Bienzyme Sensor Based on an Oxygen Reducing Bilirubin Oxidase Electrode

Based on an oxygen reducing electrode combining bilirubin oxidase and multiwalled carbon nanotubes modified gold (BOD‐MWCNT‐Au electrode) a bienzyme sensor is developed. Therefore the BOD‐MWCNT‐Au electrode is covalently coupled to enzymes catalysing oxygen‐consuming reactions (glucose oxidase and ascorbate oxidase) to result in a membrane‐free bienzyme electrode. The electrochemical characterisation of these bienzyme sensors reveals an enzyme substrate sensitivity down to 250 μM glucose and 100 μM ascorbate. In addition, the assembled sensor systems allow amperometric measurements in a potential range where the influence of interfering substances reacting directly at the transducing electrode is minimised. The results indicate that the BOD electrode provides a suitable platform for sensing analytes of medical and environmental interest for which oxidases of high activity are available.     

Anterior cingulate and cerebellar GABA and Glu correlations measured by ¹H J-difference spectroscopy

Gamma-aminobutyric acid (GABA) and glutamate (Glu) levels, normalized to total creatine (tCr), were measured in the anterior cingulate and cerebellar vermis in healthy adults (n=19, age=24.6±6.4 years) using ¹H MRS at 3 T, and metabolite correlations across regions and subjects were determined. Mean anterior cingulate and cerebellar GABA/tCr ratios were 0.31 (0.08) and 0.23 (0.06), respectively, while corresponding Glu levels were 1.16 (0.10) and 0.70 (0.07), respectively. Anterior cingulate and cerebellar glutamate levels were correlated (r=0.6103, P=.0140), although it is noted that when adjusted for multiple comparisons, all correlations reported here cluster to a P value of .0583. It is unlikely that this correlation is driven by correlations in tCr, since interregional correlations were not observed for other metabolites referenced to tCr. Correlations were also observed among metabolites in both the anterior cingulate and cerebellar vermis. In the former, N-acetylasparate was linearly dependent on glutamate (r=0.6577, P=.0063) and, at or below this significance threshold, four metabolites were correlated in the cerebellar vermis (Ins/tCh: r=0.6261, P=.0109. NAA/tCh: r=0.6426, P=.0082. NAA/Glu: r=0.6412, P=.0085. tCh/Glu: r=0.6193, P=0.0122).     

Microcalorimetric Determination of Serum Triglycerides and Cholesterol

Abstract

Reaction heats measured in a microcalorimeter between varying amounts of human serum and 700 IU of lipase (EC 3.1.1.3) and those between serum and 8 U of cholesterol oxidase (EC 1.1.3.6) were significantly linear with the contents of triglycerides (r=1.00) and cholesterol (r=0.99), respectively. However, when sera from 13 human volunteers were subject to comparative study between the micro-calorimetric and spectrophotometric methods, the correlation became less appreciable (r=0.70 and r=0.71) presumably due to the individual variations in free glycerol and cholesterol ester-constituents that were deliberately omitted in the microcalorimetric method but were routinely included in the standard (multi-enzymatic) spectro-photometric clinical method. The contribution of interfering substances, if any, is not totally ruled out.     

Measurement of the Binding of Adenosylcobalamin to Glutamate Mutase by Fluorescence Spectroscopy

Fluorescence spectroscopy is a fast, highly sensitive technique for investigating protein‐ligand interactions. Intrinsic protein fluorescence is usually occurred by exciting the proteins with 280‐295 nm ultraviolet light, and the light emission is observed approximately between 330‐350 nm. No emission light between 330‐350 nm can be observed when adenosylcobalamin (AdoCbl) is excited at 282 nm. The binding of AdoCbl to glutamate mutase was therefore investigated by fluorescence spectroscopy in this study. Our results show that direct measurement for determining the K_d of AdoCbl by fluorescence spectroscopy leads to significant errors. Here we report the source of error and a corrected method for measuring the binding of coenzyme B₁₂ to glutamate mutase using fluorescence spectroscopy.     

Enzymatic reverse FIA method for total phenols determination in urine samples

A reverse flow injection spectrophotometric enzymatic method is proposed to quantify total phenols in urine samples. The polyphenol oxidase (PPO; EC 1.14.18.1) obtained as a crude extract from sweet potato root (Ipomoea batatas) was used as enzymatic catalyze. The detection limit, the sample throughput and relative standard deviation were 7.7 mg l⁻¹ of total phenols, 49 h⁻¹ and 0.9%, respectively. The method was applied to real samples and a recovery study was carried out in order to its validation.     

Synthesis of the active sites of molybdoenzymes: MoO2(VI) and MoO(IV)-dithiolene complexes mimicking enzymatic reactions of sulphite oxidase with saturation kinetics

[Mo^VIO₂(S₂C₂(CN)₂)₂]₂₋ (┘1) and [Mo^IVO(S₂C₂(CN)₂)₂]²⁻ (2) mimick oxidoreductase enzymatic activities of sulphite oxidase with biological electron donor, SO ₃ ²⁻ , andin vitro electron acceptor, [Fe(CN)₆]³⁻, demonstrating proton coupled electron transfer reaction in water and inhibition of the oxidation of (2) in the presence of KCN. The sulphite exidizing system is characterized by substrate saturation kinetics indicating the biological significance of the reactions     

Quinone‐Catalyzed Selective Oxidation of Organic Molecules

Quinones are common stoichiometric reagents in organic chemistry. Para‐quinones with high reduction potentials, such as DDQ and chloranil, are widely used and typically promote hydride abstraction. In recent years, many catalytic applications of these methods have been achieved by using transition metals, electrochemistry, or O₂ to regenerate the oxidized quinone in situ. Complementary studies have led to the development of a different class of quinones that resemble the ortho‐quinone cofactors in copper amine oxidases and mediate the efficient and selective aerobic and/or electrochemical dehydrogenation of amines. The latter reactions typically proceed by electrophilic transamination and/or addition‐elimination reaction mechanisms, rather than hydride abstraction pathways. The collective observations show that the quinone structure has a significant influence on the reaction mechanism and has important implications for the development of new quinone reagents and quinone‐catalyzed transformations.     

Galactose Oxidase/Prussian Blue Based Biosensors

This paper describes the development of an amperometric biosensor based on galactose oxidase (GAOx) immobilization within a laponite clay film deposited on Carbon Screen‐Printed Electrodes modified by electrodeposited Prussian Blue and coated with poly‐(O‐phenylenediamine) (PPD/PB/CSPEs). Amperometric performances of GAOx@laponite/PPD/PB/CSPEs bioelectrodes were determined using several GAOx substrates. Using these modified electrodes the reduction of enzymatically generated hydrogen peroxide was performed at ?0.2 V vs. Ag‐AgCl. In an initial attempt, E.Coli transketolase activity on its immobilized form was followed using a bienzymatic GAOx‐TK biosensor.     

Practical method for the rapid screening of xanthine oxidase inhibitors in herbal extracts by high‐performance liquid chromatography based on on‐line precolumn enzymatic reaction

A high‐performance liquid chromatography method with on‐line precolumn enzymatic reaction for the screening of xanthine oxidase inhibitors in natural extracts was developed. In this method, the enzymatic reaction occurred at the capillary inlet during a predetermined waiting period, after which the reaction product, uric acid, was separated and detected by liquid chromatography using ultraviolet absorption at 295 nm. Enzyme inhibition can be read out directly from the reduced peak area of uric acid in comparison to a reference chromatogram obtained in the absence of any inhibitor. In the present study, the availability of on‐line precolumn enzymatic reaction with ultraviolet detection was firstly evaluated by determining the inhibitory mechanism and IC₅₀ values of allopurinol, a commercially available positive drug. Then, the newly developed method was applied to screening of ten natural extracts from traditional Chinese medicine and as a result, the extract of Epimedium sagittatum (Sieb. et Zucc.) Maxim was found to be most positive for xanthine oxidase inhibition. The results obtained were compared with those obtained by offline enzyme assay and the effectiveness of the present method was confirmed. A rapid, low‐cost, and fully automated method for xanthine oxidase inhibitor screening was proposed.     

Design,Synthesis, and Herbicidal Evaluation of Novel Uracil Derivatives Containing 1,3,4‐Thiadiazolyl Moiety

Uracil derivatives, such as commercial herbicides butafenacil and benzfendizone, have been identified as inhibitors of protoporphyrinogen oxidase (Protox, EC 1.3.3.4), one of the most important action targets of herbicides. In order to search for novel Protox inhibitors with high efficacy, broad‐spectrum activity, and safety to crops, commercially herbicide butafenacil was used as lead compound for further optimization; a series of title compounds 8a , 8b , 8c , 8d , 8e , 8f , 8g , 8h , 8i , 8j , 8k , 8l , 8m , 8n were designed and synthesized by introducing 1,3,4‐thiadiazole moiety into the uracil skeleton. The preliminary bioassays (in vitro) indicated that most of the target compounds displayed better inhibition against Echinochloa crus‐galli than Brassica campestris. The greenhouse bioassay results indicated that most of the compounds tested exhibited good‐to‐excellent herbicidal activities against B. campestris, A. retroflexus, E. crusgalli, and D. sanguinalis in pre‐emergence treatment at a dose of 1500 g/ha, for example, compound 8d showed 100% inhibition against the four plants tested in pre‐emergence treatment at a dose of 1500 g/ha. So, these types of skeletons can be used as valuable lead compounds for the development of a pre‐emergent herbicide.