Single-Step Partial Purification of Intracellular <Emphasis Type="Italic">β</Emphasis>-Galactosidase from <Emphasis Type="Italic">Kluyveromyces lactis</Emphasis> Using Microemulsion Droplets

Partial purification of β-galactosidase from the crude extract of Kluyveromyces lactis was carried out using water-in-isooctane microemulsions formed by the anionic surfactant, sodium di-ethylhexyl sulfosuccinate (Aerosol OT). In order to obtain the crude extract, yeast cells of K. lactis were disrupted by a cell disrupter and separated. The purification of β-galactosidase from the extract by a recently developed one-step reversed micellar (i.e., microemulsion-based) extraction method was then tested, by measuring total protein mass and enzyme activity in the product stream and by analyzing its composition using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography. Effects of salt concentration, protein concentration, and pH on the extraction were investigated. Using this approach, a 5.4-fold purification of β-galactosidase was achieved with 96 % total activity recovery, using a feed containing crude extract and 50 mM K-phosphate buffer (pH 7.5) and 50 mM KCl. Gel filtration chromatography showed that the single extraction was successful at removing low molecular weight impurity proteins (molecular weight (MW)?<?42 kDa) from the crude extract.     

Study of kinetics of complexation reaction of Co with 2-benzoylpyridine-4-phenyl-3-thiosemicarbazone and kinetic-spectrophotometric determination of cobalt

Kinetics of the complexation reaction of Co²⁺ with 2-benzoylpyridine-4-phenyl-3-thiosemicarbazone (BPPT) was spectrophotometrically examined at 421 nm. The ligand that was developed for a simple kinetic-spectrophotometric determination of Co²⁺ was based on 1:2 complex formation between Co²⁺ and BPPT. The complexation reaction was carried out in ethanol-water medium at 25 °C. Kinetic and activation parameters of the complexation reaction were calculated, and the rate equation and the reaction mechanism were proposed. The calibration graph is linear in the concentration range of 0.10−2.91 mg·L⁻¹ for the tangent method. The species that caused interference were investigated.     

Modeling signal transduction networks: a comparison of two stochastic kinetic simulation algorithms

Computational efficiency of stochastic kinetic algorithms depend on factors such as the overall species population, the total number of reactions, and the average number of nodal interactions or connectivity in a network. These size measures of the network model can have a significant impact on computational efficiency. In this study, two scalable biological networks are used to compare the size scaling efficiencies of two popular and conceptually distinct stochastic kinetic simulation algorithms--the random substrate method of Firth and Bray (FB), and the Gillespie algorithm as implemented using the Gibson-Bruck method (GGB). The arithmetic computational efficiencies of these two algorithms, respectively, scale with the square of the total species population and the logarithm of the total number of active reactions. The two scalable models considered are the size scalable model (SSM), a four compartment reaction model for a signal transduction network involving receptors with single phosphorylation binding sites, and the variable connectivity model (VCM), a single compartment model where receptors possess multiple phosphorylation binding sites. The SSM has fixed species connectivity while the connectivity between species in VCM increases with the number of phosphorylation sites. For SSM, we find that, as the total species population is increased over four orders of magnitude, the GGB algorithm performs significantly better than FB for all three SSM compartment models considered. In contrast, for VCM, we find that as the overall species population decreases while the number of phosphorylation sites increases (implying an increase in network linkage) there exists a crossover point where the computational demands of the GGB method exceed that of the FB.     

A parylene-based dual channel micro-electrophoresis system for rapid mutation detection via heteroduplex analysis

A new dual channel micro-electrophoresis system for rapid mutation detection based on heteroduplex analysis was designed and implemented. Mutation detection was successfully achieved in a total separation length of 250 microm in less than 3 min for a 590 bp DNA sample harboring a 3 bp mutation causing an amino acid change. Parylene-C was used as the structural material for fabricating the micro-channels as it provides conformal deposition, transparency, biocompatibility, and low background fluorescence without any surface treatment. A new dual channel architecture was derived from the traditional cross-channel layout by forming two identical channels with independent sample loading and waste reservoirs. The control of injected sample volume was accomplished by a new u-turn injection technique with pull-back method. The use of heteroduplex analysis as a mutation detection method on a cross-linked polyacrylamide medium provided accurate mutation detection in an extremely short length and time. The presence of two channels on the microchip offers the opportunity of comparing the sample to be tested with a desired control sample rapidly, which is very critical for the accuracy and reliability of the mutation analyses, especially for clinical and research purposes.     

Electrochemical Biosensing of DNA Immobilized Poly(Vinylferrocenium) Modified Electrode

The polymer, poly(vinylferrocenium) (PVF⁺) modified electrode was developed as the first time herein for the improved electrochemical sensing of DNA based on the oxidation signals of polymer and guanine. The morphologies of polymer film and DNA immobilized polymer film were examined using scanning electron microscope (SEM). The electrochemical behavior of polymer modified electrode was investigated by using cyclic voltammetry (CV) and differential pulse voltammetry (DPV) in the absence/presence of DNA. Experimental parameters, such as the polymeric film thickness, the DNA immobilization time, the concentration of buffer solution, pH and DNA concentration were examined in order to obtain more sensitive and selective electrochemical signals. After optimization studies, DNA hybridization was also investigated.     

Facilitated Transfer of Alkali Metal Ions by a Tetraester Derivative of Thiacalix[4]arene at the Liquid–Liquid Interface

The facilitated transfer of alkali metal ions (Na⁺, K⁺, Rb⁺, and Cs⁺) by 25,26,27,28‐tetraethoxycarbonylmethoxy‐thiacalix[4]arene across the water/1,2‐dichloroethane interface was investigated by cyclic voltammetry. The dependence of the half‐wave transfer potential on the metal and ligand concentrations was used to formulate the stoichiometric ratio and to evaluate the association constants of the complexes formed between ionophore and metal ions. While the facilitated transfer of Li⁺ ion was not observed across the water/1,2‐dichloroethane interface, the facilitated transfers were observed by formation of 1 : 1 (metal:ionophore) complex for Na⁺, K⁺, and Rb⁺ ions except for Cs⁺ ion. In the case of Cs⁺ a 1 : 2 (metal:ionophore) complex was obtained from its special electrochemical response to the variation of ligand concentrations in the organic phase. The logarithms of the complex association constants, for facilitated transfer of Na⁺, K⁺, Rb⁺, and Cs⁺, were estimated as 6.52, 7.75, 7.91 (log β₁°), and 8.36 (log β₂°), respectively.     

A Cyclic Voltammetric Study of Some Porphyrazines

Summary.  Some porphyrazines with dimethylamino- or trimethylammoniumethylsulfanyl substituents were studied in dichloromethane or dimethylsulfoxide solution by cyclic voltammetric methods. The cyclic voltammogram of metal free octakis-(dimethyl-aminoethylsulfanyl)-porphyrazine is characterized by three one-electron reduction waves which show quasi-reversible behaviour at all sweep rates observed; the same holds for its cobalt(II) derivative. The quaternized octacationic derivative, however, exhibits four one-electron reduction waves which are reversible at all sweep rates. The reaction mechanisms and diffusion coefficients were investigated. Received October 17, 2000. Accepted (revised) December 11, 2000     

Fluorogenic ferrocenyl Schiff base for Zn2+ and Cd2+ detection

Research on Chemical Intermediates - A novel sensor based on acetylferrocene-containing Schiff base (ASB) was synthesized by reaction of α-chloroacetylferrocene and...     

Organometallic chiral Schiff base for enantio-selective fluorescent recognition of methionine

Herein, the synthesis and characterization of a novel chiral Schiff bases derived from ferrocene, coded as 3, have been reported. The sensing behavior of the synthesized compound has been examined towards the enantiomers of some amino acids (methionine, alanine, serine, histidine, and threonine) by spectrofluorimetric method. The fluorescence response of compound 3 showed noticeable enhancement upon addition of d-methionine compared to l-methionine and kept nearly linear correlation with the concentration of d-methionine. The value of enantiomeric fluorescence difference ratio (ef) has been determined to be 1.54 when d - and l- methionine amount is 100 times more than compound 3. The results showed that the compound 3 can be used as a sensor for enantio-selective recognition of d-methionine.