Fibre optic fluorometric enzyme sensors for hydrogen peroxide and lactate,based on horseradish peroxidase and lactate oxidase

An optical biosensor for the determination of hydrogen peroxide based on immobilized horseradish peroxidase is described. The fluorescence of the dimeric product of the enzyme catalysed oxidation of homovanillic acid is utilized to determine the concentration of H₂O₂. The membrane-bound enzyme is attached to a bifurcated fibre bundle permitting excitation and detection of the fluorescence by a fluorometer. The response of the sensor is linear from 1 to 130 M hydrogen peroxide; the coefficient of variation is 3%. The sensor is stable for more than 10 weeks. The operating pH for maximal sensor response is 8.15. This allows the sensor to be used in combination with oxidase reactions producing hydrogen peroxide, as is demonstrated with a co-immobilized lactate oxidase-horseradish peroxidase optode for the determination of L-lactate. The fluorescence intensity of this sensor depends linearly on the concentration of lactate between 3 and 200 M and a throughput of 10 samples per hour is possible. The precision is in the same range as that of the monoenzyme optode. The lifetime of the bienzyme sensor for lactate is considerably shorter than that of the peroxidase sensor; it is limited by the stability of the immobilized lactate oxidase enzyme. The sensor has been applied to the determination of lactate in control serum.     

Catalytic properties of Phe41→His mutant of horseradish peroxidase expressed inE. coli

The recombinant horseradish peroxidase and its single-point F41H mutant have been reactivated fromE. coli inclusion bodies. The influence of the mutation on the heme entrapment, stability and activity of the enzyme was demonstrated. The catalytic rate constants for H₂O₂ cleavage and ammonium 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonate (ABTS) oxidation decrease by two and one orders of magnitude, respectively. Unlike the wild-type recombinant horseradish peroxidase, the elimination of the ABTS oxidation product is not a rate-determining step for the mutant. The F41H replacement results in significant changes of kinetics of iodide ion oxidation. The reaction rate is linear to the concentrations of iodide, H₂O₂, and the enzyme. The results suggest the direct interaction of iodide with the porphyrin ring of the heme. The decrease in ABTS oxidation activity accompanied by retention of activity in iodide oxidation in the course of low-dosage radiolysis of the F41H mutant is additional evidence of the direct electron transfer from iodide to the heme, in contrast to ABTS oxidation, in which the electron transfer chain in the protein molecule is involved.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 11, pp. 2034–2038, November, 1994.     

Voltammetric method for the determination of H2O2 in rainwater

Summary Presented below is an electrochemical method for the determination of hydrogen peroxide levels in the atmospheric liquid water. The hydrogen peroxide concentration is determined by a voltammetric method involving a rotating disk electrode. The oxidation limiting current at +0.4 V/SCE is proportional to the concentration of hydrogen peroxide found in 1 mol/l KNO₃ containing a phosphate buffer, pH 7.5. An analytical blank is prepared in situ by addition of catalase enzyme to avoid interferences. The detection limit obtained is 5×10^–9 mol/l.     

Determination of the exposed surface area of V2O5 on a V2O5/Al2O3 catalyst

NH₃, NO and CO₂ were tested as adsorbates for selective determination of exposed surface area of V₂O₅ on a V₂O₅/Al₂O₃ catalyst. The most promising appears to be CO₂ which interacts with the support Al₂O₃ only.

---

NH₃, NO CO₂ V₂O₅ V₂O₅/Al₂O₃. CO₂, Al₂O₃.

     

Horseradish peroxidase-based organic-phase enzyme electrode

An organic-phase enzyme electrode (OPEE) based on horseradish peroxidase (HRP) immobilized within Nafion on spectroscopic graphite was investigated in acetonitrile. The amperometric electrode response to hydrogen peroxide and cumene hydroperoxide present was found to be the result of the reduction of oxygen, produced upon enzymatic decomposition of both hydroperoxides (i.e., by the catalase-like activity of HRP). The electrode response was found to depend linearly on the hydroperoxide concentration up to 700 M within the range of potentials from –200 to –400 mV (versus Ag|AgCl). Detection limits of approximately 45 M for H₂O₂ and 100 M for cumene hydroperoxide were determined under the selected experimental conditions. Nernstian dependence (the open circuit voltage of HRP-based electrode versus logarithm of H₂O₂ concentration) was obtained between 0.2 and 2.0 mM, with a slope of approximately 23 mV per logarithmic unit, suggesting a catalase-like, two-electron disproportionation of the substrate in acetonitrile.     

MIITiO(C2O4)2·4H2O (MII=Mg,Ca, Sr OR Ba) as precursors in the formation of MIITiO3 powders

A synthesis route is described for the preparation of the alkaline earth metal titanyl oxalates M^IITiO(C₂O₄)₂·4H₂O (M^II=Mg, Ca, Sr or Ba). The thermal decompositions of these compounds were studied by means of DTA and TG in comparison with X-ray measurements. The final products M^IITiO₃ were characterized morphologically.

---

Zusammenfassung Ein Syntheseweg für die Darstellung der Erdalkalimetalltitanyloxalate M^IITiO(C₂O₄),·4H₂O (M ^II=Mg, Ca, Sr, Ba) wird beschrieben. Die thermische Zersetzung dieser Verbindungen wurde mittels DTA und TG sowie Röntgenbeugungsmethoden untersucht. Die entstehenden Titanate M^IITiO₃ wurden hinsichtlich ihrer morphologischen Eigenschaften charakterisiert.

---

- MⁱⁱTiO(C₂O₄)₂ · 4₂, ⁱⁱ = , , . , . ⁱⁱiO₃, .

     

Synthesis and X-Ray Crystal Structure Study of ]2-Methyl-4-(2-methylbenzoyl)-phenoxy] Acetic Acid Hydrazide

Benzophenone analog 3 has been synthesized and characterized by the X-ray diffraction (XRD) method. The compound crystallizes in a monoclinic space group P2₁/c with cell parameters a = 7.701(8) Å, b = 7.151(5) Å, c= 28.323(3) Å, = 104.639(4)°, Z = 4. The structure exhibits intra- and intermolecular hydrogen bonding of the type N–HO, C–HO, and N–HN. The molecules are interlinked through hydrogen bonds forming an infinite chain. This polymeric-like structure may play an important role in biological activity.     

An index of electrotopological state for atoms in molecules

A new method for molecular structure quantitation is described, in which both electronic and topological attributes are united. The method uses the hydrogen-suppressed skeleton to represent the structure and leads to a graph invariant index for the individual atoms and hydride groups of the molecular skeleton. An intrinsic atom value is calculated for each atom asI = ( + 1)/, in which and are the counts of valence and sigma electrons of atoms in the molecular skeleton, that is, exclusive of bonds to hydrogen atoms. The electrotopological state valueS _i for an atomi is defined asS _i =I _i + I _i, where the influence of atom j on atom i, I _i, is given as (I _i-j _j)/r ²;r is the graph separation between atoms i and j, counted as number of atoms, includingi andj. The information in the electrotopological state values is revealed by examples of various types of organic structures, including chain branching and heteroatom variation. The relation of the E-state value to NMR chemical shift is demonstrated for a series of carbonyl compounds.     

Heavy-atom tunneling in cyclobutadiene:Ab initio calculation of the intensities ofa g Raman lines

Summary GVB/[5s3p1d/3s1p] calculations were performed on the polarizability surface (₁, ₂, ₃) of cyclobutadiene. The threea _g coordinates refer to automerization (₁), symmetric CC stretch (₂), and symmetric CCH bend (₃). This surface was used together with the previously obtained variational vibrational wave functions for the calculation of Raman intensities. The calculation predicts comparable intensities for the two split components of the automerization Raman line in an isolated molecule and disagrees with observations on matrix-isolated cyclobutadiene. The disagreement is attributed to the asymmetry of the double-well potential imposed by the effect of the Ar matrix.Dedicated to Professor Ruedenberg     

Deutung der K α-satelliten bei leichten Elementen

Zusammenfassung Ausgehend von der KL-Ionisation wurden die K -Satelliten(, , ₃, ₃, ₄) der Elemente Z=8 bis Z=12 den entsprechenden Übergängen zugeordnet. Die Berechnung der Übergänge wurde nach der Z-Dependence-Theorie bis einschließlich zweiter Näherung durchgeführt.

---

Interpretation of the K -satellites of the light elements

Starting from the KL-ionisation the K -satellites(, , ₃, ₃, ₄) of the elements Z=8 to Z=12 have been assigned to the corresponding transitions. The transitions have been calculated by the Z-dependence-theory including the second approximation.

---

Résumé Les satellites K (, , ₃,₃, ₄) des éléments Z= 8 à Z=12 ont été attribués aux transitions correspondantes à partir de l'ionisation KL. Les transitions ont été calculées par la théorie dépendant de Z en tenant compte de l'approximation seconde.

---

---

Herrn Prof. Timm in dankbarer Würdigung der Hilfen, die die BASF für den Aufbau unserer theoretisch-chemischen Arbeitsgruppe geleistet hat, zum 60. Geburtstag gewidmet.

---

Der Deutschen Forschungsgemeinschaft dankt D. H. für die finanzielle Unterstützung, die die Dunchführung dieser Arbeit wesentlich erleichterte.     

Analytical applications of mixed-ligand complexes

Summary In medium of pH=1, vanadium(V) forms a 1 1 cationic complex with PAR, the instability constant of the complex beingpK=5.6. In the presence of hydrogen peroxide a 111 mixed-complex develops which is uncharged. The molar absorptivity of the complex ( ₅₄₀=1.28×10⁴ l·mole^–1·cm^–1) provides a possibility for the spectrophotometric determination of 1–65M hydrogen peroxide.

---

Zusammenfassung In saurer Lösung (pH=1) bildet Vanadin(V) mit PAR einen positiv geladenen Komplex, dessen Komplexkonstante dem pK-Wert 5,6 entspricht. In Anwesenheit von Wasserstoffperoxid bildet sich mit diesem Kation ein ungeladener Mischkomplex im Verhältnis 111. Dessen Extinktionskoef-fizient ( ₅₄₀=1,28×10⁴ l · mol^–1 · cm^–1) ermöglicht die spektrophotometrische Bestimmung von Wasserstoffperoxid im Konzentrationsbereich von 1–65Mol/l.

     

Reactions of the radical cations of aliphatic aldehydes in freon matrices

ESR spectra of -irradiated, at –196 °C, solutions of acetic and propionic aldehydes in freon-11 and freon-113 affected by aldehyde concentration, temperature, and the action of light have been studied. It has been shown that the radical cations CH₃CHO⁺ are converted into neutral radicals CH₃O and CH₃HOH and the cations CH₃CH₂CHO⁺. are converted to RO and CH₃HCHO due to ion-molecular reactions of proton transfer /CH₃O and CH₃HCHO/ of hydrogen atom transfer /CH₃HOH/.     

NMR studies of complex formation between H2PtCl4 and SnCl2 in organic solvents

Complex formation of H₂PtCl₄ with SnCl₂ in isopropanol and isopropanol-acetone solutions with the molar ratio [Sn]: [Pt]=1.0 has been studied by using¹⁹⁵Pt and¹¹⁹Sn NMR method.

---

¹⁹⁵Pt ¹¹⁹Sn H₂PtCl₄ co SnCl₂ - Sn:Pt=1,0.

     

Enhancement of H2- and retardation of O2-adsorption during O2?H2 titration cycles on Pt?Al2O3 catalyst surfaces

The title effects can be eliminated if the slow bleeding of H₂ from freshly reduced CuO-towers (often used as O₂-getters in purifying N₂ carrier gas) can be avoided, then H₂–O₂ titrations of Pt surfaces give consistent values from almost the first titration. Water injected at 25 °C also hardly affects the titer values.

---

H₂ O₂ , H₂ CuO ( O₂ N₂ — -). H₂–O₂ Pt . , 25° C, .

     

Kinetics of photoreduction of 9,10-phenanthrenequinone in the presence of amines and polymethylbenzenes

A study of the kinetics of photoreduction of 9,10-phenanthrenequinone in the presence of hydrogen donors (para-substituted N,N-dimethylanilines and polymethylbenzenes) showed that plots of the quantum yield of photoreduction (_H) and apparent reaction rate constant (k _H) vs. oxidation potential of hydrogen donors are extreme. In the presence of amines, k _H and _H increase, as a whole, whereas they decrease in the presence of polymethylbenzenes. In coordinates _H-G _e (G _e is the change in the free energy of electron transfer) for pairs quinone-H donor, _H increases with G _e approaching to zero. For the amine series, this effect is mainly in the exothermic region of G _e (G _e < 0). For the series of polymethylbenzenes, this increase is observed in the endothermic region (G _e > 0).__________Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 11, pp. 2381–2385, November, 2004.     

Some sources of defects in α-HgI2, the room temperature γ-ray detector

In the course of a systematic optimization of the materials properties of -HgI₂ crystals for room temperature - and X-ray detectors, we have investigated possible sources of defects and discuss briefly the possibility to supress them. Due to the particular structure of -HgI₂, large amount of impurities, particularly hydrocarbons, can be absorbed invan der Waals layers and lattice channels. Purification by sublimation does not work due to the affinity of hydrocarbons to iodine and their easy re-absorption in the sublimate. Lattice filtering of the large concentration of hydrocarbons contained even in suprapure iodine has been performed using the close spaced lattice of CuI. Oxidation of hydrocarbons by reaction of oxygen with HgI₂ is another possibility for their removal.Mass spectrometric investigations of a molecular beam of -HgI₂ has solved the long disputed problem of the existence of nonstoichiometry: both excess of Hg or excess of I are possible. The removal of nonstoichiometry can be achieved by suitable thermal treatment only in the case of pure crystals. In the presence of hydrocarbons, the non-stoichiometric defects are fixed showing the predominant importance of hydrocarbons for -HgI₂.Investigation of the evaporation of -HgI₂ with mass spectrometry at low temperatures (150>T>40 °C) shows a strong change of the enthalpy of evalporation at 67 °C whereas DSC does not show any peak at this temperature. It seems probable that this is due to a surface reconstruction which influences the evaporation but not the thermal bulk lattice effects which are detected by DSC.Dedicated to Prof. Dr.K. L. Komarek at the occasion his 60th birthday.     

The solid-state structure of (+)-(i)-menthyl 2-formamido-4, 4,4-trifluorobutanoate and the hydrogenation of the 2-butenoate precursor

Hydrogenation ofZ-(–)-(1R, 3R, 4S)-menthyl 2-formamido-4,4,4-trifluoro-2-butenoate catalyzed by Pd/C was performed at atmospheric pressure to yield a mixture of (2R, 1R, 3R, 4S)- and (2S, 1R, 3R, 4S)-menthyl 2-formamido-4,4,4-trifluorobutanoate diastereomers in a 5545 ratio, respectively. Repeated fractional crystallization from ethyl acetate and vapor diffusion of petroleum ether afforded (+)–(2S, 1R, 3R, 4S)-menthyl 2-formamido-4,4,4-trifluorobutanoate as clear colorless, crystalline prisms which were subjected to single-crystal X-ray diffraction analysis. The crystals belong to the orthorhombic system P2₁2₁2₁, and at 213 K:a=5.054(1),b= 10.000(2),c=32.707(1) Å,V=1652.9(4) ų,Z=4,R(F)=0.040, andR _w (F)=0.037. The finding of the (2S)-configuration for the formamido-acid portion of the (+)-ester enabled the configurational assignment of the asymmetric hydrogenation products ofZ-methyl 2-formamido-4, 4,4-trifluoro-2-butenoate catalyzed by chiral diphosphine/rhodium(I) complexes.     

Electronic spectroscopy of the state of cu2+ ions in CuO?TiO2 catalysts

Diffuse reflectance spectroscopy of supported CuO–TiO₂ catalysts have revealed that, depending on the CuO content and the calcination temperature, either Cu²⁺ ions incorporated into the TiO₂ lattice, or surface oxide clusters, primary oxide aggregates and CuO_1+X phase bonded to the support by Ti–O–Cu bridges, are formed.

---

, CuO–TiO₂ Cu²⁺, TiO₂, , CuO_1+x Ti–O–Cu .

     

Modification of the catalytic activity of Ni/SiO2 in ethane hydrogenolysis, benzene and co hydrogenation by reduction at high temperatures and restoration by O2?H2 treatments

An increase of the temperature of reduction of Ni/SiO₂ from 900 to 1120 K results in a decrease of catalytic activity per unit area in benzene hydrogenation and ethane hydrogenolysis. O₂–H₂ treatments restore the activities. This behavior is similar to that observed on Pt-based catalysts. In contrast, the CO+H₂ reaction on nickel is not affected by such treatments.

---

Ni/SiO₂ 900 1120 . O₂–H₂ . , , CO+H₂ .

     

Gibbs Energy and Entropy of Mixing in the NaNO2-KNO3 System

The activity and activity coefficients of the components of the NaNO₂-KNO₃ system, determined from the experimental data on the saturated vapor pressure at 798, 823, and 848 K, are used to calculate the relative and excess partial molar Gibbs energies (G _i and G _i ^{e x c}), entropies (S _i and S _i ^{e x c}), and integral relative and excess thermodynamic functions (G, G ^{e x c}, S, and S ^{e x c}) of the system.