Novel moving reaction boundary-induced stacking and separation of human hemoglobins in slab polyacrylamide gel electrophoresis

We developed a novel polyacrylamide gel electrophoresis (PAGE) method to stack and separate human hemoglobins (Hbs) based on the concept of moving reaction boundary (MRB). This differs from the classic isotachophoresis (ITP)-based stacking PAGE in the aspect of buffer composition, including the electrode buffer (pH 8.62 Tris–Gly), sample buffer (pH 6.78 Tris–Gly), and separation buffer (pH 8.52 Tris–Gly). In the MRB-PAGE system, a transient MRB was formed between alkaline electrode buffer and acidic sample buffer, being designed to move toward the anode. Hbs carried partial positive charges in the sample buffer due to its pH below pI values of Hbs, resulting in electromigrating to the cathode. Hbs would carry negative charges quickly when migrated into the alkaline electrode buffer and be transported to the anode until meeting the sample buffer again. Thus, Hbs were stacked within a MRB until the transient MRB reached the separation buffer and then separated by zone electrophoresis with molecular sieve effect of the gel. The experimental results demonstrated that there were three clear and sharp protein zones of Hbs (HbA_1c, HbA₀, and HbA₂) in MRB-PAGE, in contrast to only one protein zone (HbA₀) in ITP-PAGE for large-volume loading (≥15 μl), indicating high stacking efficiency, separation resolution, and good sensitivity of MRB-PAGE. In addition, MRB-PAGE was performed in a conventional slab PAGE device, requiring no special device. Thus, it could be widely used in separation and analysis of diluted protein in a standard laboratory.

Synthesis of Poly(<Emphasis Type="Italic">o</Emphasis>-anisidine)/H<Subscript>2</Subscript>SO<Subscript>4</Subscript> Film for the Development of Glucose Biosensor

The poly(o-anisidine)–sulfuric acid–glucose oxidase (POA–H₂SO₄–GOx) electrode has been investigated in the present work. Platinum electrode was used for the synthesis of poly (o-anisidine)–sulfuric acid (POA–H₂SO₄) film using galvanostatic method with 0.2 M o-anisidine, 1.0 M H₂SO₄ solution, 1.0 pH and 2 mA/cm² applied current density. The synthesized film was characterized using electrochemical technique, conductivity measurement, UV–visible spectroscopy, Fourier transform infrared spectroscopy, and scanning electron microscopy. GO_X was immobilized on synthesized POA–H₂SO₄ film by cross-linking via glutaraldehyde in phosphate and acetate buffer. The Michaelis–Menten constant ( K_\textm￠K_{\text{m}}^\prime ) was determined for the immobilized enzyme. The glucose oxidase electrode shows the maximum current response at pH 5.5 and potential 0.6 V. The sensitivity of POA–H₂SO₄–GO_X electrode in phosphate and acetate buffer has been recorded. The results of this study reveal that the phosphate buffer gives fast response as compared to acetate buffer in amperometric measurements.     

Comparative study on sample stacking by moving reaction boundary formed with weak acid and weak or strong alkali in capillary electrophoresis: I. Theory

The condensation of low abundance zwitterion substance, such as protein and peptide, has great significance to the study on proteomics. This paper develops the theory on design of online stacking conditions of zwitterion by a moving reaction boundary (MRB) in capillary electrophoresis (CE). This concerns the choice of running and sample buffers, velocity design of MRB, and salt effect on the stacking. The theoretical results unveil that: (1) the velocity of MRB formed with weak acidic buffer and strong alkali should be set between zero and the velocity of zwitterion in the alkali phase, or no stacking occurs; (2) if a strong alkali is used to prepare the sample, a much long front plug of strong base must be injected before the alkaline sample plug for complete stacking, whereas no such front plug is needed if a weak alkali with enough high concentration and pH value is used to prepare the sample buffer; (3) the existence of salt in sample matrix has a weak effect on the stacking of zwitterion if sample is prepared with weak alkaline buffer, while has a dramatic effect on the same stacking if with a strong base buffer. In addition, the concentration of weak alkali used for preparation of sample should be set at the point, at which the velocity of MRB is as much as possible close to that of negative zwitterion. The developed theory and its computation are quantitatively proved by the experiments of zwitterion stacking by the MRB as shown in the previous and the accompanying papers. The proposed theoretic results hold obvious significances on-column stacking of low abundance zwitterions, such as amino acid, or peptides or proteins, in CE.     

Improving Enzymatic Production of Ginsenoside Rh<Subscript>2</Subscript> from Rg<Subscript>3</Subscript> by using Nonionic Surfactant

In this study, several nonionic surfactants were tried to improve the enzymatic hydrolysis of ginsenoside Rg₃ into Rh₂ which was catalyzed at 50 °C and pH 5.0 by a crude glucosidase extracted from Fusarium sp. ECU2042. Among the biocompatible nonionic surfactants, polyethylene glycol 350 monomethyl ether was shown to be the best. After optimizing some influencing factors on the reaction, the conversion of Rg₃ (5 g/l) with 10 g/l crude enzyme reached almost 100% in the presence of the nonionic surfactant (7.5%, w/v), which was 25% higher than that in buffer without any surfactant. Furthermore, the enzyme stability was affected faintly by the surfactant.     

Synthesis and behaviour of hybrid polymer-silica membranes made by sol gel process with adsorbed carbonic anhydrase enzyme,in the capture of CO<Subscript>2</Subscript>

Thin nylon-SiO₂ membranes made by sol–gel SiO₂ coating of a nylon weaving were impregnated in a second step with an aqueous carbonic anhydrase solution. The biocatalytic hybrid membranes obtained were applied to the capture of CO₂ from a N₂–CO₂ gas mixture containing 10% CO₂, under a total pressure ≈ 1 atm. The CO₂ permeance of these membranes was at least similar to those previously reported for liquid membranes. When impregnated with a 0.2 mg mL⁻¹ enzyme solution in a pH ≈ 8 NaHCO₃ buffer, the permeance of a nylon-SiO₂ membrane was multiplied by a factor ≈ 3 when the buffer molarity was increased from 0.1 to 1 M. By comparison, this permeance only increased by a factor ≈ 1.3 without any enzyme in the same buffers. The permeance was also higher with the enzyme than without it: respectively ≈3.7 10⁻⁸ and ≈4.7 10⁻⁹ mol \textm_{\textmembrane} ^{- 2} {\text{m}}_{\text{membrane}}^{{^{ - 2} }} s⁻¹ Pa⁻¹ with and without enzyme, in a 1 M NaHCO₃ buffer. A maximum permeance was observed for an enzyme concentration of ≈0.2 mg mL⁻¹, possibly due to a competition between the H⁺ ions produced from CO_2,aq by the enzyme and the H⁺ captured by the buffer. Besides, when the SiO₂–CO₂ contact was enhanced by the membrane architecture, SiO₂ improved the CO₂ permeance. The influence of an in situ CaCO₃ deposit was also investigated and it improved the CO₂ permeance when no enzyme was added.     

Kinetics of the uninhibited and ethanol-inhibited CoO,Co<Subscript>2</Subscript>O<Subscript>3</Subscript> and Ni<Subscript>2</Subscript>O<Subscript>3</Subscript> catalyzed autoxidation of sulfur(IV) in alkaline medium

For getting an insight into the mechanism of atmospheric autoxidation of sulfur(IV), the kinetics of this autoxidation reaction catalyzed by CoO, Co₂O₃ and Ni₂O₃ in buffered alkaline medium has been studied, and found to be defined by Eqs. I and II for catalysis by cobalt oxides and Ni₂O₃, respectively.

Gene Cloning,Overexpression, and Characterization of a Xylanase from <Emphasis Type="Italic">Penicillium</Emphasis> sp. CGMCC 1669

A xylanase-encoding gene, xyn11F63, was isolated from Penicillium sp. F63 CGMCC1669 using degenerated polymerase chain reaction (PCR) and thermal asymmetric interlaced (TAIL)-PCR techniques. The full-length chromosomal gene consists of 724 bp, including a 73-bp intron, and encodes a 217 amino acid polypeptide. The deduced amino acid sequence of xyn11F63 shows the highest identity of 70% to the xylanase from Penicillium sp. strain 40, which belongs to glycosyl hydrolases family 11. The gene was overexpressed in Pichia pastoris, and its activity in the culture medium reached 516 U ml⁻¹. After purification to electrophoretic homogeneity, the enzyme showed maximal activity at pH 4.5 and 40°C, was stable at acidic buffers of pH 4.5–9.0, and was resistant to proteases (proteinase K, trypsin, subtilisin A, and α-chymotrypsin). The specific activity, K _m, and V _max for oat spelt xylan substrate was 7,988 U mg⁻¹, 22.2 mg ml⁻¹, and 15,105.7 μmol min⁻¹ mg⁻¹, respectively. These properties make XYN11F63 a potential economical candidate for use in feed and food industrial applications.     

Analysis of methotrexate and its eight metabolites in cerebrospinal fluid by solid-phase extraction and triple-stacking capillary electrophoresis

We establish a triple-stacking capillary electrophoresis (CE) separation method to monitor methotrexate (MTX) and its eight metabolites in cerebrospinal fluid (CSF). Three stacking methods with different mechanisms were combined and incorporated into CE separation. Complete stacking and sharp peaks were achieved. Firstly, the optimized buffer (60 mM phosphate containing 15% THF and 100 mM SDS) was filled into the capillary, which was followed by the higher conductivity buffer (100 mM phosphate, 2 psi for 45 s). The analytes extracted from CSF were injected at 2 psi for 99.9 s, which provided long sample zones and pH junction for focusing. Finally, the stacking step was performed by sweeping, and separation was achieved by micellar electrokinetic chromatography. The results of the linear regression equations indicated high linearity (r ≥ 0.9981) over the range of 0.5–7 μM. In intra- and inter-batch results, all data of RSD and RE were below 11%, indicating good precision and accuracy of this method. The LODs (S/N = 3) were 0.1 μM for MTX, 7-hydroxymethotrexate (7-OHMTX) and MTX-polyglutamates (MTX-(Glu) _{n, n = 2–5}), 0.2 μM for MTX-(Glu)₆, and 0.3 μM for 2,4-diamino-N ¹⁰-methylpteroic acid (DAMPA) and MTX-(Glu)₇. Our method was implemented for analysis of MTX and its metabolites in the CSF, and could be used for evaluation of its curative effects of acute lymphoblastic leukemia patients. The data were also confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The results showed good coincidence.     

New Thermostable Amylase from <Emphasis Type="Italic">Bacillus cohnii</Emphasis> US147 with a Broad pH Applicability

A new thermophilic bacterial strain identified as Bacillus cohnii US147 was isolated from the southern Tunisian soil. The identification was based on physiological tests and molecular techniques related to the 16S ribosomal ribonucleic acid. The isolated strain produced amylase, which was purified. This amylase had an apparent molecular mass of 30 kDa as estimated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Amylase US147 showed K _m and V _max values of 0.7 mg/ml and 2.2 U/ml, respectively, with starch as the substrate. The enzyme was active in acid and basic pH and had a maximal activity on starch at pH 9 and 70 °C. The enzyme was stable at pH 9 for 72 h and retained half of its activity after incubation at 70 °C for 150 min. A partially inhibition (15%, 25%, 23%, 20%, and 22%) was obtained with 1 mM SDS, 1 mM NaBO₃, 1 mM H₂O_2, 1 mM Zn⁺², and 5 mM ethylenediamine tetraacetic acid (EDTA), respectively. The amylase recovered its original activity by the addition of 10 mM Ca ²⁺ to the 5 mM EDTA. These properties indicated a possible use of this amylase in starch saccharification, in detergent, and in other industrial applications.     

Thermochemical properties of MnNb<Subscript>2</Subscript>O<Subscript>6</Subscript>

Homogeneous manganocolumbite (MnNb₂O₆) was synthesized from Nb₂O₅ and MnO oxides. Powder sample was orthorhombic with unit cell parameters: α = 0.5766 nm, b = 1.4439 nm, c = 0.5085 nm and V = 0.4234 nm³. Heat capacity over the temperature range of 313–1253 K was measured in an inert atmosphere with combined thermogravimetry and calorimetry using NETZSCH STA 449C Jupiter thermoanalyzer. Melting point was 1767 ± 3 K, enthalpy of melting was 144 ± 4 kJ mol⁻¹. Experimental heat capacity of MnNb₂O₆ is fitted to polynomial C _pm = 221.46 + 3.03 · 10⁻³ T + −39.79 · 10⁵ T ⁻² + 40.59 · 10⁻⁶ T ².     

Photodegradation of aqueous methyl orange on MnTiO<Subscript>3</Subscript> powder at different initial pH

The photodegradation of aqueous methyl orange on MnTiO₃ powder was studied in this work. The effects of initial pH and H₂O₂ on the photodegradation of aqueous methyl orange were studied. Single-phase MnTiO₃ powder was synthesized by a sol–gel method. X-ray diffraction analysis shows powders have a particle size of 43.7 nm and rhombohedral pyrophanite structure. The photocatalysis experiments revealed that the efficiency of decolorization of the aqueous methyl orange (1 × 10⁻⁵ M) on the MnTiO₃ powder is very fast in sunlight. The rate of decolorization appears larger at initial pH 5 and 9 than at pH 7. H₂O₂ (2%) can remarkably accelerate the decolorization at low initial pH.     

Biochemical Properties and PCR Performance of a Family B DNA Polymerase from Hyperthermophilic Euryarchaeon <Emphasis Type="Italic">Thermococcus peptonophilus</Emphasis>

The Thermococcus peptonophilus (Tpe) DNA polymerase gene was expressed under the control of the T7lac promoter on pET-22b(+) in Escherichia coli BL21-CodonPlus(DE3)-RIL in order to fully elucidate its biochemical properties and evaluate its feasibility in polymerase chain reaction (PCR) application. The expressed enzyme was then purified by heat treatment followed by two steps of column chromatography after which optimum pH and temperature of the enzyme were evaluated to be 7.0 and 75 °C, respectively. The optimal buffer for PCR with Tpe DNA polymerase consisted of 50 mM Tris–HCl (pH 8.0), 2 mM MgCl₂, 80 mM KCl, and 0.02% Triton X-100. Tpe DNA polymerase revealed a 3.6-fold higher fidelity (3.37 × 10⁻⁶) than Taq DNA polymerase (12.13 × 10⁻⁶) and performed significantly more efficiently in PCR amplification than both Taq and Pfu DNA polymerases. Ratios of 31:1 of Taq to Tpe DNA polymerases allowed PCR amplification of targets up to 15 kb in length with a 2.2-fold higher fidelity than Taq DNA polymerase. The results of the PCR experiments indicate that Tpe DNA polymerase may provide a higher fidelity DNA amplification in a shorter reaction time.     

Stacking and quantitative analysis of lovastatin in urine samples by the transient moving chemical reaction boundary method in capillary electrophoresis

A simple, sensitive, and useful concentration method for lovastatin (Lvt) in urine has been developed based on the transient moving chemical reaction boundary method (tMCRBM) in capillary electrophoresis. The MCRB is formed with acidic sample buffer (Gly-HCl) and alkaline running buffer (Gly-NaOH). The following optimal conditions were determined for stacking and separation: electrophoretic buffer of 100 mM Gly- NaOH (pH 11.52), sample buffer of 20 mM Gly-HCl (pH 4.93), fused-silica capillary of 76 cm × 75-μm i.d (67 cm from detector), sample injection at 14 mbar for 3 min. A 21- to 26-fold increase in peak height was achieved for detection of Lvt in urine under the optimal conditions compared with normal capillary zone electrophoresis. By combining the sample pretreatment procedure with the stacking method, the sensitivity of Lvt in urine was increased by 105- to 130-fold. The limits of detection (LOD) and quantification (LOQ) for Lvt in urine were decreased to 8.8 ng/mL and 29.2 ng/mL, respectively. The intra-day and inter-day precision values (expressed as RSD) were 2.23–3.61% and 4.03–5.05%, respectively. The recoveries of the analyte at three concentration levels changed from 82.65 to 100.49%.     

Preparation and electrical properties of Nd and Mn co-doped Bi<Subscript>4</Subscript>Ti<Subscript>3</Subscript>O<Subscript>12</Subscript> thin films

Ferroelectric thin films of Nd and Mn co-doped bismuth titanate, Bi_3.15Nd_0.85Ti_3−x Mn _x O₁₂ (BNTM) (x = 0–0.1), were fabricated on Pt/TiO₂/SiO₂/Si(100) substrates by a sol–gel technique. The BNTM films had a polycrystalline perovskite structure and uniform and dense surface morphologies. A lattice distortion was observed in the BNTM films due to Mn ion doping. The ferroelectric measurement of the films indicated that the values of coercive field (E _c ) decreased gradually with the increase of the Mn content (x), however, the remanent polarization (P _r ) increase firstly and then decrease with the increase of x. The sample with x = 0.05 had optimum electrical properties and a maximum 2P _r value. The 2P _r and 2E _c values of the film at a maximum applied electric field of 400 kV/cm were 38.3 μC/cm² and 180 kV/cm, respectively. Moreover, this BNTM capacitors did not show fatigue behaviors after 1.0 × 10¹⁰ switching cycles at a frequency of 1 MHz, suggesting a fatigue-free character. The main reason for the increase of the 2P _r and the decrease of the 2E _c might be attributed to the lattice distortion in BNTM films due to Mn ion doping.     

Voltammetric determination of the trace amounts of bromate in drinking water after pre-concentration on hydrous γ-Al<Subscript>2</Subscript>O<Subscript>3</Subscript>

A differential pulse voltammetric (DPV) method for the determination of bromate in drinking water, after pre-concentration on γ-Al₂O₃, is proposed. The reduction peak of bromate has been observed at the potential E _p -−1.6 V in an ammonia buffer as a supporting electrolyte. The method has been successfully applied to determine a bromate concentration of 2.5 μg·l⁻¹ in drinking water (RSD=6.1%, n=7). A sample pre-treatment with a column filled with mixed cation-exchange resin in Ag, Ba and H forms was needed before pre-concentration of bromate on alumina.     

Importance of DMSA stability,its consequence for Sn(DMSA)<Subscript>2</Subscript> complex formation and relevance to <Superscript>99m</Superscript>Tc-DMSA radipharmacs preparation

The dimercaptosuccinic acid (DMSA) stability at pH = 3.8, 7.1, 8.8 and the interaction with SnCl₂·2H₂O were investigated. The degradation of DMSA is the most significant at alkali pH. There where observed oxidized and dimerized forms of DMSA and fumaric acid presence. Progress of instability is less substantial and slower under neutral reaction conditions. At acidic pH the concentration of soluble DMSA decreases with the time. The ligand forms with SnCl₂ complex under alkali pH = 8.8 and its chemical formula was determined as Sn(DMSA)_2.     

Synthesis and colour properties of pigments based on terbium-dopped Mg<Subscript>2</Subscript>SnO<Subscript>4</Subscript>

The main aim of this work was to synthesize the magnesium orthostannate doped by terbium cations and tested whether these materials can be used for colouring of the different materials, e.g. organic binder and ceramic glazes. Initial composition of pigments was counted according the general formula 2MgO(1 − x)SnO₂–xTbO₂, where values of x varied from 0.1 to 0.5 in 0.1 steps. The simultaneous TG/DTA measurements of mixture containing tin oxide, magnesium carbonate hydroxide and terbium oxide showed that the formation of a new compound started at temperature 1,029 °C, but single-phase system was not prepared. Granulometric compositions of samples that were prepared by calcining at temperatures 1,300–1,400 °C are characterized by values of median (d ₅₀) in range 4–8 μm. The calcining temperature 1,500 °C caused the increase of the particle sizes at around 12 μm. The composition of sample 2MgO–1.5SnO₂–0.5TbO₂ and heating temperature 1,500 °C are the most suitable conditions for preparation of colourfully interesting pigment that can be recommended also for colouring of ceramic glazes. Especially, for colouring of decorative lead containing glaze G 07091 containing 5 wt% of PbO and 8 wt% of Al₂O₃.     

A Temperature and Salt-Tolerant <Emphasis Type="SmallCaps">l</Emphasis>-Glutaminase from Gangotri Region of Uttarakhand Himalaya: Enzyme Purification and Characterization

Purification and characterization of halotolerant, thermostable alkaline l-glutaminase from a Bacillus sp. LKG-01 (MTCC 10401), isolated from Gangotri region of Uttarakhand Himalaya, is being reported in this paper. Enzyme has been purified 49-fold from cell-free extract with 25% recovery (specific activity 584.2 U/mg protein) by (NH₄)₂SO₄ precipitation followed by anion exchange chromatography and gel filtration. Enzyme has a molecular weight of 66 kDa. l-Glutaminase is most active at pH 11.0 and stable in the pH range 8.0–11.0. Temperature optimum is 70 °C and is completely stable after 3 h pre-incubation at 50 °C. Enzyme reflects more enhanced activity with 1–20% (w/v) NaCl, which is further reduced to 80% when NaCl concentration was increased up to 25%. l-Glutaminase is almost active with K⁺, Zn²⁺, and Ni²⁺ ions and K _m and V _max values of 240 μM and 277.77 ± 1.1 U/mg proteins, respectively. Higher specific activity, purification fold, better halo-tolerance, and thermostability would make this enzyme more attractive for food fermentation with respect to other soil microbe derived l-glutaminase reported so far.     

High Level Expression of an Acid-Stable Phytase from <Emphasis Type="Italic">Citrobacter freundii</Emphasis> in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

To obtain a high level expression of phytase with favorable characteristics, a codon-optimized phytase gene from Citrobacter freundii was synthesized and transferred into Pichia pastoris. Small-scale expression experiments and activity assays were used to screen positive colonies. After purified by Ni²⁺–NTA agarose affinity column, the characterizations of the recombinant phytase were determined. The recombinant phytase (r-phyC) had two distinct pH optima at 2.5 and 4.5 and an optimal temperature at 50 °C. It retained more than 80% activity after being incubated under various buffer (pH 1.5–8.0) at 37 °C for 1 h. The specific activity, Km, and Vmax values of r-phyC for sodium phytate were 2,072 ± 18 U mg⁻¹, 0.52 ± 0.04 mM, and 2,380 ± 84 U mg⁻¹ min⁻¹, respectively. The enzyme activity was significantly improved by 1 mM of K⁺, Ca²⁺, and Mg²⁺. These characteristics contribute to its potential application in feed industry.     

Interfacial and aggregation properties of aqueous sodium <Emphasis Type="Italic">N</Emphasis>-dodecanoylsarcosinate solutions at different pH

The pH dependence of an anionic surfactant, sodium N-dodecanoylsarcosinate (SLAS), has been studied by measuring interfacial tension, fluorescence, dynamic light scattering, etc., in aqueous solutions with phosphate and borate buffers. The interfacial tension (γ) of SLAS decreases remarkably with a pH decrease and is constant at pH > 7.3. The observed values for the critical micelle concentration (cmc) and the surfactant concentration at which its γ value is reduced by 20 mN/m from that of pure water (C ₂₀) decrease with a pH decrease, while those also become constant at pH > 6.5 and >7.3, respectively. On the other hand, the interfacial excess of SLAS increases at pH < 7.3. These interfacial behaviors have been further investigated by the addition of Tl⁺ which replaces Na⁺ of SLAS. The observed γ values of LAS⁻ with the different counter cations are in the order of H⁺ < Tl⁺ < Na⁺. In order to reveal aggregation properties of SLAS, the aggregation number (N _agg), the micropolarity, the hydrodynamic radius (R _h) of micelle, and the fluorescence anisotropy of Rhodamine B (r) have been evaluated at various pHs. The N _agg value shows a decreasing tendency with a pH increase. The I ₁/I ₃ ratio and the R _h values do not strongly depend on pH. The r value decreases until pH 7 and remains constant at pH > 7.0. These interfacial and micelle properties have been discussed in detail considering the electrostatic interaction and the molecular structures of the hydrophilic headgroup.