Native and modified glucose oxidase in reversed micelles

The enzymatic activity of the native and modified glucose oxidase (GOx) from Aspergillus niger in the system of reversed micelles of Aerosol OT in octane was investigated. Two forms of the modified enzyme were studied: a hydrophobized form obtained by the attachment of palmitic chains to lysine amino groups by the reaction with palmitic acid ester of N-hydroxysuccinimide and a glycosylated (hydrophilized) form obtained by the attachment of the cellobiose moieties. The native glucose oxidase and its derivatives, while incorporated into micelles in a surfactant concentration range from 0.05 to 0.3 M, display an enzymatic activity, which is comparable with the activity in aqueous solution. The dependence of the enzymatic activity on hydration degree of surfactant (the molar ratio of water to surfactant, W₀) does not indicate the formation of qualitatively new associated forms of the enzyme subunits inside the micelles. The apparent size of Aerosol OT micelles obtained by dynamic light scattering gradually increases from 10±3 nm at low W₀ up to 25±5 nm at high W₀. Incorporation of the native and hydrophobized glucose oxidase into micelles does not affect their mean size. Kinetic analysis shows that the enzyme specificity is about an order of magnitude greater in the system of reversed micelles as compared with aqueous solution.     

The formula of anchoring energy for a nematic liquid crystal

The interface energy for a nematic liquid crystal (NLC) is considered as the sum of potential energy between LC molecules and molecules of the substrate surface, and a formula for anchoring energy is derived by elementary principles. The anchoring energy for a NLC should have two terms, the first term is the same as the Rapini-Papoular expression, the second is related to the normal of interface and resultes from the biaxial property of a NLC induced by interface. Hence there are two anchoring coefficients, W₁ and W₂. We demonstrate that W₁ is equal to the tilt angle strength A_θ, and W₂ corresponds to the difference between A_θ and the azimuthal strength A_ϕ. Thus A_θ-A_ϕ is due to the biaxial property of the NLC near the interface. Applying this formula to the twisted NLC cell, we discuss the threshold and saturation field, as well as the maximal tilt angel θ_m with respect to A_θ/A_ϕ. Previously proposed formulae are discussed from our point view.     

Interaction of iron(III) tetrasulphonated phthalocyanine with cytochrome c oxidase apoprotein

A complex of bovine cytochrome c oxidase protein with iron(III) tetrasulphonated phthalocyanine in place of hemes has been prepared. Its structure and properties have been investigated by difference spectroscopy, electrophoresis, molecular weight estimation, potentiometric measurements and polypeptide fragments examination. The visible absorption spectrum of Fe^III-apo-oxidase shows the main intense peak at 657 nm and weaker one at 700 nm. Molecular weight estimation demonstrated that one mole of the complex includes three functional units of MW 130,000 per unit. Spectroscopic examination of dithionite reduced Fe^III-apo-oxidase suggests the open crevice structure of the subunits containing iron tetrasulphonated phthalocyanine which is supported by the results of circular dichroism studies. Deep conformational changes of the protein upon displacement of hemes a and a₃ are not reversed upon Fe^IIIL⁺ incorporation into the protein. The molar ratio of the protein to Fe^IIIL in the complex (MW 130,000) was found to be 1:2. In the reduced form Fe^IIIL-apo-oxidase reacts with CN⁻, N^-₃ imidazole and molecular oxygen. Oxygen binding is irreversible, which indicates that the oxygen adduct is not of the oxyhemoglobin type. Electrophoretic and gel filtration studies of the SDS-urea dissociation products of cytochrome c oxidase and its phthalocyanine derivative suggest that Fe^IIIL and hemes a and a₃ are located on the same polypeptide fragments of the protein. Fe^IIIL-apo-oxidase is reduced by ferrous cytochrome c in agreement with their midpoint potentials which are 315.5 and 260 mV, respectively. However, the rate of the reaction of Fe^IIIL-apo-oxidase with ferrous cytochrome c is markedly lower than that of the native cytochrome c oxidase suggesting different mechanisms for this process in both cases.     

核糖核酸酶A在丁酸十二铵-环己烷反胶束溶液中的催化动力学

研究了核糖核酸酶A(RNaseA)在丁酸十二铵(DAB)-环己烷反胶束溶液中催化水解胞苷2',3'-环单磷酸酯的动力学,数据符合Michaelis-Menten酶催化机理.以k_cat/K_m表示酶催化活性时,Rnase A在反胶束溶液中的催化活性是在水溶液中的14～30倍.无论是固定DAB浓度还是固定H₂O与DAB浓度之比,随增溶水量的增加,k_cat/K_m呈下降趋势.     

反胶团中漆树酶催化氧化性能与反应产物鉴定

研究了漆树酶在AOT/正辛烷/水反胶团中邻氨基苯酚的催化氧化性能。发现在40℃、pH=7.6、R=[H₂O]/[AOT]≈18的条件下,漆树酶的活力最高,在含水量较低的反胶团中较稳定。当R=6.2,于30℃保存15h活力仍保持82%;"水池"内存在的1.0×10^-3mol/L的Zn²⁺、Fe³⁺、Mg²⁺等离子对漆树酶有一定的抑制作用。漆树酶对邻氨基苯酚在反胶团和水溶液介质中,其主要氧化产物均为2-氨基-吩嗪-3-酮,但在反胶团中其反应产率比在水溶液中约高0.5倍。     

Syntheses and structural determination of nine-coordinate K3[Nd(nta)2(H2O)]·6H2O and K3[Er(nta)2(H2O)]·5H2O

The syntheses and structural determination of Nd^III and Er^III complexes with nitrilotriacetic acid (nta) were reported in this paper. Their crystal and molecular structures and compositions were determined by single-crystal X-ray structure analyses and elemental analyses, respectively. The crystal of K₃[Nd^III(nta)₂(H₂O)]·6H₂O complex belongs to monoclinic crystal system and C2/c space group. The crystal data are as follows: a=1.5490(11) nm, b=1.3028(9) nm, c=2.6237(18) nm, β=96.803(10)°, V=5.257(6) nm³, Z=8, M=763.89, D_c=1.930 g cm⁻³, μ=2.535 mm⁻¹ and F(000)=3048. The final R₁ and wR₁ are 0.0390 and 0.0703 for 4501 (I>2σ(I)) unique reflections, R₂ and wR₂ are 0.0758 and 0.0783 for all 10474 reflections, respectively. The Nd^IIIN₂O₇ part in the [Nd^III(nta)₂(H₂O)]³⁻ complex anion has a pseudo-monocapped square antiprismatic nine-coordinate structure in which the eight coordinate atoms (two N and six O) are from the two nta ligands and a water molecule coordinate to the central Nd^III ion directly. The crystal of the K₃[Er^III(nta)₂(H₂O)]·5H₂O complex also belongs to monoclinic crystal system and C2/c space group. The crystal data are as follows: a=1.5343(5) nm, b=1.2880(4) nm, c=2.6154(8) nm, b=96.033(5)°, V=5.140(3) nm³, Z=8, M=768.89, D_c=1.987 g cm⁻³, μ=3.833 mm⁻¹ and F(000)=3032. The final R₁ and wR₁ are 0.0321 and 0.0671 for 4445 (I>2σ(I)) unique reflections, R₂ and wR₂ are 0.0432 and 0.0699 for all 10207 reflections, respectively. The Er^IIIN₂O₇ part in the [Er^III(nta)₂(H₂O)]³⁻ complex anion has the same structure as Nd^IIIN₂O₇ part in which the eight coordinate atoms (two N and six O) are from the two nta ligands and a water molecule coordinate to the central Nd^III ion directly.     

Micellar solubilization of biopolymers in hydrocarbon solvents. I. a structural model for protein-containing reverse micelles

Three proteins (horse liver alcohol dehydrogenase, ribonuclease, lysozyme) were solubilized in hydrocarbon with the help of reverse micelles formed by aqueous di(2-ethyl-hexyl) sodium sulfosuccinate (AOT). Sedimentation and diffusion coefficients of the micellar aggregates were measured with an analytical ultracentrifuge. Partial specific volumes were also evaluated from density measurements. The molecular weight of the protein-containing reverse micelles (M _t ) could thus be determined for each protein system at various w₀ values (w₀ - [H₂O]/[AOT]). For horse liver alcohol dehydrogenase at w₀ = 46.4, for example,M _t is ca. 2,670,000 Daltons; for lysozyme at wo = 22.5,M _t is ca. 323,000 Daltons and increases by increasing w₀. On the basis of these experimentally determined molecular weights, a structural model for the protein-containing reverse micelle is proposed. The model is based upon the assumption that the protein is confined in the water pool of a spherical micelle, and that the inner core volume is the sum of the protein volume and the volume of all water molecules present in a micelle. It is possible then to calculate the micellar structure at each w₀ value. For example, in the case of ribonuclease at w₀ = 20, the inner core radius is ca. 37.5 A, and a layer of water of ca. 22 A separates the protein surface from the surfactant layer. The possible implications of this model for the reactivity of enzymes solubilized in hydrocarbons by reverse micelles are discussed.     

Reverse micellar aggregates: effect on ketone reduction. 2. Surfactant role

Kinetics of the reduction of 3-chloroacetophenone (CAF) with sodium borohydride (NaBH(4)) were followed by UV-vis spectroscopy at 27.0 degrees C in different reverse micellar media, toluene/BHDC/water and toluene/AOT/water, and compared with results in an isooctane/AOT/water reverse micellar system. AOT is sodium 1,4-bis-2-ethylhexylsulfosuccinate, and BHDC is benzyl-n-hexadecyl dimethylammonium chloride. The kinetic profiles were investigated as a function of variables such as surfactant and NaBH(4) concentration and the amount of water dispersed in the reverse micelles, W(0) = [H(2)O]/[surfactant]. In all cases, the first-order rate constant, k(obs), increases with the concentration of surfactant as a consequence of incorporating the substrate into the interface of the reverse micelles where the reaction takes place. The reaction is faster at the cationic interface than at the anionic one probably because the negative ion BH(4)(-) is part of the cationic interface. The effect of the external solvent on the reaction shows that reduction is favored in the isooctane/AOT/water reverse micellar system than that with an aromatic solvent. This is probably due to BH(4)(-) being more in the water pool of the toluene/AOT/water reverse micellar system. The kinetic profile upon water addition depends largely on the type of interface. In the BHDC system, k(obs) increases with W(0) in the whole range studied while in AOT the kinetic profile has a maximum at W(0) approximately 5, probably reflecting the fact that BH(4)(-) is part of the cationic interface while, in the anionic one, there is a strong interaction between water and the polar headgroup of AOT below W(0) = 5 and, above that, BH(4)(-) is repelled from the interface once the water pool has formed. Application of a kinetic model based on the pseudophase formalism, which considers the distribution of the ketone between the continuous medium and the interface and assumes that reaction takes place only at the interface, has enabled us to estimate rate constants at the interface of the reverse micellar systems. At W(0) < 10, it was considered that NaBH(4) is wholly at the interface and, at W(0) >/= 10, where there are free water molecules, also the partitioning between the interface and the water pool was taken into account. The results were used to evaluate CAF and NaBH(4) distribution constants between the different pseudophases as well as the second-order reaction rate constant of the reduction reaction in the micellar interface.     

Electrochemical extraction of proteins by reverse micelle formation

The transfer of proteins by the anionic surfactant bis(2-ethylhexyl) sulfosuccinate (AOT) at a polarized 1,2-dichloroethane/water (DCE/W) interface was investigated by means of ion-transfer voltammetry. When the tetrapentylammonium salt of AOT was added to the DCE phase, the facilitated transfer of certain proteins, including cytochrome c (Cyt c), ribonuclease A, and protamine, could be controlled electrochemically, and a well-defined anodic wave for the transfer was obtained. At low pH values (e.g., pH 3.4), the anodic wave was usually well-separated from the wave for the formation of protein-free (i.e., unfilled) reverse micelles. The anodic wave for the protein transfer was analyzed by applying the theory for facilitated transfer of ions by charged ligands and then supplying information regarding the number of AOT anions reacting with one protein molecule and the total charge carried by the protein transfer. However, controlled-potential electrolyses performed for the transfer of Cyt c, which is red, revealed that the protein-AOT complexes were unstable in DCE and liable to aggregate at the interface when the pH of the W phase was 3.4. At pH 7.0, when formation of unfilled reverse micelles occurred simultaneously, the protein-AOT complexes appeared to be stabilized, probably via fusion with unfilled reverse micelles.     

Structural and theoretical studies of Xe(OChF5)2 and [XeOChF5][AsF6] (Ch = Se, Te)

The XeOSeF₅⁺ cation has been synthesized for the first time and characterized in solution by ¹⁹F, ⁷⁷Se and ¹²⁹Xe NMR spectroscopy and in the solid state by X-ray crystallography and Raman spectroscopy with AsF₆⁻ as its counter anion. The X-ray crystal structures of the tellurium analogue and of the Xe(OChF₅)₂ derivatives have also been determined: [XeOChF₅][AsF₆] crystallize in tetragonal systems, P4/n, a=6.1356(1) Å, c=13.8232(2) Å, V=520.383(14) Å³, Z=2 and R₁=0.0453 at −60°C (Te) and a=6.1195(7) Å, c=13.0315(2) Å, V=488.01(8) Å³, Z=2 and R₁=0.0730 at −113°C (Se); Xe(OTeF₅)₂ crystallizes in a monoclinic system, P2₁/c, a=10.289(2) Å, b=9.605(2) Å, c=10.478(2) Å, β=106.599(4)°, V=992.3(3) Å³, Z=4 and R₁=0.0680 at −127°C; Xe(OSeF₅)₂ crystallizes in a triclinic system, , a=8.3859(6) Å, c=12.0355(13) Å, V=732.98(11) Å³, Z=3 and R₁=0.0504 at −45°C. The energy minimized geometries and vibrational frequencies of the XeOChF₅⁺ cations and Xe(OChF₅)₂ were calculated using density functional theory, allowing for definitive assignments of their experimental vibrational spectra.     

Enhanced stabilization of reverse micelles by compressed CO2

The effect of compressed CO2 on the solubilization capacity of water in reverse micelles of sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in longer chain n-alkanes was studied at different temperatures and pressures. It was found that the amount of solubilized water is increased considerably by CO2 in a suitable pressure range. The suitable CO2 pressure range in which the solubilization capacity of water could be enhanced decreased with increasing W0 (water-to-AOT molar ratio). The microenvironments in the CO2-stabilized reverse micelles were investigated by UV/Vis adsorption spectroscopy with methyl orange (MO) as probe. The mechanism by which the reverse micelles are stabilized by CO2 is discussed in detail. The main reason is likely to be that CO2 has a much smaller molecular volume than the n-alkane solvents studied in this work. Therefore, it can penetrate the interfacial film of the reverse micelles and stabilize them by increasing the rigidity of the micellar interface and thus reducing the attractive interaction between the droplets. However, if the CO2 pressure is too high, the solvent strength of the solvents is reduced markedly, and this induces phase separation in the micellar solution.     

Disruption of reverse micelles and release of trapped ribonuclease A photochemically induced by Malachite Green leuconitrile derivative

Photoinduced disruption of a sodium bis(2-ethylhexyl) sulfosuccinate (AOT) reverse micelle is triggered by a Malachite Green leuconitrile derivative (MGL). UV irradiation of MGL solubilized in an AOT-water-chloroform mixture creates a cationic surfactant that interacts electrostatically with the anionic AOT. We investigated the disruption of the reverse micelle by using proton nuclear magnetic resonance spectroscopy and found that UV irradiation of MGL decreases the number of water molecules solubilized in the interior of the AOT reverse micelles. Furthermore, the photoinduced disruption of the reverse micelle is shown to release ribonuclease A, which is trapped in the water in the interior of the AOT reverse micelle. This photoinduced release may offer a desirable transport system of biopolymers.     

Comparative Study on the Structure of Water in Reverse Micelles Stabilized with Sodium Bis(2-ethylhexyl) Sulfosuccinate or Sodium Bis(2-ethylhexyl) Phosphate in n-Heptane

The microstructure of water solubilized in H(2)O/surfactant/n-heptane ternary systems has been investigated by employing (1)H-NMR and FT-IR spectroscopic techniques. Two reverse micellar systems were prepared and studied, i.e., sodium bis(2-ethylhexyl) sulfosuccinate in n-heptane (H(2)O/AOT/n-heptane) and sodium bis(2-ethylhexyl) phosphate in n-heptane (H(2)O/NaDEHP/n-heptane). (1)H-NMR data showed that the chemical shift of water protons for the AOT and NaDEHP reverse micelles varied downfield and upfield, respectively, with an increase of the water content. The opposite shift directions with increasing water content are interpreted as due to a composition change of the solubilized water associated with head-groups and sodium counterions in reverse micellar systems. On the basis of deconvolution results of FT-IR spectra, a four-component model is proposed to interpret the FT-IR and (1)H-NMR results. Copyright 2000 Academic Press.     

Reaction between cerium trifluoride and elemental fluorine

Reactions between cerium trifluoride and elemental fluorine have been examined by means of the kinetic and crystallographic method. The reactions were performed at 503–573 K under fluorine pressures of 6.7–26.7 kPa for reaction time up to 3 h. Two kinds of CeF₄ were synthesized by controlling the reaction condition. One is the metastable CeF₄-I synthesized at 523–538 K which returns to CeF₃ to release elemental fluorine, and the other is the stable CeF₄-II obtained at temperatures higher than ca. 540 K, which never returns to CeF₃. X-ray diffraction–Rietveld analysis revealed that the structure of CeF₄-I and CeF₄-II were monoclinic structure (C12/c1, Z=12, CeF₄-I/CeF₄-II: a₀, 1.26 nm/1.25; b₀, 1.06/1.05; c₀, 0.82/0.82; β, 126.1/126.3°; cell volume, 8.91×10⁻²⁸/8.78×10⁻²⁸ m³) and the cell volume of the CeF₄-I was 1.4% larger than that of the CeF₄-II. Bond lengths between Ce and F in CeF₄-I were somewhat longer than those in CeF₄-II. When CeF₄ and NaCl were put separately in the reaction container, CeF₄-I changed to CeF₃ with converting NaCl to NaF, however, CeF₄-II never reacted with NaCl.     

Do probe molecules influence water in confinement?

The water inside reverse micelles can differ dramatically from bulk water. Some changes in properties can be attributed to the interaction of water molecules with the micellar interface, forming a layer of shell water inside the reverse micelle. The work reported here monitors changes in intramicellar water through chemical shifts and signal line widths in 51V NMR spectra of a large polyoxometalate probe, decavanadate, and from infrared spectroscopy of isotopically labeled water, to obtain information on the water in the water pool in AOT reverse micelles formed in isooctane. The studies reveal several things about the reverse micellar water pool. First, in agreement with our previous measurements, the proton equilibrium of the decavanadate solubilized within the reverse micelles differs from that in bulk aqueous solution, indicating a more basic environment compared to the starting stock solutions from which the reverse micelles were formed. Below a certain size, reverse micelles do not form when the polyoxometalate is present; this indicates that the polyanionic probe requires a layer of water to solvate it in addition to the water that solvates the surfactant headgroups. Finally, the polyoxometalate probe appears to perturb the water hydrogen-bonding network in a fashion similar to that in the interior surface of the reverse micelles. These measurements demonstrate the dramatic differences possible for water environments in confined spaces.     

盐酸胍对反胶束中RNase A活性的影响

以胞嘧啶核苷酸2',3'-环单磷酸酯为底物研究了变性剂盐酸胍对十二胺丁酸盐-环己烷反胶束溶液中核糖核酸酶A活性的影响,同水溶液相比,盐酸胍对反胶束中酶活性的抑制作用很小。反胶束的大小限制了酶分子天然态构象的改变,从而保证了其活性中心的完整性。核糖核酸酶A的内源荧光研究发现,同水溶液相比,反胶束中蛋白的最大发射波长没有发生变化,但荧光偏振极化度增加,也表明了在反胶束中酶分子的运动自由度较在水溶液中有所降低。     

Hydrothermal synthesis and structure of one-dimensional Co(dien)2(VO3)3·(H2O) (dien=diethylenetriamine)

One-dimensional Co(dien)₂(VO₃)₃·(H₂O) was prepared from the hydrothermal reaction of NH₄VO₃, Co₂O₃, diethylenetriamine (dien) and H₂O at 130 °C. The compound crystallizes in the monoclinic system, space group P2₁/c with a=16.1581(6) Å, b=8.7006(3) Å, c=13.9893(4) Å, β=103.1483(11)°, V=1915.13(11) Å³, Z=4, and R₁=0.0268 for 3060 observed reflections. Single crystal X-ray diffraction revealed that the structure is composed of infinite one-dimensional chains formed by corner-sharing VO₄ tetrahedra with Co(dien)³⁺ complex cations and crystallization water molecules occupying the interchain positions, which are held together to a three-dimensional network via extensive hydrogen-bonding interactions. The compound, with a new zig-zag conformation of metavanadate chains, is the first example of vanadium oxides incorporating trivalent transition metal coordination groups. Other characterizations by elemental analysis, IR and thermal analysis are also described.     

Structural characterization of cyclopentadienyl(duroquinone)cobalt dihydrate

The structure of cyclopentadienyl(duroquinone)cobalt dihydrate, (C₅H₅)Co-[(CH₃)₄C₆O₂]·2H₂O, has been determined by three-dimensional X-ray analysis. The crystal structure consists of discrete cyclopentadienyl(duroquinone)cobalt molecules linked together by a complex network of hydrogen bonds between water molecules and duroquinone oxygen atoms. Each (C₅H₅)Co[(CH₃)₄C₆O₂] molecule consists of a cobalt atom sandwiched between a cyclopentadienyl ring and a duroquinone ring. A detailed comparison of the molecular parameters of this complex with those of closely related complexes is given. Crystallographic evidence that the metal---duroquinone interaction in cyclopentadienyl(duroquinone)cobalt dihydrate is considerably stronger than that in the electronically-equivalent 1,5-cyclooctadiene(duroquinone)nickel complex is given not only by the metal---C(olefin) distances being 0.12 Å (av) shorter in the duroquinone---cobalt complex [viz., 2.104(8) Å vs. 2.222(7) Å] but also by the much greater C_2v-type distortion of the duroquinone ring from the planar D_2h configuration in free duroquinone. The compound crystallizes with two formula species in a triclinic unit cell of symmetry P and reduced cell dimensions á = 8.60 Å, b = 9.00 Å, c = 10.15 Å, = 87° 34′, β = 84° 10′, γ = 73° 44′. Least-squares refinement yielded final unweighted and weighted discrepancy factors of R₁ = 10.8% and R₂ = 12.0%, respectively, for 2481 independent diffraction maxima collected photographically.     

Specifics of the formation of J-aggregates of cyanine dyes in solutions of AOT/water/hexane reverse micelles

The behavior of a cyanine dye (3,3′-di-(gamma-sulfopropyl)-4,5,4′,5′-dibenzo-9-ethylthiacarbocyanine betaine pyridinium salt) was studied in AOT/water/hexane reverse micelles over a wide range of W at various concentrations of the dye, AOT, and reverse micelles. The processes occurring during the formation of the AOT/water/hexane micellar solution were studied in detail. It has been shown that, before the formation of the stable microemulsion, the dye aggregation processes occur by virtue of the interaction of the dye with the AOT anion. The amount of J-aggregates is proportional to the logarithm of the ratio of the amount of AOT molecules to the amount of dye molecules. The time behavior of J-aggregates after the formation of a micellar structure depends on the concentration of reverse micelles, thereby indicating an important role of intermicellar exchange.     

Phase behaviors of AOT/longer chain n-alkanes reverse micelles in the presence of compressed ethylene

It was found that, in a suitable pressure range, ethylene could increase the amount of solubilized water in reverse micelles of sodium bis-2-ethylhexylsulfosuccinate (AOT) in longer chain n-alkanes considerably, where the phase separation was induced by a micelle-micelle interaction mechanism. The microenvironments in the ethylene-stabilized reverse micelles were investigated by the UV-vis adsorption spectra using methyl orange (MO) as a probe. The maximum absorption of MO decreased with the increase of ethylene pressure at constant W0 value. Conductivity measurements demonstrated that the percolation temperature of the reverse micellar system increased considerably after compressed ethylene was added. The results of this work confirm that some small-molecule gases have the function of cosurfactants to stabilize reverse micelles.