Kinetic and mechanistic investigation of the ozonolysis of 2,4-xylidine (2,4-dimethyl-aniline) in acidic aqueous solution

The ozonolysis of 2,4-xylidine (2,4-dimethyl-aniline) in acidic aqueous solution was investigated by determining the major reaction products and their evolution as a function of the reaction time and their dependence on the pH of the reaction system. 2,4-Dimethyl-nitrobenzene and 2,4-dimethyl-phenol were found to be primary reaction products; their formation might be explained by electron transfer and substitution reactions. 2,4-Dimethyl-phenol was further oxidized yielding 2,4-dimethyl- and/or 4,6-dimethyl-resorcinol by electrophilic addition of HO(center dot) radicals. The best fitting phenomenological kinetic model and the good convergence of calculated and experimentally determined rate constants imply two additional competitive pathways of substrate oxidation: (i) electrophilic addition of HO(center dot) radicals and fast subsequent substitution would also yield the resorcinol derivatives. (ii) Substrate and isolated products are thought to be oxidized by hydrogen abstraction at the benzylic sites, but the corresponding products (alcohols, aldehydes, and carboxylic acids) could not be identified. Fe(II) was added to probe for the presence of H(2)O(2), but had no or only a minor effect on the kinetics of the ozonolysis. (c) 2009 Elsevier B.V. All rights reserved.

Catalytic properties of thioredoxin immobilized on superparamagnetic nanoparticles

Thioredoxin (Trx1), a very important protein for regulating intracellular redox reactions, was immobilized on iron oxide superparamagnetic nanoparticles previously coated with 3-aminopropyltriethoxysilane (APTS) via covalent coupling using the EDC (1-ethyl-3-{3-dimethylaminopropyl}carbodiimide) method. The system was extensively characterized by atomic force microscopy, vibrational and magnetic techniques. In addition, gold nanoparticles were also employed to probe the exposed groups in the immobilized enzyme based on the SERS (surface enhanced Raman scattering) effect, confirming the accessibility of the cysteines residues at the catalytic site. For the single coated superparamagnetic nanoparticle, by monitoring the enzyme activity with the Ellman reagent, DTNB=5,5`-dithio-bis(2-15 nitrobenzoic acid), an inhibitory effect was observed after the first catalytic cycle. The inhibiting effect disappeared after the application of an additional silicate coating before the AFTS treatment, reflecting a possible influence of unprotected iron-oxide sites in the redox kinetics. In contrast, the doubly coated system exhibited a normal in-vitro kinetic activity, allowing a good enzyme recovery and recyclability. (C) 2011 Elsevier Inc. All rights reserved.

Fabrication and integration of planar electrodes for contactless conductivity detection on polyester-toner electrophoresis microchips

In this report, we describe the microfabrication and integration of planar electrodes for contactless conductivity detection on polyester-toner (PT) electrophoresis microchips using toner masks. Planar electrodes were fabricated by three simple steps: (i) drawing and laser-printing the electrode geometry on polyester films, (ii) sputtering deposition onto substrates, and (iii) removal of toner layer by a lift-off process. The polyester film with anchored electrodes was integrated to PT electrophoresis microchannels by lamination at 120 degrees C in less than 1 min. The electrodes were designed in an antiparallel configuration with 750 mu m width and 750 gm gap between them. The best results were recorded with a frequency of 400 kHz and 10 V-PP using a sinusoidal wave. The analytical performance of the proposed microchip was evaluated by electrophoretic separation of potassium, sodium and lithium in 150 mu m wide x 6 mu m deep microchannels. Under an electric field of 250 V/cm the analytes were successfully separated in less than 90 s with efficiencies ranging from 7000 to 13 000 plates. The detection limits (S/N = 3) found for K+, Na+, and Li+ were 3.1, 4.3, and 7.2 mu mol/L, respectively. Besides the low-cost and instrumental simplicity, the integrated PT chip eliminates the problem of manual alignment and gluing of the electrodes, permitting more robustness and better reproducibility, therefore, more suitable for mass production of electrophoresis microchips.

Expression, purification and kinetic characterization of His-tagged glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi

Trypanosomes are flagellated protozoa responsible for serious parasitic diseases that have been classified by the World Health Organization as tropical sicknesses of major importance. One important drug target receiving considerable attention is the enzyme glyceraldehyde-3-phosphate dehydrogenase from the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease (T. cruzi Glyceraldehyde-3-phosphate dehydrogenase (TcGAPDH); EC 1.2.1.12). TcGAPDH is a key enzyme in the glycolytic pathway of T. cruzi and catalyzes the oxidative phosphorylation of D-glyceraldehyde-3-phosphate (G3P) to 1,3-bisphosphoglycerate (1,3-BPG) coupled to the reduction of oxidized nicotinamide adenine dinucleotide, (NAD(+)) to NADH, the reduced form. Herein, we describe the cloning of the T. cruzi gene for TcGAPDH into the pET-28a(+) vector, its expression as a tagged protein in Escherichia coli, purification and kinetic characterization. The His(6)-tagged TcGAPDH was purified by affinity chromatography. Enzyme activity assays for the recombinant His(6)-TcGAPDH were carried out spectrophotometrically to determine the kinetic parameters. The apparent Michaelis-Menten constant (K(M)(app)) determined for D-glyceraldehyde-3-phosphate and NAD(+) were 352 +/- 21 and 272 +/- 25 mu M, respectively, which were consistent with the values for the untagged enzyme reported in the literature. We have demonstrated by the use of Isothermal Titration Calorimetry (ITC) that this vector modification resulted in activity preserved for a higher period. We also report here the use of response surface methodology (RSM) to determine the region of optimal conditions for enzyme activity. A quadratic model was developed by RSM to describe the enzyme activity in terms of pH and temperature as independent variables. According to the RMS contour plots and variance analysis, the maximum enzyme activity was at 29.1 degrees C and pH 8.6. Above 37 degrees C, the enzyme activity starts to fall, which may be related to previous reports that the quaternary structure begins a process of disassembly. (C) 2010 Elsevier Inc. All rights reserved.