NaH2PO4 Buffer Research Articles

An immunonanogold resonance scattering spectral probe for rapid assay of human chorionic gonadotrophin

BackgroundSeveral assays for human chorionic gonadotrophin (hCG) such as radioimmunoassay and fluorescence immunoassays were reported. So far, there was no report about the immunonanogold resonance scattering spectral (ING-RSS) assay based on the immunoreaction and resonance scattering (RS) effect. MethodsThree ING-RSS probes for hCG were prepared, using nanogold in size of 8, 10, 13 nm to label rabbit hCG antiserum (anti-hCG). ResultsIn citric acid–Na2HPO4 buffer solution and in the presence of polyethylene glycol (PEG)6000, the immunoreaction between hCG and nanogold-labeled anti-hCG took place, the immunonanogold-complex was formed and deposited. The decreased intensity ΔIRS for the nanogold in sizes of 8, 10, 13 nm was proportional to hCG concentration in the ranges of 40–10000, 40–10000 and 40–8000 mIU/ml, with the detection limits of 19.4, 15.1 and 13.6 mIU/ml, respectively. The hCG in urine samples were assayed by the ING-RSS assay, and by immunospectrophotometry. The results of both assays showed a close correlation. ConclusionThis assay has simplicity, sensitivity and good selectivity for quantitative determination of hCG.

Determination of highly soluble L-Carnitine in biological samples by reverse phase high performance liquid chromatography with fluorescent derivatization

This study was performed in order to validate an effective high performance liquid chromatograpy (HPLC) method to determine L-carnitine in biological samples such as plasma, milk and muscle in cows. An L-carnitine derivative for fluorescence absorption was synthesized with 1-aminoanthracene (16 mg/mL in acetone) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC; 160 mg/mL in 0.01 M NaH2PO4 buffer) as a precolumn fluorescent derivative reagent. gamma-Butyrobetaine HCI was used as an internal standard. A reversed-phase column with fluorescence detection at the excitation and emission wavelengths of 248 and 418 nm were used. The mobile phase consisted of 30% acetonitrile with 0.1 M ammonium acetate in water (pH 3.5) adjusted with acetic acid and delivered at a flow rate of 1.5 mL/ min. The L-carnitine concentration in plasma, milk and muscle samples of cows after oral feeding with 24 g L-carnitine/day for 2 months was then determined. All biological samples were deproteinated by barium hydroxide and zinc sulfate heptahydrate before the derivative reaction. Blank cow plasma was dialyzed using cellulose membrane for standard calibration. The calibration curve showed good linearity (r2 > 0.999) over the concentration range of 50 to 5000 ng/mL. The precision and accuracy were also satisfactory with less than 15% intra- and interday coefficiency of variations. The peaks of L-carnitine and internal standard in HPLC chromatography were successfully separated in plasma, milk and muscle samples of cows. The current derivatization method of L-carnitine for fluorescence detection was simple and adequately sensitive and could be applied to determine L-carnitine in biological samples.

Molecular Changes in the Vasculature of Injured Tissues

We have explored molecular specialization of the vasculature of regenerating wound tissue in the skin and tendons to identify a different repertoire of markers from that obtained by studying tumor vasculature. We screened a phage-displayed peptide library for peptides that home to wounds in mice and identified two peptides that selectively target phage to skin and tendon wounds: CARSKNKDC (CAR) and CRKDKC (CRK). CAR is homologous to heparin-binding sites in various proteins and binds to cell surface heparan sulfate and heparin. CRK is similar to a segment in thrombospondin type 1 repeat. Intravenously injected CAR and CRK phage, as well as fluorescein-labeled CAR and CRK peptides, selectively accumulated at wound sites, where they partially co-localized with blood vessels. The CAR peptide showed a preference for early stages of wound healing, whereas the CRK favored wounds at later stages of healing. The CAR peptide was internalized into the target cells and delivered the fluorescent label into the cell nuclei. These results identify new molecular markers in wound tissues and show that the expression of these markers in wound vasculature changes as healing progresses. The peptides recognizing these markers may be useful in delivering treatments into regenerating tissues.

Identification of Mouse Prp19p as a Lipid Droplet-associated Protein and Its Possible Involvement in the Biogenesis of Lipid Droplets

Prp19p is an integral component of the heteromeric protein complex (the NineTeen complex) in the nucleus, and it is essential for the structural integrity of NineTeen complex and its subsequent activation of the spliceosome. We identified Prp19p, which has never been reported in relation to any function outside of the nucleus, as a member of proteins associated with lipid droplets. Down-regulation of Prp19p expression with RNA interference in 3T3-L1 cells repressed lipid droplet formation with the reduction in the level of expression of perilipin and S3-12. The levels of expression of SCD1 (stearoyl-CoA desaturase-1), DGAT-1 (acyl-CoA diacylglycerol acyltransferase-1), and glycerol-3-phosphate acyltransferase were also reduced in Prp19p down-regulated cells, and a significant decrease in triglycerides was observed. Unlike perilipin, which is one of the most extensively studied lipid droplet-associated proteins, Prp19p is not essential for cAMP- and hormone-sensitive lipase-dependent lipolysis pathways, even though Prp19p is a component of the lipid droplet phospholipid monolayer, and down-regulation of Prp19p represses fat accretion significantly. These results suggest that Prp19p or Prp19-interacting proteins during lipid droplet biogenesis in adipocytes may be considered as another class of potential targets for attacking obesity and obesity-related problems.

Crystal structure of dihydrodipicolinate synthase (BA3935) from Bacillus anthracis at 1.94 Å resolution

Introduction. As part of a structural genomics program, we are determining structures of proteins from the causative agent of anthrax, Bacillus anthracis, a Grampositive spore-forming bacterium. Among our initial candidates for crystallographic analysis are the products of essential genes based on knock-out studies in Bacillus subtilis. The BA3935 gene of B. anthracis (www.tigr.org), annotated as DapA2, encodes a putative protein consisting of 292 amino acid residues with a subunit molecular weight of 31,233 Da. The predicted protein has 60% identity with dihydrodipicolinate synthase (DHDPS or DapA) from B. subtilis and 40% amino acid sequence identity to its orthologue in Escherichia coli. dapA is one of only 271 out of the total of 4118 genes in B. subtilis that are indispensable for growth of the organism on standard laboratory media. 1 DHDPS catalyses the condensation of aspartate semialdehyde and pyruvate and is the first committed step on the pathway to diaminopimelate and L-lysine in prokaryotes, some fungi, and higher plants (Scheme 1). The product released from the E. coli enzyme has been shown to be 4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid (HTDPA) rather than L-2,3-dihydrodipicolinic acid (DHDPA); as the name of the enzyme suggests, 2 DHDPA can be formed spontaneously from HTDPA by elimination of water. The steps of aspartate semialdehyde synthesis from aspartate are shared with the biosynthetic pathways leading methionine and threonine. The diaminopimelate/ lysine pathway is thought to be of particular importance in Gram-positive bacteria because diaminopimelate makes up a higher proportion of the dry cell weight than it does in Gram-negative bacteria, a consequence of the thicker cell wall in the former. In Bacilli, the product of the DHDPS reaction is further reduced by dipicolinate synthase to dipicolinate, which makes up 10% of the dry weight of spores. Crystal structures have been determined of DHDPS from E. coli, 3 Nicotiana sylvestris, 4 and most recently from Thermotoga maritima. 5 In this article, we report the crystal structure of DHDPS from B. anthracis (Ba DHDPS), the first structure of a dihydrodipicolinate synthase from a Gram-positive bacterium. The structure of Ba DHDPS was determined by molecular replacement using the coordinate set for the E. coli orthologue (PDB code 1DHP) as the search model. 3 Two structures have been determined to 1.9 and 2.2 A resolution in different orthorhombic crystal forms. Data collection, refinement, and model-building statistics are summarized in Table I. The structures in the two crystal forms are very similar and unless otherwise stated, the commentary here will refer to the structure refined to higher resolution

Separation and determination of seven fluoroquinolones by pressurized capillary electrochromatography

In this paper, pressurized CEC was used for the separation and determination of seven fluoroquinolones (FQs). The effect of different experimental conditions, such as the concentration and pH of the buffer, the organic modifier concentration, the surfactant and ion-paring agents added to the electrolyte, and applied voltage were studied. All the seven FQs were baseline separated using mobile phase containing 27% v/v ACN, 5 mmol/L Na2HPO4 buffer (pH 4.0 adjusted using citric acid), 11 mmol/L SDS, and 0.01% TEA v/v at detection wavelength of 287 nm and at an applied voltage of -10 kV. The calibration curves were linear (r>0.9991) over a concentration range of 1.0-50.0 mg/L for norfloxacin (NFLX); 2.5-50.0 mg/L for fleroxacin (FLX), ciprofloxacin (CPFX), and lomefloxacin (LMX); and 5.0-50.0 mg/L for enoxacin (ENX), ofloxacin (OFLX), and gatifloxacin (GFLX). The detection limits (S/N = 3) for ENX, OFLX, FLX, NFLX, CPFX, LMX, and GFLX were 0.5, 0.8, 0.4, 0.2, 0.4, 0.5, and 1.0 mg/L, respectively. The method is simple, rapid, and reproducible. It was successfully applied to the analysis of fish muscle samples spiked with FQs. Mean recoveries ranged from 81.6 to 97.6%.

Crystal structure of PurE (BA0288) from Bacillus anthracis at 1.8 Å resolution

Crystal structure of PurE (BA0288) from <i>Bacillus anthracis</i> at 1.8 Å resolution

An Improved Method for the Quantitation of Flavonoids in Herba Leonuri by Capillary Electrophoresis

A capillary electrophoresis system coupled with wall-jet amperometric detection was used to determine five flavonoids (kaempferol, rutin, hyperoside, quercitrin, quercetin) in Herba Leonuri. Several important effect factors, such as the pH and concentration of running buffer, separation voltage, injection time, and detection potential, were investigated to acquire the optimum conditions. With 50 mmoL/L Na(2)B(4)O(7)-100 mmoL/L NaH(2)PO(4) buffer (pH=7.50) as the running buffer, the five flavonoids were baseline separated within 15 min in a 60 cm length capillary at a separation voltage of 15 kV. The relationship between peak currents and analyte concentrations was linear over about 2 orders of magnitude, with detection limits (S/N = 3) ranging from 0.03 to 0.08 microg/mL for all analytes. The use of this method for the quantitation of the above flavonoids present in the real sample of Herba Leonuri was reported.

Discovery of Pectin-degrading Enzymes and Directed Evolution of a Novel Pectate Lyase for Processing Cotton Fabric

There is a growing need in the textile industry for more economical and environmentally responsible approaches to improve the scouring process as part of the pretreatment of cotton fabric. Enzymatic methods using pectin-degrading enzymes are potentially valuable candidates in this effort because they could reduce the amount of toxic alkaline chemicals currently used. Using high throughput screening of complex environmental DNA libraries more than 40 novel microbial pectate lyases were discovered, and their enzymatic properties were characterized. Several candidate enzymes were found that possessed pH optima and specific activities on pectic material in cotton fibers compatible with their use in the scouring process. However, none exhibited the desired temperature characteristics. Therefore, a candidate enzyme was selected for evolution. Using Gene Site Saturation Mutagenesistrade mark technology, 36 single site mutants exhibiting improved thermotolerance were produced. A combinatorial library derived from the 12 best performing single site mutants was then generated by using Gene Reassemblytrade mark technology. Nineteen variants with further improved thermotolerance were produced. These variants were tested for both improved thermotolerance and performance in the bioscouring application. The best performing variant (CO14) contained eight mutations and had a melting temperature 16 degrees C higher than the wild type enzyme while retaining the same specific activity at 50 degrees C. Optimal temperature of the evolved enzyme was 70 degrees C, which is 20 degrees C higher than the wild type. Scouring results obtained with the evolved enzyme were significantly better than the results obtained with chemical scouring, making it possible to replace the conventional and environmentally harmful chemical scouring process.

Differential Pulse Voltammetry for Determination of Benorilate in Pharmaceutical Formulations at Carbon Paste Electrode

Benorilate was determined by the differential pulse voltammetry (DPV) using a carbon paste electrode in 1.25×10−3 mol·l−1 KH2PO4 and Na2HPO4 buffer solution (pH=6.88, 25°C). The anodic peak potential was +1.020 V (versus saturated calomel electrode (SCE)). A good linear relationship was realized between the anodic peak currents and benorilate concentrations in the range of 2.0×10−7 to 4.5×10−4 mol·l−1 with the detection limit of 5.0×10−8 mol·l−1. The recovery was 94.3 to 104.6% with the relative standard deviation of 3.5% (n=9). The pharmaceutical preparations, benorilate tablets samples, were determined with desirable results. This work was supported by the National Natural Science Foundation of China (No. 20175023, 20375034), and the Natural Science Foundation of Jiangsu Provincial Education Department (No. 02KJB150013).

Determination of Artemisinin in Artemisia annua L. by Reversed Phase HPLC

Artemisinin is a compound of current interest in the treatment of drug‐resistant malaria. An accurate, sensitive, and reproducible reversed‐phase HPLC with UV detection method is described for the determination of artemisinin in the herb of Artemisa annua L. Artemisinin in the extracts was converted into a UV‐absorbing compound, Q260 by being treated with 0.2% (by weight) NaOH solution and then 0.08 M acetic acid. A reversed‐phase C18 silica column (Discovery 250 × 4.6 mm, 5 µm) was selected as a fixed phase, and kept at 30°C. The mobile phase was 45/10/45 (by volume) methanol/acetonitrile/0.9' mM Na2HPO4‐3.6 mM NaH2PO4 buffer (pH 7.76). The flow rate was 0.5 mL/min. The detection wavelength was 260 nm. The standard calibration curve was linear with correlation coefficient 0.9996. The artemisinin content in the herb of Artemisa annua L. was 0.652%. The relative standard deviation and recovery were 1.1% and 98.9%, respectively.

Adsorption of Bovine Serum Albumin on Chromium and Molybdenum Surfaces Investigated by Fourier-Transform Infrared Reflection−Absorption Spectroscopy (FT-IRRAS) and X-ray Photoelectron Spectroscopy

The adsorption of bovine serum albumin (BSA) was investigated on two metal surfaces, chromium and molybdenum, in pure water and in a phosphate buffer solution (pH 7). Fourier-transform infrared reflection−absorption spectroscopy (FT-IRRAS) and X-ray photoelectron spectroscopy (XPS) were used to characterize the surfaces after immersion times ranging from 2 min to 1 h. In pure water, short times of immersion (a few minutes) resulted in higher BSA adsorption on Cr than on Mo; for longer times (20 min, 1 h), the amount of BSA was similar on both surfaces. Changes in the conformation of the BSA molecules when the coverage increases were evidenced by the position and shape of the IR amide band. A more compact structure was observed on chromium. On molybdenum, the amideI/amide II intensity ratio did not change significantly with time or BSA concentration, indicating limited change in the conformation upon adsorption. In a Na2HPO4 + NaH2PO4 buffer solution, both FT-IRRAS and XPS data show that the amount of prot...

Simultaneous determination of twelve tea catechins by high-performance liquid chromatography with electrochemical detection.

A high-performance liquid chromatographic method with electrochemical detection was developed for the determination of twelve tea catechins including four major catechins: epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG) and epigallocatechin gallate (EGCG); four of their epimers at the C-2 position, C, GC, CG and GCG; and four methylated catechin derivatives, epigallocatechin-3-O-(3-O-methyl)gallate, gallocatechin-3-O-(3-O-methyl)gallate, epigallocatechin-3-O-(4-O-methyl)gallate and epicatechin-3-O-(3-O-methyl)gallate. These catechins were separated on an ODS C18 reversed-phase column by isocratic elution with 0.1 M NaH2PO4 buffer (pH 2.5)-acetonitrile (87:13) containing 0.1 mM EDTA.2Na. The detection limits (S/N = 3) of these catechins were approximately 10-40 pmol ml-1 at an applied voltage of 600 mV. Extracting these catechins from tea leaf powder with H2O-acetonitrile (1:1) at 30 degrees C for 40 min inhibited the epimerization at C-2 significantly from these epicatechins compared to extraction with hot water at 90 degrees C. This analytical method is sensitive to and appropriate for the simultaneous determination of various biologically active catechins in green tea.

Direct determination of sialic acids in serum by capillary electrophoresis with UV detection

A novel method for the determination of N-acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) has been developed using high-performance capillary electrophoresis with UV detection at 195 nm, without pre or post-column derivatisation. The acids were separated in a 50-cm, fused-silica capillary (50μ i.d, 45.5-cm effective length) with Na2B4O7−Na2HPO4 buffer. The detection limit for NANA is a concentration of 9.6×10−6 M or, in terms of mass:3.879×10−14 mol (39 fmol). This method is applicable to determination of NANA in normal human serum. The results were also compared with those of the colorimetrie method.

N-Hydroxysuccinimidyl Fluorescein-O-acetate as a Fluorescent Derivatizing Reagent for Catecholamines in Liquid Chromatography

A new amine-reactive derivatizing reagent, N-hydroxysuccinimidyl fluorescein-O-acetate (SIFA), was developed for catecholamine (CA) analysis in liquid chromatography. The reactivity of this reagent with the CAs norepinephrine (NE), epinephrine (E), and dopamine (DA) was investigated in detail. In aqueous methanol containing 32 mmol/L pH 9.0 H3BO3–Na2B4O7 buffer, SIFA reacted with NE, E, and DA under mild conditions. The derivatives were separated in 20 min on a C18 column with a mobile phase of methanol/water (38:62, v/v) containing 10 mmol/L pH 5.0 H3cit–Na2HPO4 buffer. At λex/λem = 490/516 nm, the detection limits were 3.2, 12, and 56 fmol, respectively, with a signal-to-noise ratio of 3, which were comparable to those using 1,2-diphenylethylenediamine as the derivatizing reagent for CA analysis. Amino acids, aliphatic amines, and alcohols had no obvious interference with the determination. The proposed method has been applied to the determination of CAs in human urine, with recoveries of 95.3–103.9%.

Determination of Ethanol Based on Polarographic Catalytic Current of Sulfate Radical Anion Using Ethanol as Catalyst

ABSTRACT A new method for the determination of ethanol is proposed based on the catalytic current of sulfate radical anion SO4 −. In 0.1 mol/L KH2PO4–Na2HPO4 buffer (pH5.6±0.1)–1.6×10−2 mol/L K2S2O8 solution, the first order derivative peak current is proportional to ethanol concentration in the range of 1×10−4∼9×10−4 (r = 0.9998). The limit of detection is 6×10−5 mol/L. The proposed method is applied to determine ethanol directly content in ardent spirits. The fundamental principle of the proposed method is that ethanol is oxidized by sulfate radical anion SO4 − electrogenerated on the electrode surface to ethanol α-radical, and the reduction of the ethanol α-radical produces a polarographic reduction wave.

Immunoblotting of gliadins separated by acid PAGE: Analysis of electrotransference conditions

Several transference buffers and conditions (time and voltage) were tested to establish a suitable procedure for immunoblotting of gliadins after acid polyacrylamide gel electrophoresis (A‐PAGE). Ethanolic extracts of wheat flour were separated by A‐PAGE, transferred to nitrocellulose membranes and detected using an anti‐gliadin serum. The blot resolution was highly dependent on the buffer employed for electrotransference. The best results were obtained when a 10 mM‐H3PO4/NaH2PO4 buffer, pH 2.7, was employed. All gliadin fractions were transferred using this buffer. Sharp bands were obtained and good resolution was achieved.

Immunoaffinity purification of juvenile hormone-binding protein from Galleria mellonella hemolymph.

Previously described methods of purification of hemolymph juvenile hormone-binding protein (hJHBP) from Lepidoptera were tedious and required multiple steps. These methods resulted in low protein yield (Kramer et al., 1976; Goodman et al., 1978; Peterson et al., 1982; Park et al., 1993; Ozyhar & Kochman, 1987). In this report a simple method of purification of hJHBP from Galleria mellonella (L.) larvae is described. Monoclonal antibodies against hJHBP were obtained and crosslinked to CNBr-activated Sepharose 4B. The hemolymph of G. mellonella was centrifuged and then chromatographed on Sephadex G-200 gel filtration column. Juvenile-hormone-binding activity containing material from Sephadex G-200 column was subjected to purification on an immunoaffinity column. Bound protein was eluted from anti-hJHBP Sepharose 4B gel by lowering pH to 3.0 with 200 mM citric acid 200 mM Na2HPO4 buffer. This method resulted in 320-fold purification of G. mellonella hJHBP with 56% yield.

Improved method for quantification of the bacteriocin nisin

Nisin, a bacteriocin produced by Lactococcus lactis subsp. lactis, is used in some types of food preservation due to its inhibitory action on Gram-positive bacteria and their spores. A commonly used agar diffusion bioassay technique for quantification of nisin in food samples was modified to increase its sensitivity, accuracy and precision. Several variables were evaluated. Results showed Micrococcus luteus as the most sensitive organism tested, a lower agar concentration (0 x 75% compared 1 x 5%) increased the sensitivity of the assay (21% improvement over standard method), and incorporation of 1% Na2HPO4 buffer into the bioassay agar made it possible to prevent false inhibitory zones from developing due to the low pH of the test solutions. This resulted in a 57% improvement in accuracy and a 12% improvement in precision compared to the standard method.

A Helical Epitope in the C4 Domain of HIV Glycoprotein 120

The fourth conserved domain of the human immunodeficiency virus type 1 (HIV-1) envelope, the C4 region of glycoprotein 120 (gp120), is an amphipathic stretch of amino acids that, based on mutational analyses, constitutes a major component of the CD4 binding region in gp120. In the absence of crystallographic and NMR data on C4 in intact gp120, we sought to gain insight into C4's conformation and accessibility in gp120 by taking an immunochemical approach. In this study, a peptomer composed of a repeat peptide of C4 amino acids 419-436 from gp120 of HIV-1MN was synthesized for use as a conformationally constrained immunogen. Circular dichroism studies disclosed that the polymerized peptide, peptomer-(419-436), in 0.01 M Na2HPO4 buffer, pH 7.4, at 25 degrees C contained a dominant alpha-helical structure (53 +/- 1%) compared with 2 +/- 4% alpha-helical content for the monomeric peptide-(419-436). The peptomer in Ribi's adjuvant induced the production of rabbit antibodies that recognized recombinant and native gp120 but, consistent with the literature, the C4 peptide having no conformational constraints did not. The experimental results show that only those antibodies formed against a helical immunogen from C4 will recognize recombinant and native gp120, and, therefore, the results support the notion that C4 is an alpha-helix in gp120.