Superactivity of peroxidase solubilized in reversed micellar systems

Vaccinium mirtyllus peroxidase solubilized in reversed micelles was used for the oxidation of guaiacol. Some relevant parameters for the enzymatic activity, such as pH,w _o (molar ratio water/surfactant), surfactant type and concentration, and cosurfactant concentration, were investigated. The peroxidase showed higher activities in reversed micelles than in aqueous solution. The stability of the peroxidase in reversed micelles was also studied, namely, the effect ofw _o and temperature on enzyme deactivation. The peroxidase displayed higher stabilities in CTAB/hexanol in isooctane reversed micelles, with halflife times higher than 500 h.     

Electrochemical transformations and anti/prooxidant activity of sterically hindered <Emphasis Type="Italic">o</Emphasis>-benzoquinones

The electrochemical properties of a series of sterically hindered o-benzoquinones in aprotic solvents were examined. The reaction of O₂ and KO₂ with o-benzoquinones and electrogenerated o-semiquinone radical anions was studied. The formation of o-semiquinone radical anions in the reactions of KO₂ with the studied o-quinones was detected using UV-visible and ESR spectroscopy. The influence of the test compounds on lipid peroxidation was considered at the molecular and tissue levels in vitro. In the most cases, o-quinones exhibit antioxidant activity, which varies depending on the type of lipid substrate.     

A comparative study of immobilization methods of a tyrosinase enzyme on electrodes and their application to the detection of dichlorvos organophosphorus insecticide

The use of several designs of amperometric enzymatic biosensors based on the immobilized tyrosinase enzyme (Tyr) for determining dichlorvos organophosphate pesticide are described. The biosensors are based on the reversible inhibition of the enzyme and the chronocoulometric measurement of the charge due to the charge-transfer mediator 1,2-naphthoquinone-4-sulfonate (NQS). Tyr becomes active when reducing the quinone form of the mediator molecule (NQS) to the reactive o-diol form substrate of Tyr (H₂NQS) at the working electrode, thus permitting modulation of the catalytic activity of the enzyme and measurement of the inhibition produced by the pesticide. The full activity of the enzyme reversibly recovers after removal of the pesticide and re-oxidation of H₂NQS.Tyr was immobilized onto electrodes using different procedures: (i) entrapment within electropolymerized conducting and non-conducting polymers, (ii) covalent attachment to self-assembled monolayers (SAM), (iii) cross-linking with glutaraldehyde (and nafion covering) and (iv) dispersion within carbon-paste electrodes. The mediator was co-immobilized onto the working electrode next to the enzyme and reagentless biosensors were subsequently constructed. In the SAM design (ii) NQS was added to the solution. The analytical properties of the different biosensors based on the competitive inhibition produced by dichlorvos were then compared. A detection limit of about 0.06 μM was obtained for dichlorvos with entrapment of NQS and Tyr within electropolymerized poly(o-phenylenediamine) polymer (oPPD), which was the design that proved to have the best analytical performance.     

Electrochemical theory of the transfer of protons across a biological membrane facilitated by weak acid uncouplers added to the medium

An electrochemical theory of the transport of protons across a biological membrane facilitated by uncouplers of oxidative phosphorylation added to the medium has been developed. The interfacial potential differences at the two membrane/solution interfaces are explicitly taken into account. The derived equations are applied to explain the uncoupling activity of uncouplers like 2,4,6-trinitrophenol, 2,4-dinitrophenol, carbonylcyanide p-trifluoromethoxyphenylhydrazine, and 3,5-di(tert-butyl)-4-hydroxy-benzylidenemalononitrile, in terms of their protonic dissociation constants in aqueous solution (K^W) as well as in the membrane medium (K^M) and their standard ion-transfer potential (of anionic form) at the membrane/solution interface (ΔΦ_A^o); the (relative) values of K^M and ΔΦ_A^o are estimated from data determined by ion-transfer voltammetry of the uncouplers at the nitrobenzene/water interface. The equations can also explain the unique behaviour of picrate as uncoupler that it is ineffective when added to intact mitochondria but effective when added to submitochondrial particles with inside-out mitochondrial inner membranes.     

Heat revolution on photophysical properties and electroluminescent performance of Ir(ppy)3-doped bipolar host of oxadiazole derivatives attaching with inert group of tert-butyl moiety

Two bipolar materials,2,5-bis(2-(9H-carbazole-9-yl)phenyl)-1,3,4-oxadiazole(o-CzOXD)and 2,5-bis(2-(3',6'-di-tert-butyl-9H-carbazole-9-yl)phenyl)-1,3,4-oxadiazole(tBu-o-CzOXD),were synthesized according to reported methods.In parallel study,it was demonstrated that introduction of inert tert-butyl group improved material thermal stability,even though this modification only had a slight influence to the photophysical and electrochemical properties of these materials.A comparative study focusing on effects of heat treatment was carried out on the quartz glass substrates with vacuum deposited films containing one of the bipolar host doped with 6 wt%fac-tris(2-phenylpyridinato-N,C2’)iridium(Ir(ppy)3).Results show that when the two samples were heated,the absorption,emission,and photo images of the host:dopant system changed,with the o-CzOXD suffering more severe degradation under high temperature,which is consistent with their thermal stability.In addition,it was proved that the high temperature-annealed host:dopant system can enhance the emission of the dopant.This finding was used as a guideline to improve our device performance.We fabricated two types of phosphorescent organic light-emitting devices(PhOLEDs),one was based on o-CzOXD,the other was based on tBu-o-CzOXD.They had analogous structure.We investigated the effect of heat on device performance by selectively annealing.Although these two freshly prepared devices exhibited similar performance,when annealed at 90°C for 10 min,the OLEDs based on tBu-o-CzOXD showed significant performance enhancement,which can be attributed to the observation that annealing Ir(ppy)3 doped host can change film morphology and enhance the dopant emission.The maximum efficiencies of the freshly prepared tBu-o-CzOXD device were 25.8 cd A-1,23.1lm W-1,and 9.3%;whereas those for annealed device were 47.0 cd A-1,42.2 lm W-1,and 13.4%.     

Reversed micelles as matrix microreactors for chemical processing of macromolecules

Being solubilized in the systems of the surfactant reversed micelles (RM), the macromolecules incorporate into the inner cavity of RM, whose size can be changed by varying the surfactant hydration degree W_o, i.e. [H₂O]/[Surfactant] molar ratio. The conjugates of macromolecules (protein-protein, protein-linear polyelectrolyte) are synthesized in RM of AOT (Aerosol OT, sodium bis[2-ethylhexyl]sulfosuccinate) in octane. The yield critically depends on the hydration degree: the reaction does not proceed at low W_o, but at W_o exceeding threshold value (which differs for various macromolecules) the yield increases sharply and reaches 100%. Using the ultracentrifugation it was demonstrated that at W_o lower than threshold the polyelectrolyte represents a compact globule compressed by the micellar matrix in the RM inner cavity. Under these conditions there is no space for the protein in the RM, containing polyelectrolyte, and therefore reaction does not proceed. At W_o higher than threshold RM become large enough to entrap the conjugated macromolecules simultaneously. The possibility of regulation of the conjugate composition by variation of W_o (size of micellar matrix) was demonstrated. The RM are applied as universal matrix microreactors, for modification of macromolecules with water insoluble reagents and for regulation of supramacromolecular composition of oligomeric enzymes.     

Hydrolysis of palm kernel olein in AOT-isooctane-water reversed micelles

Rhizopus arrhizus lipase (EC 3.1.1.3; triacylglycerol hydrolase) was used in this study to investigate the hydrolysis of palm kernel olein in AOT-isooctane-water reversed micelle system at W_o = 13, pH = 7, and T = 30?C. The hydrolytic reaction obeys Michaelis-Menten kinetics for substrate concentrations in the range (0.175 and 0.877M). The apparentK _m and V_max for the substrate were 0.397M (equivalent to 9.06% w/v) and 5523 U/mg protein, respectively. Product inhibition with a dissociation constant of the enzyme-product complex,K _I = 9.74 mM, was confirmed. Experimental results from the change of product concentrations with respect to time correlated sufficiently with those predicted theoretically from the rate equation for a reaction time up to 100 min. However, the discrepancy between the observed results and the predicted ones would increase with reaction time. Possible reasons for this deviation were discussed.     

The ion transfer of the basic dye rhodamine B across the liquid-liquid interface of two immiscible solvents

The ion transfer of the basic dye rhodamine B at the interface between water and nitrobenzene, water and 1, 2-dichloroethane, as well as water and nitrobenzene - chlorobenzene mixtures has been studied by cyclic voltammetry and chronopotentiometry with linear current scanning. A transfer mechanism of rhodamine B is proposed in terms of its electrochemical behavior, dissociation and distribution equilibria, and is ascribed as diffusion-controlled reversible process of rhodamine B. The experimental data obtained for the relationship between interracial half-wave potential Δ^W_oφ° and pH are in agreement with the theoretical equation based on the mechanism, and the standard interfacial potential differences Δ^W_oφ° and standard Gibbs energies Δ^W_oG° are calculated by extrapolation. The effect of the nature of solvent on the transfer behavior and the stability of the interface have been discussed.     

Ion-pair extractions by the use of planar and non-planar extracting solvents

The effect of extracting solvent was studied on the ion-pair extraction reactions with a special interest in the effect of molecular shape (planar or non-planar) of the solvent. The following two reactions were investigated: (HNN)_o + (Q⁺)_W (Q·NN)_o + (H⁺)_W (Extraction I); (Q-ClO₄)_o + (Pi^–)_w (Q·Pi)_o + (ClO ₄ ^– )_w (Extraction II), where HNN, NN^–, Pi^–, and Q⁺ represent 1-nitro-2-naphthol, its base form (1-nitro-2-naphtholate), picrate, and a cationic species, respectively. Above extraction equilibria were confirmed to hold in cases of various extracting solvents including planar solvents (1-chloronaphthalene, etc.) and non-planar solvents (1,2-dichloroethane, chloroform). An approximately linear relationship was found to exist between the extraction constants of Extraction I (logK _ex ^I ) and the Kosower's Z-value of extracting solvents. It was also found that the compatibility between the molecular shape of the ion-pair complexes and that of the extracting solvents affected the extractability to a considerable extent.     

A model for two-electron four-center mixed valence systems. Application to [W4O8Cl8(H2O)4]2−

The energy levels of a two-electron four-center mixed-valence system are calculated as functions of a resonance integral (β′) and an electron repulsion parameter (k′_o). Using configuration interaction, exact solutions are given for all intermediate cases between the valence-bond limit (small β′/k′_o) and the molecular-orbital limit (large β′/k′_o). The results are applied to the [W₄O₈Cl₈(H₂O)₄]^2? case. The main predictions are the occurrence of a diamagnetic ground state with a large singlet-triplet gap, and the existence of an intense intervalence band polarized in the molecular plane. These predictions are supported by experiment. From the analysis of the optical spectrum it is concluded that this system is nearer to the molecular-orbital limit than to the valence-bond limit.     

Trialkylantimony(V) o-amidophenolates: Electrochemical transformations and antiradical activity

The electrochemical transformations and antiradical activity of trialkylantimony(V) o-amidophenolate derivatives, (AP)SbR₃ (AP = 4,6-di-tert-butyl-N-(2,6-diisopropylphenyl)-o-amidophenolate); R = CH₃ (I), C₂H₅ (II), and C₆H₁₁ (III), are studied. The electrochemical oxidation of compounds I–III proceeds successively to form mono- and dicationic forms of the complexes. The presence of the donor hydrocarbon groups at the antimony(V) atom shifts the oxidation potentials to the cathodic range and decreases the stability of the monocationic complexes formed in electrochemical oxidation. The second anodic process is irreversible and accompanied by o-iminoquinone decoordination. The antiradical activity of compounds I–III is studied in the reaction with the diphenylpicrylhydrazyl radical and oleic acid autooxidation. The values obtained for indices EC₅₀ and IC₅₀ indicate the antiradical activity of the studied compounds. Complexes I–III were found to be the efficient inhibitors of oleic acid oxidation and act as efficient destructors of hydroperoxides.     

2-Acetylpyridine- and 2-benzoylpyridine-derived thiosemicarbazones and their antimony(III) complexes exhibit high anti-trypanosomal activity

Complexes [Sb(2Ac4oClPh)Cl₂] (1), [Sb(2Ac4oFPh)Cl₂] (2), [Sb(2Ac4oNO₂Ph)Cl₂] (3), [Sb(2Bz4oClPh)Cl₂] (4), [Sb(2Bz4oFPh)Cl₂] (5) and [Sb(2Bz4oNO₂Ph)Cl₂] (6) were obtained with 2-acetylpyridine-N(4)-ortho-chlorophenyl thiosemicarbazone (H2Ac4oClPh) and its N(4)-ortho-fluor (H2Ac4oFPh) and N(4)-ortho-nitro (H2Ac4oNO₂Ph) analogues, and with the corresponding 2-benzoylpyridine-derived thiosemicarbazones (H2Bz4oClPh, H2Bz4oFPh, H2Bz4oNO₂Ph). The studied compounds are excellent inhibitors of Trypanosoma cruzi growth. H2Bz4oClPh and complexes (4) and (1) were the most trypanosomicidal.Upon coordination of H2Ac4oClPh to antimony(III) in 1, the therapeutic index (TI) goes from 10.58 to 14.35. However, the best values of TI were found for H2Bz4oClPh (TI = 1240) and H2Ac4oNO₂Ph (TI = 773). Structure-activity relationship (SAR) studies did not allow the establishment of correlations between the anti-trypanosomal activity and physico-chemical parameters, but correlations were found between the cytotoxicities and physico-chemical properties.     

Kinetics of oxidative ammonolysis of 4-bromo-o-xylene: III. Conversion of 4-bromo-o-tolunitrile as a substrate

Kinetics of oxidative ammonolysis of 4-bromo-o-tolunitrile on V-Sb-Bi-Zr/γ-Al₂O₃-oxide catalyst in the temperature range 633–673 K were studied. We found that the rate of conversion of 4-bromo-o-tolunitrile to the target 4-bromphthalonitrile and CO₂ was described by the half-order equation with respect to the substrate concentration and was independent of the partial pressures of oxygen and ammonia. The byproducts are 4-bromophthalimide formed through the hydrolysis of 4-bromophthalonitrile, CO₂ produced by oxidation of 4-bromo-o-tolunitrile and decarboxylation of 4-bromophthalimide, and 4-brombenzonitrile produced from 4-bromo-o-tolunitrile and 4-bromophthalimide.     

Simultaneous detection of ascorbate and uric acid using a selectively catalytic surface

The direct and selective detection of ascorbate at conventional carbon or metal electrodes is difficult due to its large overpotential and fouling by oxidation products. Electrode modification by electrochemical reduction of diazonium salts of different aryl derivatives is useful for catalytic, analytical and biotechnological applications. A monolayer of o-aminophenol (o-AP) was grafted on a glassy carbon electrode (GCE) via the electrochemical reduction of its in situ prepared diazonium salts in aqueous solution. The o-aminophenol confined surface was characterized by cyclic voltammetry. The grafted film demonstrated an excellent electrocatalytic activity towards the oxidation of ascorbate in phosphate buffer of pH 7.0 shifting the overpotential from +462 to +263 mV versus Ag/AgCl. Cyclic voltammetry and d.c. amperometric measurements were carried out for the quantitative determination of ascorbate and uric acid. The catalytic oxidation peak current was linearly dependent on the ascorbate concentration and a linear calibration curve was obtained using d.c. amperometry in the range of 2-20 μM of ascorbate with a correlation coefficient 0.9998, and limit of detection 0.3 μM. The effect of H₂O₂ on the electrocatalytic oxidation of ascorbate at o-aminophenol modified GC electrode has been studied, the half-life time and rate constant was estimated as 270 s, and 2.57 × 10⁻³ s⁻¹, respectively. The catalytically selective electrode was applied to the simultaneous detection of ascorbate and uric acid, and used for their determination in real urine samples. This o-AP/GCE showed high stability with time, and was used as a simple and precise amperometric sensor for the selective determination of ascorbate.     

The standard enthalpies of formation of alkoxy-NNO-azoxy compounds

The standard enthalpies of combustion Δ_c H ^o and formation Δ_f H ^o of seven alkoxy-NNO-azoxy compounds containing the-N⁺(O^?)=NO-characteristic group were determined by combustion in a calorimetric bomb in the atmosphere of oxygen. The contribution of this group to the Δ_f H ^o enthalpies of the substances studied was calculated. The Δ_f H ^o enthalpies found by the method of group contributions were in satisfactory agreement with experimental data.     

Determination of the Water/AOT/Isooctane Reverse Micelles Size Parameters from Their Refractive Index Data

The present paper aims to study the proprieties of water confined in water/AOT/isooctane reverse micelles from their refractive indices at 298.5 K. The refractive indices of the microemulsions were investigated at increasing water concentration and at different micellar volume fractions, Φ _d. The refractive index of micellar water was deduced for a large water to AOT molar ratio, W ₀. The refractive index of interfacial water was also calculated. Then, the molar faction of interfacial water, α, was deduced for different W ₀ values and compared with those measured by NMR as reported in the literature. The results show qualitative agreement. Finally, the average aggregation number, n _agg, the area per surfactant headgroup, σ, as well as the interfacial thickness, d _i, of the reverse micelles were determined for the W ₀ values studied. The values of these structural parameters also show good qualitative agreement with NMR and small angle X-ray scattering data reported in the literature. Thus, the refractive index method can be a cheap and fast alternative for these two methods.     

Hydrogen-bonded clusters and solvate structures in the supercritical CO2-water-o-hydroxybenzoic acid system: the car-parrinello molecular dynamics

Hydrogen-bonded clusters and solvate structures formed by o-hydroxybenzoic acid (o-HBA) and water in supercritical CO₂ were studied (T = 318 K, 348 K, ρ = 0.7 g/cm³). The atom-atom radial distribution functions, coordination numbers, average numbers of hydrogen bonds for individual atomic groups, and power spectrum were calculated by the Car-Parrinello molecular dynamics. Despite the high polarity of the cosolvent, the hydroxyl group of o-HBA predominantly forms intramolecular hydrogen bond, while hydrogen bonds with water involve only the atoms of carboxyl groups. The temperature effect on the stability of these bonds showed itself in different ways. The intermolecular interactions of o-HBA with carbon dioxide were found to be weaker than those with water. It was established that the Lewis acid-Lewis base interactions between CO₂ and the hydroxyl group of the solute increase with increasing temperature. Instantaneous configurations illustrating the temperature effects on the molecular structures were obtained.     

Water-in-Oil-in-Water Double Emulsions as Protective Carriers for Sambucus nigra L. Coloring Systems

The use of natural colorants is needed to overcome consumer concerns regarding synthetic food colorants′ safety. However, natural pigments have, in general, poor stability against environmental stresses such as temperature, ionic strength, moisture, light, and pH, among others. In this work, water-in-oil-in-water (W₁/O/W₂) emulsions were used as protective carriers to improve color stability of a hydrophilic Sambucus nigra L. extract against pH changes. The chemical system comprised water and corn oil as the aqueous and oil phases, respectively, and polyglycerol polyricinoleate (PGPR), Tween 80, and gum Arabic as stabilizers. The primary emulsion was prepared using a W₁/O ratio of 40/60 (v/v). For the secondary emulsion, W₁/O/W₂, different (W₁/O)/W₂ ratios were tested with the 50/50 (v/v) formulation presenting the best stability, being selected as the coloring system to test in food matrices of different pH: natural yogurt (pH 4.65), rice drink (pH 6.01), cow milk (pH 6.47), and soy drink (pH 7.92). Compared to the direct use of the extract, the double emulsion solution gave rise to higher color stability with pH change and storage time, as corroborated by visual and statistical analysis.     

Structural and catalytic aspects of cutinase in w/o microemulsions

 Structural and catalytic properties of cutinase were studied in bis(2-ethylhexyl) sodium sulfo-succinate (AOT)-isooctane microemulsion systems. The effect of the water content of the microemulsions on the cutinase activity on an esterification reaction of lauric acid with pentanol showed that cutinase followed a bell-shaped profile presenting a maximum at w _o=9, with w _o=[H₂O]/[AOT]. Kinetic studies allowed the determi-nation of the apparent parameters K _m and V _max. Electron paramagnetic resonance (EPR) spectroscopy studies of active site labeled cutinase in microemulsions with varying w _o values showed that in all microemulsions, the mobility of the label is higher than in the aqueous solution. Furthermore, it was found that the maximum of the enzyme activity did not correspond to a reduced active site mobility. Up to w _o=9 there was an increase of both activity and active site mobility. As the water content of the system became higher, the mobility of the bound spin label further increased whereas the enzymatic activity dropped considerably. Received: 20 December 1996 Accepted: 24 February 1997     

Preyssler catalyst-promoted rapid,clean, and efficient condensation reactions for 3H-1,5-benzodiazepine synthesis in solvent-free conditions

A simple, convenient, and environmentally benign synthesis of 1,5-benzodiazepines is developed by condensing different o-phenylenediamines and 1,3-aryl-1,3-propanodiones. The reaction is catalyzed by a Preyssler (NaH₁₄P₅W₃₀O₁₁₀, HPA) heteropolyacid as a safe, clean, and recyclable catalyst. The method is operationally simple and provides access to a variety of 1,5-benzodiazepines in good to excellent yields.