Effect of the composition of the medium on the characteristics of polydisulfide bioantioxidants

Characteristics of polydisulfides of gallic acid (PDSG), 2-amino-4-nitrophenol (PDSANP), and biuret (PDSB) depending on the composition of the aqueous medium were studied. In contrast to PDSANP and PDSB, there was oxidation of PDSG with accumulation of products of polydisulfide transformation in the medium. The rate of PDSG autoxidation depended on pH on the concentration of polydisulfide and buffers and increased at high pH. The rate of oxidation significantly increased in the presence of DMPA, ethanol, or CTAB (surfactant). Decreasing the pH of the solution and adding ovalbumin and/or Triton X-100 to the medium can decrease the rate of autoxidation of PDSG in an aqueous medium. Exogenous H₂O₂ inhibited the oxidation of PDSG. The secondary structure of catalase was changed by PDSG. Electrical conductance of PDSG and PDSANP solutions was studied. Possible mechanisms of PDSG autoxidation and polydisulfides-protein interaction due to forces of cooperative electrostatic interaction, thiol-disulfide exchanges, and nucleophilic replacements were discussed.     

Intramolecular synergism of the inhibiting action of polydisulfide antioxidants in the system ferritin--H2O2--tetramethylbenzidine

Polydisulfides of urea (PDSU), thiourea (PDSTU), biuret (PDSB), and gallic acid (PDSG) and their monomer analogues (urea, biuret, and gallic acid) inhibited (in a competitive manner) tetramethylbenzidine (TMB) peroxidation catalyzed by ferritin in 0.1 M acetate buffer, pH 4.2, containing 10% dimethylformamide. Their efficiency characterized in terms of inhibition constants, Ki, increased in the following order PDSU < PDSB approximately PDSTU < PDSG. This order is determined by the reactivity of monomers with respect to HO* radicals which are the main oxidizing agents in the system ferritin--H2O2. Polydisulfide antioxidants exhibit the intramolecular synergism of the inhibiting action (non-additivity of antiradical activity relative to their monomers) that was quantitatively characterized by alpha = (Ki)pol/(Ki)mon x n, where n is the number of monomers in the polymeric inhibitors. The alpha values increased from 1.5 up to 5.18 in the following order: PDSG < PDSU < PDSB. Significantly higher inhibiting efficiency of polydisulfide antioxidants as compared to monomer forms and synergism of the inhibitory action offer promising opportunities of their use as quenchers of free radical processes in biochemical systems.     

Effect of gallic acid polydisulfide on activity and stability of catalase in various media

The effect of the active bioantioxidant polydisulfide of gallic acid (PDSG) on the catalytic activity and operational and thermal stability of catalase was studied in three media: distilled water (pH approximately 5.6), phosphate buffer, pH 7.4, and reversed micelles of Aerosol OT (AOT) in heptane of varied hydration degree w0. PDSG inhibited the catalase-induced decomposition of H2O2 by the mixed or noncompetitive mechanism: in various media the inactivation constant Ki varied in the range of (0.63-2.32).10-5 M. PDSG nearly twofold decreased the rate constant of interaction of the complex I of catalase with H2O2 (k2, M-1.sec-1) in water and reversed micelles of AOT and 3-5 times increased the effective rate constant of catalase thermal inactivation, k*in, sec-1, depending on the reaction medium. PDSG significantly decreased the rate constant of catalase inactivation during the enzymatic reaction, kin, sec-1, and thus increased the enzyme operational stability in water and reversed AOT micelles in heptane. The interaction of PDSG with catalase in water and in phosphate buffer was accompanied by significant changes in CD spectra in the far UV-region that indicated disturbances in the secondary structure of catalase subunits induced by the bioantioxidant; the latter was suggested to initiate the reaction of thiol--disulfide exchange with the enzyme. The problem of the compatibility of catalase with disulfide bioantioxidants is discussed.     

Determining role of media in peroxide oxidation of aromatic type antioxidants with participation of methemalbumins

Participation of the complexes of hemin and albumins (or delipidated albumins) in peroxidation of aromatic free radical scavengers and antioxidants was studied at varying hemin/albumin ratios. The radical-scavenging amines included o-phenylenediamine (OPD) and tetramethylbenzidine (TMB); the antioxidants, gallic acid (GA) and GA polydisulfide (GAPD). Peroxidation reactions were carried out in buffered physiological saline (BPS) supplemented with 2% dimethylsulfoxide(DMSO), pH 7.4 (medium A), or in 40% aqueous dimethylformamide (DMF), pH 7.4 (medium B). In all systems involving methemalbumins, kinetic constants (kcat), Michaelis constants (kM), and the ratios thereof (kcat/kM) were determined for OPD oxidation in medium A and TMB oxidation in medium B. Oxidation of OPD, GA, and GAPD in medium A was characterized by a decrease in the catalytic activity of hemin after the formation of hemin-albumin complexes. Conversely, oxidation of TMB and OPD in medium B was distinguished by pronounced activation of hemin present within methemalbumins.     

Key role of the medium in methemalbumin-catalyzed peroxidation of aromatic free radical scavengers and antioxidants

Participation of the complexes of hemin and albumins (or delipidated albumins) in peroxidation of aromatic free radical scavengers and antioxidants was studied at varying hemin/albumin ratios. The radicalscavenging amines includedo-phenylenediamine (OPD) and tetramethylbenzidine (TMB); the antioxidants were gallic acid (GA) and GA polydisulfide (GAPD). Peroxidation reactions were carried out in buffered physiological saline (BPS) supplemented with 2% dimethylsulfoxide (DMSO), pH 7.4 (medium A), or in 40% aqueous dimethylformamide (DMF), pH 7.4 (medium B). In all systems involving methemalbumins, kinetic constants (k_cat), Michaelis constants(K _M), and the ratios thereof(k _cat/K_M) were determined for OPD oxidation in medium A and TMB oxidation in medium B. Oxidation of OPD, GA, and GAPD in medium A was characterized by a decrease in the catalytic activity of hemin after the formation of hemin-albumin complexes. Conversely, oxidation of TMB and OPD in medium B was distinguished by pronounced activation of hemin present within methemalbumins.     

Inhibition of peroxidase oxidation of 3,3',5,5'-tetramethylbenzidine and o-phenylenediamine by 1-amino-2-naphthol-4-sulfonic acid and its polysulfide]

The kinetics of coupled peroxidation of 3,3',5,5'-tetramethylbenzidine and 1-amino-2-naphtol-4-sulfonic acid (ANSA) or its polydisulfide (poly(ADSNSA)) was studied in 0.01 M phosphate buffer (pH 6.4) at 20 degrees C. Both ANSA and poly(ADSNSA) strongly inhibited the TMB oxidation resulting in a marked delay in the product formation. Stoichiometric inhibition coefficients f, i.e., the average numbers of free-radical particles terminated by one inhibitor molecule, were estimated. The free-radical trapping effect of poly(ADSNSA) was 7.5 times greater than that of ANSA. Kinetics of coupled o-phenylenediamine (PhDA) and ANSA or poly(ADSNSA) oxidation was studied in phosphate-citrate buffers at pH 3 to 7. No lag periods in oxidation product accumulation were observed under any of the reaction conditions. A weak activation of PhDA conversion depending on pH and PhDA/ANSA ratios was observed at low ANSA concentrations, whereas increased ANSA or poly(ADSNSA) concentrations were inhibitory. The degree of PhDA inhibition was maximal in acid media, reached minimum at pH 5 to 6, and than again increased at pH above 6. Tentative mechanism of coupled aromatic amine phenol bi-substrate system peroxidation is discussed.     

Peroxidase-Catalyzed Co-oxidation of 3,3",5,5"-Tetramethylbenzidine with 2-Amino-4-nitrophenol, 4,4"-Dihydroxydiphenylsulfone, and Their Polydisulfides in Aqueous and Micellar Media

The effects of different concentrations of 2-amino-4-nitrophenol (ANP) and of its polydisulfide (poly(ADSNP)) on peroxidase-catalyzed oxidation of 3,3"5,5"-tetramethylbenzidine (TMB) were studied at 20°C in reversed micelles of AOT (0.2 M) in heptane and in mixed reversed micelles of AOT (0.1 M)–Triton X-100 (0.1 M) in isooctane supplemented with 15% hexanol. The oxidation of TMB was activated nearly twofold in the presence of ANP and nearly fourfold in the presence of poly(ADSNP) in reversed micelles of AOT, whereas in the mixed micelles oxidation of the TMB–ANP pair was associated with inhibition of TMB conversion and poly(ADSNP) activated oxidation of TMB. The co-oxidation of TMB with 4,4"-dihydroxydiphenylsulfone (DDS) and with its polydisulfide (poly(DSDDS)) at different concentrations of phenol components was accompanied by activation of TMB conversion in 0.01 M phosphate buffer (pH 6.4) supplemented with 5% DMF and in reversed micelles of AOT in heptane. The effect of pH of the aqueous solution on the initial oxidation rate of the TMB–DDS and TMB–poly(DSDDS) pairs and also the effect of hydration degree of reversed micelles of AOT on conversion of the same pairs by peroxidase were studied. A scheme of peroxidase-dependent co-oxidation of aromatic amine–phenol pairs is proposed and discussed. A significant part of this scheme is a nonenzymatic exchange of phenoxyl radicals with amines and of aminyl radicals with phenols.     

Characterisation of the major autoxidation products of 3-hydroxykynurenine under physiological conditions

3-Hydroxykynurenine (3-OHKyn) is a tryptophan metabolite that is readily autoxidised to products that may be involved in protein modification and cytotoxicity. The oxidation of 3-OHKyn has been studied here with a view to characterising the major products as well as determining their relative rates of formation and the role that H₂O₂ and hydroxyl radical (HO^·) may play in modifying the autoxidation process. Oxidation of 3-OHKyn generated several compounds. Xanthommatin (Xan), formed by the oxidative dimerisation of 3-OHKyn, was the major product formed initially. It was, however, found to be unstable, particularly in the presence of H₂O₂, and degraded to other products including the p-quinone, 4,6-dihydroxyquinolinequinonecarboxylic acid (DHQCA). A compound that has a structure consistent with that of hydroxy-xanthommatin (OHXan) was also formed in addition to at least two minor species that we were unable to identify. Hydrogen peroxide was formed rapidly upon oxidation of 3-OHKyn, and significantly influenced the relative abundance of the different autoxidation species. Increasing either pH (from pH 6 to 8) or temperature (from 25°C to 35°C) accelerated the rate of autoxidation but had little impact on the relative abundance of the autoxidation species. Using electron paramagnetic resonance (EPR) spectroscopy, a clear phenoxyl radical signal was observed during 3-OHKyn autoxidation and this was attributed to xanthommatin radical (Xan^·). Hydroxyl radicals were also produced during 3-OHKyn autoxidation. The HO^· EPR signal disappeared and the Xan^· EPR signal increased when catalase was added to the autoxidation mixture. The HO^· did not appear to play a role in the formation of the autoxidation products as evidenced using HO^· traps/scavengers. We propose that the cytotoxicity of 3-OHKyn may be explained by both the generation of H₂O₂ and by the formation of reactive 3-OHKyn autoxidation products such as the Xan^· and DHQCA.     

Autoxidation of naphthohydroquinones: effects of pH, naphthoquinones and superoxide dismutase

The rates of autoxidation of a number of pure naphthohydroquinones have been determined, and the effects of pH, superoxide dismutase (SOD) and of the parent naphthoquinone on the oxidation rates have been investigated. Most compounds were slowly oxidised in acid solution with the rates increasing with increasing pH, although 2-hydroxy-, 2-hydroxy-3-methyl- and 2-amino-1,4-naphthohydroquinone were rapidly oxidised at pH 5 and the rates of oxidation of these substances were comparatively unresponsive to changes in pH. At pH 7.4, autoxidation rates decreased in the order 2,3-dichloro-1,4-naphthohydroquinone > 5-hydroxy > 2-bromo > 2-hydroxy-3-methyl > 2-amino > 2-hydroxy > 2-methoxy > 2,3-dimethoxy > 2,3-dimethyl > 2-methyl > unsubstituted hydroquinone. The autoxidation rates of the alkyl, alkoxy, hydroxy and amino derivatives were decreased in the presence of SOD, but this enzyme had no effect on the rate of autoxidation of the 2,3-dichloro and 2-bromo derivatives while that of the 5-hydroxy derivative was increased. The rates of autoxidation of all compounds except the halogen derivatives and 5-hydroxy-1,4-naphthohydroquinone were increased by addition of the parent naphthoquinone, and quinone addition partially or completely overcame the inhibitory effect of SOD. There is evidence that the reduction of quinones to hydroquinones in vivo may lead either to detoxification or to activation. This may be due to differences in the rate or mechanism of autoxidation of the hydroquinones that are formed, and the data gained in this study will provide a framework for testing this possibility.     

Autooxidation and oxygenation of human hemoglobin]

The processes of reversible oxygen binding and nonreversible autoxidation of human hemoglobin were studied. The activation energy of the oxygen binding, as determined by the temperature dependence of the P50 parameter, was 26 +/- 4 kJ/mol, the activation energy of the autoxidation, as determined by the temperature dependence of the apparent rate constant of autoxidation, was 120 +/- 15 kJ/mol. Pyridoxal phosphate decreased the oxygen affinity of hemoglobin, slightly diminished the cooperativity of the oxygenation process and unaffected the activation energy of the oxygen binding. Pyridoxal phosphate slightly reduced the Bohr coefficient value from 0.70 to 0.65. Pyridoxal phosphate, but not pyridoxal, raised the apparent rate constant of autoxidation reaction. The rate of autoxidation significantly increased as the pH value of the medium decreased, reflecting, probably, protonation of the distal histidine of the hemoglobin. The activation energy of autoxidation was independent of pH. Aliphatic alcohols also increased the rate of the autoxidation process, probably, either by stabilization of the hemoglobin T-state, or by direct nucleophilic displacement of the oxygen molecule.     

Spectrophotometric and fluorometric study of albumins and hemin interactions with aromatic antioxidants

Difference spectrophotometry and fluorescence quenching of human and bovine serum albumins were used to determine their association constants (Ka) with hemin in buffered physiological saline (pH 7.4) supplemented with 2% dimethylsulfoxide or in 40% aqueous dimethylformamide (pH 7.4). Ka values depended on the medium, the extent of albumin delipidation, and on the method of determination. The formation of hemin complexes with o-phenylenediamine, tetramethylbenzidine, gallic acid, its polydisulfide, and two substituted di-tert-butyl pyrocatechols was studied by difference spectrophotometry in the same media; Ka values for the complexes were calculated and compared to each other. The formation of complexes of these aromatic ligands with albumins was studied fluorometrically; Ka values were of order of approximately 10(-5) M-1 and decreased with the ligand hydrophobicity.     

Inhibition of 3,3",5,5"-Tetramethylbenzidine and o-Phenylenediamine Peroxidation with 1-Amino-2-Naphtol-4-Sulfonic Acid and Its Polydisulfide

The kinetics of coupled peroxidation of 3,3",5,5"-tetramethylbenzidine (TMB) and 1-amino-2-naphtol-4-sulfonic acid (ANSA) or its polydisulfide (poly(ADSNSA)) was studied in a 0.01 M phosphate buffer (pH 6.4) at 20°C. Both ANSA and poly(ADSNSA) strongly inhibited the TMB oxidation resulting in a marked delay in the product formation. Stoichiometric inhibition coefficients f, i. e., the average numbers of free-radical particles terminated by one inhibitor molecule, were estimated. The free-radical trapping effect of poly(ADSNSA) was 7.5 times greater than that of ANSA. Kinetics of coupled o-phenylenediamine (PhDA) and ANSA or poly(ADSNSA) oxidation was studied in phosphate–citrate buffers at pH 3 to 7. No lag periods in oxidation product accumulation were observed under any of the reaction conditions. A weak activation of PhDA conversion depending on pH and PhDA/ANSA ratios was observed at low ANSA concentrations, whereas increased ANSA or poly(ADSNSA) concentrations were inhibitory. The degree of PhDA-inhibition was maximal in acid media, reached minimum at pH 5 to 6, and than again increased at pH above 6. A tentative mechanism of coupled aromatic amine–phenol bi-substrate system peroxidation is discussed.     

Autoxidation of naphthohydroquinones: Effects of pH,naphthoquinones and superoxide dismutase

The rates of autoxidation of a number of pure naphthohydroquinones have been determined, and the effects of pH, superoxide dismutase (SOD) and of the parent naphthoquinone on the oxidation rates have been investigated. Most compounds were slowly oxidised in acid solution with the rates increasing with increasing pH, although 2-hydroxy-, 2-hydroxy-3-methyl- and 2-amino-1,4-naphthohydroquinone were rapidly oxidised at pH 5 and the rates of oxidation of these substances were comparatively unresponsive to changes in pH. At pH 7.4, autoxidation rates decreased in the order 2,3-dichloro-1,4-naphthohydroquinone > 5-hydroxy > 2-bromo > 2-hydroxy-3-methyl > 2-amino > 2-hydroxy > 2-methoxy > 2,3-dimethoxy > 2,3-dimethyl > 2-methyl > unsubstituted hydroquinone. The autoxidation rates of the alkyl, alkoxy, hydroxy and amino derivatives were decreased in the presence of SOD, but this enzyme had no effect on the rate of autoxidation of the 2,3-dichloro and 2-bromo derivatives while that of the 5-hydroxy derivative was increased. The rates of autoxidation of all compounds except the halogen derivatives and 5-hydroxy-1,4-naphthohydroquinone were increased by addition of the parent naphthoquinone, and quinone addition partially or completely overcame the inhibitory effect of SOD. There is evidence that the reduction of quinones to hydroquinones in vivo may lead either to detoxification or to activation. This may be due to differences in the rate or mechanism of autoxidation of the hydroquinones that are formed, and the data gained in this study will provide a framework for testing this possibility.     

The alpha 1 beta 1 contact of human hemoglobin plays a key role in stabilizing the bound dioxygen.

When the alpha and beta chains were separated from human oxyhemoglobin (HbO(2)), each individual chain was oxidized easily to the ferric form, their rates being almost the same with a very strong acid-catalysis. In the HbO(2) tetramer, on the other hand, both chains become considerably resistant to autoxidation over a wide range of pH values (pH 5-11). Moreover, HbA showed a biphasic autoxidation curve containing the two rate constants, i.e. k(f) for the fast oxidation due to the alpha chains, and k(s) for the slow oxidation to the beta chains. The k(f)/k(s) ratio increased from 3.2 at pH 7.5-7.3 at pH 5.8, but became 1 : 1 at pH values higher than 8.5. In the present work, we used the valency hybrid tetramers such as (alpha(3+))2(beta O(2))(2) and (alpha O(2)(2)(beta(3+))(2), and demonstrated that the autoxidation rate of either the alpha or beta chains (when O2- ligated) is independent of the valency state of the corresponding counterpart chains. From these results, we have concluded that the formation of the alpha 1 beta 1 or alpha 2 beta 2 contact suppresses remarkably the autoxidation rate of the beta chain and thus plays a key role in stabilizing the HbO(2) tetramer. Its mechanistic details were also given in terms of a nucleophilic displacement of O(2)(-) from the FeO(2) center, and the emphasis was placed on the proton-catalyzed process performed by the distal histidine residue.     

The Mechanism of Iron (III) Stimulation of Lipid Peroxidation

A study conducted on Fe²⁺ autoxidation showed that its rate was extremely slow at acidic pH values and increased by increasing the pH; it was stimulated by Fe³⁺ addition but the stimulation did not present a maximum at a Fe²⁺/Fe³⁺ ratio approaching 1:1. The species generated during Fe³⁺-catalyzed Fe²⁺ autoxidation was able to oxidize deoxyribose; the increased Fe²⁺ oxidation observed at higher pHs was paralleled by increased deoxyribose degradation. The species generated during Fe³⁺-catalyzed Fe²⁺ autoxidation could not initiate lipid peroxidation in phosphatidylcholine liposomes from which lipid hydroperoxides (LOOH) had been removed by treatment with triph-enylphosphine. Neither Fe²⁺ oxidation nor changes in the oxidation index of the liposomes due to lipid peroxidation were observed at pHs where the Fe³⁺ effect on Fe²⁺ autoxidation and on deoxyribose degradation was evident. In our experimental system, a Fe²⁺/Fe³⁺ ratio ranging from 1:3 to 2:1 was unable to initiate lipid peroxidation in LOOH-free phosphatidylcholine liposomes. By contrast Fe³⁺ stimulated the peroxidation of liposomes where increasing amounts of cumene hydroperoxide were incorporated. These results argue against the participation of Fe³⁺ in the initiation of LOOH-independent lipid peroxidation and suggest its possible involvement in LOOH-dependent lipid peroxidation.     

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.     

Pyruvate but not lactate prevents NADH-induced myoglobin oxidation

In this work, we investigated the influence of NADH on the redox state of myoglobin and the roles of pyruvate and lactate in this process. NADH increased the autoxidation rate of myoglobin. Both a drop in pH and partial deoxygenation markedly stimulated the autoxidation process and the influence of NADH. A correlation between met-Mb formation rate and NADH oxidation rate was always observed. The increased rate of Mb autoxidation caused by NADH was inhibited by catalase and pyruvate but not by l-lactate. The antioxidant activity versus H2O2 of both pyruvate and lactate was evidenced by chemiluminescence experiments. The antioxidant activity of lactate disappeared completely in the presence of myoglobin or apo-myoglobin, whereas it was only reduced for pyruvate. These results could be of interest in preventing autoxidation of myoglobin that can contribute to ischemia-reperfusion injury during infarction or high-intensity exercise.     

Biphasic nature in the autoxidation reaction of human oxyhemoglobin

In comparison with myoglobin molecule as a reference, we have studied the autoxidation rate of human oxyhemoglobin (HbO₂) as a function of its concentration in 0.1 M buffer at 35°C and in the presence of 1 mM EDTA. At pH 6.5, HbA showed a biphasic autoxidation reaction that can be described completely by a first-order rate equation containing two rate constants — k_f, for fast autoxidation of the α-chain, and k_s, for slow autoxidation of the β-chain, respectively. When tetrameric HbO₂ was dissociated into αβ-dimers by dilution, the value of k_f increased markedly to an extent comparable with the autoxidation rate of horse heart oxymyoglobin (MbO₂). The rate constant k_s, on the other hand, was found to remain at an almost constant value over the whole concentration range from 1.0 × 10⁻³ M to 3.2 × 10⁻⁶ M in heme. At pH 8.5 and pH 10.0, however, the autoxidation of HbO₂ was monophasic, and no enhancement in the rate was observed by diluting hemoglobin solutions. Taking into consideration the effects of 2,3-diphosphoglyceric acid and chloride anion on the autoxidation rate of HbO₂, we have characterized the differential susceptibility of the α- and β-chains to the autoxidation reaction in aqueous solution.     

Peroxidase-catalyzed oxidation of 3,3',5,5'-tetramethylbenzidine in the presence of 2,4-dinitrosoresorcinol and polydisulfide derivatives of resorcinol and 2,4-dinitrosoresorcinol

A comparative study of the kinetics of peroxidase-catalyzed oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of 2,4-dinitrosoresorcinol (DNR), its polydisulfide derivative [poly(DNRDS)], and resorcinol polydisulfide [poly(RDS)], substances that competitively inhibit the formation of TMB conversion product, was carried out. The inhibition constants, Ki for DNR, poly(DNRDS), and poly(RSD) were determined at 20 degrees C and pH 6.4 to be 110, 13.5, and 0.78 microM, respectively. The stoichiometric coefficients of inhibition were calculated to be 0.38 and 76 for poly(DNRDS) and poly(RDS), respectively. In the pH range 6.4-7.0, the initial rates of the peroxidative oxidation of TMB, and its mixtures with DNR and poly(DNRDS) and the Ki value for poly(RDS) substantially decreased with increasing pH. The kinetic parameters of poly(RDS) (Ki 0.22-0.78 microM and f76) suggest that it is the most efficient inhibitor of peroxidase oxidation of TMB: in micromolar concentrations, it completely stops this process and can be used in EIA.     

Characteristics of tyrosinase in AOT-isooctane reverse micelles

Isooctane-AOT-H(2)O is a suitable system for studying enzyme behavior in organic solvents. Tyrosinase was able to catalyze a well-known reaction in aqueous medium: oxidation of 4-methylcatechol to yield 4-methyl-o-benzoquinone. This reaction was studied using the preceding ternary system with adequate amounts of each component to make up reverse micelles. 4-Methyl-o-benzoquinone stability was demonstrated in isooctane even at alkaline pH values. Apparent K(m) and V(max) were similar to those in water, but substrate inhibition was more evident. The pH and temperature appear to be shifted toward high and low values, respectively. Characteristic parameters of reverse micelles, omega(0) (= H(2)O/AOT) and percentage of H(2)O (v/v), were investigated. The results obtained showed that the steady-state rate varies either with omega(0) or with percentage of H(2)O. The variation observed with omega(0) showed an optimal value while an increase in percentage of H(2)O can lead to decreased or increased activity depending on substrate concentration.