Alkaline Borate Research Articles

Comparison of activity indexes for recognizing enzyme mutants of higher activity with uricase as model

BackgroundFor screening a library of enzyme mutants, an efficient and cost-effective method for reliable assay of enzyme activity and a decision method for safe recognition of mutants of higher activity are needed. The comparison of activity concentrations of mutants in lysates of transformed Escherichia coli cells against a threshold is unsafe to recognize mutants of higher activity due to variations of both expression levels of mutant proteins and lysis efficiency of transformed cells. Hence, by a spectrophotometric method after verification to measure uricase activity, specific activity calculated from the level of total proteins in a lysate was tested for recognizing a mutant of higher activity.ResultsDuring uricase reaction, the intermediate 5-hydroxyisourate interferes with the assay of uric acid absorbance, but the measurement of absorbance at 293 nm in alkaline borate buffer was reliable for measuring uricase initial rates within a reasonable range. The level of total proteins in a lysate was determined by the Bradford assay. Polyacrylamide gel electrophoresis analysis supported different relative abundance of uricase mutant proteins in their lysates; activity concentrations of uricase in such lysates positively correlated with levels of total proteins. Receiver-operation-curve analysis of activity concentration or specific activity yielded area-under-the-curve close to 1.00 for recognizing a mutant with > 200% improvement of activity. For a mutant with just about 80% improvement of activity, receiver-operation-curve analysis of specific activity gave area-under-the-curve close to 1.00 while the analysis of activity concentration gave smaller area-under-the-curve. With the mean plus 1.4-fold of the standard deviation of specific activity of a starting material as the threshold, uricase mutants whose activities were improved by more than 80% were recognized with higher sensitivity and specificity.ConclusionSpecific activity calculated from the level of total proteins is a favorable index for recognizing an enzyme mutant with small improvement of activity.

Environmental friendly anodizing of AZ91D magnesium alloy in alkaline borate–benzoate electrolyte

A kind of environmental friendly anodizing routine for AZ91D magnesium alloy, based on an alkaline borate–sodium benzoate electrolyte (NaBz) was studied. The effect of NaBz on the properties of the anodized film was investigated by scanning electron microscopy (SEM), X-ray diffraction (XRD), energy dispersive spectrometry (EDS), potentiodynamic polarization and electrochemical impedance spectroscopy (EIS), respectively. The results showed that the anodizing process, surface morphology, thickness, phase structure and corrosion resistance of the anodized film were strongly dependent on the concentration of NaBz. In the presence of adequate NaBz, a thick, compact and smoothing anodized film with excellent corrosion resistance was produced. Moreover, the forming mechanism of the anodized film in the presence of NaBz additive was also approached, which was a suppression of arc discharge process by the adsorption of Bz − on the surface of magnesium alloy substrate.

Capillary electrophoresis methods for the determination of covalent polyphenol–protein complexes

The bioactivities and bioavailability of plant polyphenols including proanthocyanidins and other catechin derivatives may be affected by covalent reaction between polyphenol and proteins. Both processing conditions and gastrointestinal conditions may promote formation of covalent complexes for polyphenol-rich foods and beverages such as wine. Little is known about covalent reactions between proteins and tannin, because suitable methods for quantitating covalent complexes have not been developed. We established capillary electrophoresis methods that can be used to distinguish free protein from covalently bound protein-polyphenol complexes and to monitor polyphenol oxidation products. The methods are developed using the model protein bovine serum albumin and the representative polyphenol (-)epigallocatechin gallate. By pairing capillaries with different diameters with appropriate alkaline borate buffers, we are able to optimize resolution of either the protein-polyphenol complexes or the polyphenol oxidation products. This analytical method, coupled with purification of the covalent complexes by diethylaminoethyl cellulose chromatography, should facilitate characterization of covalent complexes in polyphenol-rich foods and beverages such as wine.

Application of renewable silver amalgam annular band electrode to voltammetric determination of vitamins C, B1 and B2

In this work, the design and results of applying silver liquid amalgam film–modified silver solid amalgam annular band electrode (AgLAF–AgSAE), refreshed before each measurement, to voltammetric determination of vitamins C (VC), B1 (VB1) and B2 (VB2) are presented. The method is based on adsorptive accumulation of analytes at the AgLAF–AgSAE in a phosphate buffer (VB1), phosphate buffer with Triton X-100 (VB2) and an alkaline borate buffer with Triton X-100 (VC). The analytical parameters and procedure of electrode activation were optimized. The calibration graphs obtained for vitamins C, B1 and B2 are linear, respectively, for concentration range 0.05–12, 0.01–0.1 and 0.05–3mgL−1. The detection limits were calculated and equaled 0.02, 0.003 and 0.009mgL−1, while repeatability of the peak current was 2%, 1% and 3%, respectively. These results are comparable with results obtained for polarographic determination of the same vitamins using mercury electrodes. Finally, the AgLAF–AgSAE was applied to the determination of vitamins in pharmaceutical samples and fruit juices with satisfactory results.

Isolation of high-quality RNA from various tissues of Jatropha curcas for downstream applications

A method for isolating transcriptionally active RNA for downstream applications from diverse tissues of Jatropha curcas, a plant rich in latex, lipids, waxes, polysaccharide, polyphenols, and secondary metabolites, is described. The described method uses alkaline borate buffer during tissue homogenization to negate the formation of viscous gel observed in guanidium-salt-containing methods. By this method, quality RNA was extracted from leaf, immature inflorescence, endosperm, and root tissues with yields ranging from 1.80 to 7.80 mg/100 mg fresh weight (FW). The total RNA obtained was found to be suitable for poly(A) +RNA purification, complementary DNA (cDNA) synthesis, cloning of full-length cDNA, and cDNA library construction.

Influence of electric parameters on MAO of AZ91D magnesium alloy using alternative square-wave power source

Micro-arc oxidation (MAO) process was carried out on AZ91D alloy in alkaline borate solution using an alternative square-wave power source with different parameters. The effects of voltage, frequency and duty cycle on the coatings were investigated by orthogonal experiment. It is found that the thickness of coatings increases with the increase of voltage and duty cycle, but decreases with the increase of frequency. The structure and morphology of the coatings also depend on voltage, frequency and duty cycle. The coatings become more porous and crack with increasing voltage and duty cycle. The coating is thin and transparent when the voltage is lower than 120 V. The corrosion resistances of different coatings were evaluated by polarization curves and electrochemical impedance spectroscopy (EIS) in 3.5% NaCl (mass fraction) solution. When the optimized values of voltage, frequency and duty cycle are 140 V, 2 000 Hz and 0.4, respectively, the anodic coating shows the best corrosion resistance.

NONCOMPETITIVE IMMUNOASSAY FOR FOLLICLE-STIMULATING HORMONE IN HUMAN SERUM USING CAPILLARY ELECTROPHORESIS WITH CHEMILUMINESCENCE DETECTION

A noncompetitive immunoassay based on capillary electrophoresis (CE) with chemiluminescence (CL) detection has been developed for the determination of follicle-stimulating hormone (FSH) in human serum. The work involved the development of separation and CL conditions allowing for routine analysis of serum samples. In this study, horseradish peroxidase (HRP)-labeled monoclonal anti-FSH can catalyze the luminol–hydrogen peroxide reaction. The determined FSH can react with an excessive amount of HRP-labeled anti-FSH. Within 15 min, free enzyme conjugate and immune complex could be separated in alkaline borate buffer by a high voltage of 15 kV. To improve the sensitivity, a series of measures was adopted, including the choice of sodium tetraphenylboron as a new CL enhancer, with a unique design in the detect window. Under optimal conditions, the calibration curve for FSH was established in the concentration range of 1–150 IU/L and the detection limit was 0.08 IU/L. Compared with enzyme-linked immunosorbent assay (ELISA), this method decreased the detection limit by about 25-fold, and it has been successfully employed on determination of the FSH in human serum.

A user‐friendly alternative to formaldehyde‐based DNA silver‐staining method on polyacrylamide gels

We have developed a practical, cost-effective and user-friendly protocol to meet the needs of nucleic acids research, particularly in respect of DNA detection on polyacrylamide gels. In this method, the most commonly used alkaline formaldehyde developer in DNA silver stain, which does harm to operator, is first replaced by glucose in alkaline borate buffer. In addition, the effects of six reducing sugars on the quality of DNA visualization were investigated. Consequently, the optimal protocol using glucose takes about 45 min to complete all the procedures, with a detection limit of 5 pg of single DNA band on polyacrylamide gels, was developed. The results indicate that this user-friendly and economic protocol could be a good choice for routine use in DNA visualization on polyacrylamide gels.

Relation between Low-Temperature Thermal Conductivity and the Specific Heat of Cesium Borate Glasses

Low-temperature specific heat and thermal conductivity measurements have been performed on cesium borate glasses as a function of cesium oxide content. We have found experimental evidence of a concurrent growth of specific heat and thermal conductivity with increasing Cs(+) content. This finding shows the existence of an uncommon relationship between the peak in C(p)/T(3) and the plateau in thermal conductivity in glasses and represents the most intriguing result for these alkaline borate glasses. The role of local modes associated with heavy cations on the vibrational dynamics in oxide glasses has been considered. Furthermore, a possible correlation between low-temperature thermal properties and the structure on the nanometer length scale of these glasses is put forward.

Role of Halides on the Passivation of Iron in Alkaline Buffer Solutions

Cyclic voltammetric studies were carried out on pure iron in alkaline borate and phosphate buffer solutions at pH 10.8. At higher potentials, on anodic polarization, iron forms FeB 4O7 and FeOOH in borate buffer, and FeHPO 4 in phosphate buffer which got converted to higher valency phosphates. In phosphate solutions, in presence of halides, the interfacial diffusion layer turned to be cation selective outer sublayer and an anion selective inner sublayer, and in borate solutions a precipitate layer of metal oxyhydroxide was formed, which was anion selective, and anions adsorb on this.

Influence of Nb 2O 5 on the structure, optical and electrical properties of alkaline borate glasses

Glasses with composition xNb 2O 5·(30 − x)MO·70B 2O 3 [where M = Ca, Sr, Ba] have been prepared by using normal melt-quench technique. The density ( D), molar volume ( V M), structure, optical and electrical properties have been investigated in order to understand the role of Nb 2O 5 in these glasses. Values of D and V M increase with increasing concentration of Nb 2O 5 in these glasses. The increase in V M indicates that the volume of non-bridging oxygens (NBOs) sites produced by the Nb 2O 5 is greater than that produced by alkaline oxides in present glasses. The results of optical basicity ( Λ th) show that the ionic character of these glasses is improving with increase in Nb 2O 5 content. The analysis of IR spectra reveals the presence of NbO 4 and NbO 6 units. The band gap energy ( E g) of the present glasses is found to decrease with increase in Nb 2O 5 content. A theoretical approach has been used to calculate the refractive index ( n r), molar refraction ( R m) and optical polarizability ( α 0) for the glass samples having D in between 1 and 3 g cm −3. The conductivity results support the IR analysis and indicate the presence of NbO 4 and NbO 6 units in these glasses.

Boson peak in alkaline borate glasses: Raman spectroscopy, neutron scattering, and specific-heat measurements

The dependence of the boson peak on the alkaline ion in modified borate glasses ${({M}_{2}\mathrm{O})}_{0.14}{({\mathrm{B}}_{2}{\mathrm{O}}_{3})}_{0.86}$ $({M}^{+}={\mathrm{Li}}^{+},{\mathrm{Na}}^{+},{\mathrm{K}}^{+},{\mathrm{Cs}}^{+})$ was analyzed by performing Raman spectroscopy, inelastic neutron scattering, and low-temperature specific-heat measurements. It is found that the distribution of vibrations merging into the boson peak shifts to higher frequency by going from cesium to lithium. A linear correlation between the boson peak frequency and the transverse sound velocity is evidenced. The dependence on the polarizing power of the metallic cation is analyzed, stemming from considerations about elastic moduli. These findings suggest a mainly transverse character of the excess vibrational modes in glasses.

Sweet silver: A formaldehyde‐free silver staining using aldoses as developing agents, with enhanced compatibility with mass spectrometry

Protein detection methods after electrophoresis have to be sensitive, homogeneous, and not to impair downstream analysis of proteins by MS. Speed, low cost, and user friendliness are also favored features. Silver staining combines many of these features, but its compatibility with MS is limited. We describe here, a new variant of silver staining that is completely formaldehyde-free. Reducing sugars in alkaline borate buffer are used as developers. While keeping the benefits of silver staining, this method is shown to afford a much better performance in terms of compatibility with MS, both in PMF by MALDI and in LC/ESI/MS/MS.

Sorghum Protein Extraction by Sonication and Its Relationship to Ethanol Fermentation

ABSTRACTThe objectives of this research were to develop a rapid method for extracting proteins from mashed and nonmashed sorghum meal using sonication (ultrasound), and to determine the relationships between the levels of extractable proteins and ethanol fermentation properties. Nine grain sorghum hybrids with a broad range of ethanol fermentation efficiencies were used. Proteins were extracted in an alkaline borate buffer using sonication and characterized and quantified by size‐exclusion HPLC. A 30‐sec sonication treatment extracted a lower level of proteins from nonmashed sorghum meal than extracting the proteins for 24 hr with buffer only (no sonication). However, more protein was extracted by sonication from the mashed samples than from the buffer‐only 24‐hr extraction. In addition, sonication extracted more polymeric proteins from both the mashed and nonmashed samples compared with the buffer‐only extraction method. Confocal laser‐scanning microscopy images showed that the web‐like protein microstructures were disrupted during sonication. The results showed that there were strong relationships between extractable proteins and fermentation parameters. Ethanol yield increased and conversion efficiency improved significantly as the amount of extractable proteins from sonication of mashed samples increased. The absolute amount of polymeric proteins extracted through sonication were also highly related to ethanol fermentation. Thus, the SE‐HPLC area of proteins extracted from mashed sorghum using sonication could be used as an indicator for predicting fermentation quality of sorghum.

P.435 Evaluation of meniscus position during orthodontic treatment

In this work, the design and results of applying silver liquid amalgam film–modified silver solid amalgam annular band electrode (AgLAF–AgSAE), refreshed before each measurement, to voltammetric determination of vitamins C (VC), B1 (VB1) and B2 (VB2) are presented. The method is based on adsorptive accumulation of analytes at the AgLAF–AgSAE in a phosphate buffer (VB1), phosphate buffer with Triton X-100 (VB2) and an alkaline borate buffer with Triton X-100 (VC). The analytical parameters and procedure of electrode activation were optimized. The calibration graphs obtained for vitamins C, B1 and B2 are linear, respectively, for concentration range 0.05–12, 0.01–0.1 and 0.05–3 mg L−1. The detection limits were calculated and equaled 0.02, 0.003 and 0.009 mg L−1, while repeatability of the peak current was 2%, 1% and 3%, respectively. These results are comparable with results obtained for polarographic determination of the same vitamins using mercury electrodes. Finally, the AgLAF–AgSAE was applied to the determination of vitamins in pharmaceutical samples and fruit juices with satisfactory results.

Kinetic Spectrophotometric Analysis and Spectrofluorimetric Analysis of Ciprofloxacin Hydrochloride and Norfloxacin in Pharmaceutical Preparations Using 4‐Chloro‐7‐Nitrobenzo‐2‐Oxa‐1,3‐Diazole (NBD‐Cl)

AbstractSimple, reliable, sensitive and accurate kinetic spectrophotometric and spectrofluorimetric methods were proposed for the determination of ciprofloxacin hydrochloride (CPX) and norfloxacin (NRX) in pure form and in pharmaceuticals. The methods are based on coupling the studied drugs with 4‐chloro‐7‐nitrobenzo‐2‐oxa‐1,3‐diazole (NBD‐Cl) in the presence of alkaline borate buffer. Spectrophotometric measurement was achieved by recording the absorbance at 477 nm after a fixed time of 20 and 15 min on a water bath adjusted at 70 ± 1 °C for CPX and NRX, respectively. The same product exhibited emission peaks at 540 nm. The different experimental parameters affecting the development and stability of the color were carefully studied and optimized. The absorbance concentration plots were linear over the ranges 3‐18 and 2.5‐15.0 μg/mL for CPX and NRX, respectively, while the fluorescence concentration plots were linear over the ranges 0.06‐0.36 and 0.05‐0.30 μg/mL for CPX and NRX, respectively. The limit of detection of the kinetic method was about 0.2 μg/mL for both drugs while the fluorescence measurement enabled their detection at a concentration of about 0.012 μg/mL. The proposed methods were successfully applied for the assay of the two drugs in their commercial products. The results obtained were statistically compared with those obtained by reference HPLC and spectrophotometric methods. The stoichiometry of the reaction was determined and the reaction pathway was postulated.

Environmental friendly plasma electrolytic oxidation of AM60 magnesium alloy and its corrosion resistance

Plasma electrolytic oxidation of Mg-based AM60 alloys was investigated using 50 Hz AC anodizing technique in an alkaline borate solution, which contained a new kind of organic. The anodic film is relatively smooth with some micro pores and cracks, while the anodic film consists of MgO, MgAl 2O 4 and MgSiO 3. The electrochemical behavior of anodic film was studied by electrochemical impedance spectroscopy and potentiodynamic polarization. Polarization results indicate the PEO treatment can decrease corrosion current by 3–4 magnitude compared with blank AM60 alloy. The anodic film presents a good level of corrosion protection for AM60 magnesium alloy, over 272 h of the salt spray test based on ASTM B117. The effect of micro-structure and composition on corrosion protection efficiency was also investigated.

Impact of Mashing on Sorghum Proteins and Its Relationship to Ethanol Fermentation

Nine grain sorghum cultivars with a broad range of ethanol fermentation efficiencies were selected to characterize the changes in sorghum protein in digestibility, solubility, and microstructure during mashing and to relate those changes to ethanol fermentation quality of sorghum. Mashing reduced in vitro protein digestibility considerably, and a large amount of polymers cross-linked by disulfide bonds were developed during mashing. As a marker of cross-linking, protein digestibility of the original samples was highly related to conversion efficiency. gamma-Kafirin (%) neither correlated to ethanol yield nor conversion efficiency significantly. Solubility of proteins in an alkaline borate buffer in conjunction with SDS decreased substantially after mashing. Solubility and the SE-HPLC area of proteins extracted from mashed samples were highly correlated with ethanol fermentation. Ethanol yield increased and conversion efficiency improved notably with the increase of extracted proteins from mashed samples. SE-HPLC total area could be used as an indicator to predict ethanol fermentation. CFLSM images proved that sorghum proteins tended to form highly extended, strong web-like microstructures during mashing. The degree of protein cross-linking differed among samples, and more open microstructures were observed in samples with higher conversion efficiencies. The web-like protein matrix was found to hold not only starch granules but also some oligosaccharides or polysaccharides inside. The formation of web-like microstructures because of cross-linking reduced conversion efficiency.

Oxide/hydroxide films on tin. Part I: Kinetic aspects of the electroformation and electroreduction of the films

The anodic behaviour of tin in borate solutions has been studied by linear potential sweep voltammetry and triangularly modulated triangular potential sweeps. It is shown that the active to passive transition involves the formation of intermediate soluble species followed by the precipitation of a tin oxide/hydroxide layer that passivates the electrode. The potentiodynamic current-potential behaviour is adequately described by using the Layer-pore-resistance model. A good agreement between experimental and theoretical curves is obtained which allow us to demonstrate that primary passivation of tin in alkaline borate solutions (pH 8.9) as well as the film electroreduction is mainly controlled by the resistance of the electrolyte in the pores of the layer. In addition, the triangularly modulated triangular potential sweep method is applied in order to analyze the existence of reaction intermediates.

Plasma electrolytic oxidation of AZ91D magnesium alloy with different additives and its corrosion behavior

AbstractPlasma electrolytic oxidation (PEO) of Mg‐based AZ91D alloys was investigated using 50 Hz AC anodizing technique in an alkaline borate solution, which contained a new kind of organic additive and without F, P, and Cr. The anodizing technological parameters have been optimized and a kind of ivory‐white smooth anodic film with high corrosion resistance was obtained. It was found that the formation of the anodic films was always coupled with sparking and oxygen evolution, whose intensity changed with the additive and anodizing voltage. All EIS plots have two capacitive loops and one low frequency inductive component. Two capacitive arcs present the barrier and porous layer of the PEO film and the inductive component in the low frequency domain is a complex behavior due to the porous structure connected to the electrolyte. EIS plots and fitting results show that a self‐sealing process of the PEO firm with different additives takes place in the beginning of immersion time, then corrosion attack becomes a preponderant process to promote the degradation of the film. Tafel results show that PEO treatment decreases the corrosion current density by four, even five orders of magnitude, while additives content does not affect strongly the electrochemical corrosion behavior. Salt spray test shows that the PEO film formed with NaAlO2 and Na2SiO3 presents good corrosion resistance, over 600 h without any sealing treatment. The difference of corrosion resistance arose by additives examined by electrochemical techniques and salt spray test does not show strict corresponding relationship.