peroxidase is proposed, which represents a chain of successive and parallel reversible and irreversible elementary steps.     

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).     

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.     

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.     

The effect of hydrophobization of glucose-6-phosphate dehydrogenase with progesterone on its thermal inactivation

Thermal inactivation of glucose-6-phosphate dehydrogenase (G6PDH) and its conjugates with progesterone containing 3, 7 and 35 molecules of the modifier was studied in bidistilled water over a temperature range 35-47 degrees. At different temperatures and initial concentrations of the enzyme and its modified forms, thermal inactivation is described by the equation of the first order up to a significant degree of enzyme deactivation. The effective Kin values are decreased with the increase of the native G6PDH concentration and changed in a complicated manner with the increase of the conjugate concentration depending on the enzyme modification degree, which reflects a great role of the enzyme hydrophobicity in its inactivation. The role of hydrophobicity of the modified G6PDH in changes of its specific activity is discussed.