Selective Determination of Catechol in the Presence of Hydroquinone at Bare Indium Tin Oxide Electrodes via Peak‐Potential Separation and Redox Cycling by Hydrazine

The electrochemical, selective determination of catechol (CT) in the presence of hydroquinone (HQ) is not readily achieved, because the formal potentials of two phenolic compounds are very close. Here, we have developed a simple electrochemical method for the selective determination of CT by using bare indium tin oxide electrodes and employing CT redox cycling by hydrazine. The cyclic voltammetry of CT and HQ was investigated in Tris buffer (pH 9.0), phosphate buffered saline buffer (pH 7.4), and acetate buffer (pH 4.5). Especially in Tris buffer, the anodic peak potential of CT is much lower than that of HQ, resulting in a large difference between two peak potentials (ca. 0.4 V). The difference allows the selective determination of CT in the presence of excess HQ. The anodic current of CT is amplified using CT redox cycling by hydrazine, which also helps to stabilize CT and HQ in Tris buffer for several hours. The detection limits of CT in Tris buffer containing 0.1 mM HQ are 1 μM and 10 μM in the presence and absence of hydrazine, respectively.     

A metallic cobalt electrode for the indirect potentiometric determination of calcium and magnesium in natural waters using flow injection analysis

A flow injection analysis of Ca(2+) and Mg(2+) using indirect potentiometric detection in natural waters is proposed, where Ca(2+) or Mg(2+) are injected into a buffer carrier containing phosphate, resulting in the formation of Ca(3)(PO(4))(2) or Mg(3)(PO(4))(2). The consequent reduction in free phosphate in the carrier solution is detected using a metallic cobalt wire electrode. Indirect electrode response was used and the experimental conditions affecting electrode response were optimized. Responses were linear in the concentration range 5x10(-4) to 5x10(-3) M with a detection limit of 1x10(-5) M in 20 mM phosphate buffer at pH 8.0. The relative standard derivation at 1 mM of Ca(2+) and Mg(2+) were 3.9 and 3.7% (n=10), respectively. EGTA and 8-hydroxyquinoline were used as the masking agents for Ca(2+) and Mg(2+), respectively. Concentrations of Ca(2+) and Mg(2+) in natural waters were successfully determined by the proposed method.     

Cation-dependent phosphatase activites in a rat pancreatic islet plasma membrane fraction prepared by one-step gradient centrifugation.

A plasma membrane-enriched fraction was prepared from homogenized rat pancreatic islets by a one-step sucrose gradient centrifugation. Using 125I-wheat germ agglutinin as a plasma membrane probe, a fraction was obtained at a sucrose density of about 1.10 that was enriched in 5'-nucleotidase, Mg2+-ATPase and alkaline phosphatase. The fraction contained little, if any, monoamino oxidase activity, insulin or DNA. Hydrolysis of 3-0-methyl-fluoresceinphosphate was stimulated by K+ (10mM) at a pH optimum of pH 8.2. Hydrolysis of ATP-gamma-32P in the presence of MgCl2 was of high specific activity and was optimum at pH 7.0 and 8.2. K+ did not affect ATP-hydrolysis. At pH 8.2, a small fraction of the total Mg2+-ATPase activity was inhibited by ouabain in the presence of Na+ and K+. Since K+-stimulated phosphatase activity does not correlate with Mg2+-ATPase, the two assay systems define separate enzymatic processes.     

Phospholipid-lysozyme coating for chiral separation in capillary electrophoresis

A phospholipid coating with lysozyme as chiral recognition reagent permeated into the phospholipid membrane was developed for the chiral capillary electrophoretic (CE) separation of D- and L-tryptophan. As a kind of carriers, coated as phospholipid membranes onto the inner wall of a fused-silica capillary, liposomes are able to interact with basic proteins such as lysozyme, which may reside on the surface of the phospholipid membrane or permeate into the middle of the membrane. The interaction results in strong immobilization of lysozyme in the capillary. Coatings prepared with liposomes alone did not allow stable immobilization of lysozyme into the phospholipid membranes, as seen from the poor repeatability of the chiral separation. When 1-(4-iodobutyl)-1,4-dimethylpiperazin-1-ium iodide (M1C4) was applied as a first coating layer in the capillary, the electroosmotic flow (EOF) was effectively suppressed, the phospholipid coating was stabilized, and the lysozyme immobilization was much improved. The liposome composition, the running buffer, and the capillary inner diameter all affected the chiral separation of D- and L-tryptophan. Coating with 4 mM M1C4 and then 1 mM phosphatidylcholine (PC)/phosphatidylserine (PS) (80:20 mol%), with 20 mM (ionic strength) Tris at pH 7.4 as the running buffer, resulted in optimal chiral separation with good separation efficiency and resolution. Since lysozyme was strongly permeated into the membrane of the phospholipids on the capillary surface, the chiral separation of D- and L-tryptophan was achieved without lysozyme in the running buffer. The effects of different coating procedures and separation conditions on separation were evaluated, and the M1C4-liposome and liposome-lysozyme interactions were elucidated. The usefulness of protein immobilized into phospholipid membranes as a chiral selector in CE is demonstrated for the first time.     

Improved method for the simultaneous determination of whey proteins, caseins and para-kappa-casein in milk and dairy products by capillary electrophoresis

A capillary electrophoresis method for the simultaneous determination of whey proteins, caseins and their degradation products, such as para-kappa-casein, was proposed. The effect of several parameters (pH, ionic strength and concentration of urea in the electrophoresis buffer and applied voltage) on the analysis time and on the separation efficiency of the major milk proteins was studied. Using a hydrophilically coated capillary, in combination with electrophoresis buffer 0.48 M citric acid-13.6 mM citrate-4.8 M urea at pH 2.3, and a separation voltage of 25 kV, a complete separation of beta-lactoglobulin and para-kappa-casein was achieved, permitting the quantification of both components.     

Properties of (Mg2 + Ca2+)-ATPase of erythrocyte membranes prepared by different procedures: influence of Mg2+, Ca2+, ATP, and protein activator.

Erythrocyte membranes prepared by three different procedures showed (Mg2+ + Ca2+)-ATPase activities differing in specific activity and in affinity for Ca2+. The (Mg2+ + Ca2+)-ATPase activity of the three preparations was stimulated to different extents by a Ca2+-dependent protein activator isolated from hemolysates. The Ca2+ affinity of the two most active preparations was decreased as the ATP concentration in the assay medium was increased. Lowering the ATP concentration from 2 mM to 2-200 microM or lowering the Mg:ATP ratio to less than one shifted the (Mg2+ + Ca2+)-ATPase activity in stepwise hemolysis membranes from mixed "high" and "low" affinity to a single high Ca2+ affinity. Membranes from which soluble proteins were extracted by EDTA (0.1 mM) in low ionic strength, or membranes prepared by the EDTA (1-10 mM) procedure, did not undergo the shift in the Ca2+ affinity with changes in ATP and MgCl2 concentrations. The EDTA-wash membranes were only weakly activated by the protein activator. It is suggested that the differences in properties of the (Mg2+ + Ca2+)-ATPase prepared by these three procedures reflect differences determined in part by the degree of association of the membrane with a soluble protein activator and changes in the state of the enzyme to a less activatable form.     

Simultaneous electrophoretic analysis of proteins of very high and low molecular mass using Tris‐acetate polyacrylamide gels

To separate and analyze giant and small proteins in the same electrophoresis gel, we have used a 3–15% polyacrylamide gradient gel containing 2.6% of the crosslinker bisacrylamide and 0.2 M of Tris‐acetate buffer (pH 7.0). Samples were prepared in a sample buffer containing lithium dodecyl sulphate and were run in the gel described above using Tris‐Tricine‐SDS‐sodium bisulfite buffer, pH 8.2, as electrophoresis buffer. Here, we show that this system can be successfully used for general applications of SDS‐PAGE such as CBB staining and immunoblot. Thus, by using Tris‐acetate 3–15% polyacrylamide gels, it is possible to simultaneously analyze proteins, in the mass range of 10–500 kDa, such as HERC1 (532 kDa), HERC2 (528 kDa), mTOR (289 kDa), Clathrin heavy chain (192 kDa), RSK (90 kDa), S6K (70 kDa), β‐actin (42 kDa), Ran (24 kDa) and LC3 (18 kDa). This system is highly sensitive since it allows detection from as low as 10 μg of total protein per lane. Moreover, it has a good resolution, low cost, high reproducibility and allows for analysis of proteins in a wide range of weights within a short period of time. All these features together with the use of a standard electrophoresis apparatus make the Tris‐acetate‐PAGE system a very helpful tool for protein analysis.     

1,3-dialkylimidazolium-based room-temperature ionic liquids as background electrolyte and coating material in aqueous capillary electrophoresis

The 1-ethyl-3-methylimidazolium (EMIM) cation was found to have constant mobility of 4.5 x 10(-4) cm2 V(-1) s(-1) over the pH range of 3 to 11. The electroosmotic flow of bare silica capillary was reversed by the covalently bonded room-temperature ionic liquid (RTIL) coating. With run buffer of 5 mM EMIM (pH 8.5), NH4+ in human urine was separated from the K+ matrix and was detected to be 0.37 +/- 0.012%. K+, Na+, Li+, Ca2+, Mg2+ and Ba2+ were baseline separated in RTIL-coated capillary with run buffer of 10 mM EMIMOH-acetic acid at pH 5, and the concentration of the above ions in a red wine were detected to be 907, 27.9, 0, 71.0, 83.4 and 31.1 microg/ml, respectively. The RTIL-coated capillary showed stable electroosmotic flow for at least 80 h in the run buffer.     

Size exclusion chromatography coupled to electrospray ionization mass spectrometry for analysis and quantitative characterization of arsenic interactions with peptides and proteins

Arsenic‐binding proteins are of toxicological importance since enzymatic activities can be blocked by arsenic interactions. In the present work, a novel methodology based on size exclusion chromatography coupled to electrospray ionization mass spectrometry (SEC‐ESI‐MS) was developed with special emphasis to preserve the intact proteins and their arsenic bindings. The eluent composition of 25 mM Tris/HCl, pH 7.5, with the addition of 100‐mM NaCl optimized for SEC with UV detection provided the highest SEC separation efficiency, but was not compatible with the ESI‐MS because of the non‐volatility of the buffer substance and of the salt additive. In order to find the best compromise between chromatographic separation and ionization of the arsenic‐binding proteins, buffer type and concentration, pH value, portion of organic solvent in the SEC eluent as well as the flow rate were varied. In the optimized procedure five different arsenic‐binding peptides and proteins (glutathione, oxytocin, aprotinin, α‐lactalbumin, thioredoxin) covering a molar mass range of 0.3–14 kDa could be analyzed using 75% 10‐mM ammonium formate, pH 5.0/25% acetonitrile (v : v) as eluent and a turbo ion spray source operated at 300 °C and 5.5 kV. A complete differentiation of all peptides and proteins involved in the arsenic‐binding studies as well as of their arsenic‐bound forms has become feasible by means of the extracted ion chromatograms (XIC) of the mass spectrometric detection. The new method offered the possibility to estimate equilibrium constants for the reaction of phenylarsine oxide with different thiol‐containing biomolecules by means of the XIC peak areas of reactants and products. Limits of detection in the range of 2–10 µM were obtained by SEC‐ESI‐MS for the individual proteins. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous determination of digoxin and digitoxin by micellar electrokinetic chromatography and application to drug formulations

A simple micellar electrokinetic chromatographic method is described for simultaneous determination of digoxin and digitoxin. The simultaneous analysis of digoxin and digitoxin was performed in Tris buffer (10 mM; pH 9) with 90 mM sodium dodecyl sulfate and 10% isopropyl alcohol as an anionic surfactant and organic modifier. Under these conditions, good separation with high efficiency is achieved in short analysis times. Several parameters affecting the separation of the drugs were studied, including the pH and concentrations of the Tris buffer and sodium dodecyl sulfate. The linear range of the method for the determination of digoxin and digitoxin was over 0.01–0.3 mg/mL; the detection limit (signal to noise ratio = 3; injection 3.5 kPa 3 s) was 4 and 6 μg/mL, respectively. Application of the proposed method to the determination of digoxin in commercial tablets and in injections proved to be feasible.     

Application of the extended solvation model for thermodynamic study of copper ion binding to Jack bean urease

A Thermodynamic study on the interaction Jack bean urease, JBU, with Cu²⁺ ion was studied by isothermal titration calorimetry (ITC) at 300 and 310 K in 30 mM Tris buffer solution, pH 7.0. The heats of JBU + Cu²⁺ interactions are reported and analyzed in terms of the extended solvation theory. It was indicated that there are a set of 12 identical and non-cooperative sites for Cu²⁺ ion. The binding of Cu²⁺ ion with JBU is exothermic with dissociation equilibrium constants of 284.883 and 345.855 μM at 300 and 310 K, respectively.     

DNA and DNAzyme-mediated 2D colloidal assembly

DNA-mediated interactions present a significant opportunity for controlling colloidal self-assembly. Using microcontact printing to achieve spatial control of DNA-surface patterning and DNA-functionalized polystyrene colloids, we report that DNA hybridization can be utilized for sequence-specific reversible self-assembly of well-ordered 2D colloidal arrays. Two essential indicators of DNA-hybridization mediated assembly were confirmed: thermal reversibility and sequence specificity. The arrays melted at 50 degrees C and reassembled when introduced to fresh colloid suspension, and sequence specificity with <1% nonspecific binding was confirmed using fluorescent polystyrene colloids. The real-time assembly of the colloids onto the periodically patterned substrate was monitored by simple laser diffraction to obtain assembly kinetics. Maximum surface coverage of DNA-mediated assembly was determined to be 0.593 for DNA-functionalized 100 nm polystyrene colloids, and 90% of the assembly was complete after 6.25 h of hybridization in 50 mM NaCl Tris buffer. We also demonstrate that DNAzymes, catalytic DNA molecules, can be incorporated into the design, and in the presence of 10 microM Pb(2+), the hybridization-induced array assembly can be disrupted via DNAzyme activity.     

Separate metal requirements for loop interactions and catalysis in the extended hammerhead ribozyme

An extended hammerhead ribozyme derived from Schistosoma mansoni, including conserved loops in stems I and II, has been examined to directly monitor the relationship between docking of loops and its activity using site-directed spin labeling (SDSL) and EPR spectroscopy. Dynamics with EPR spectroscopy and fast-quench kinetics measurements have shown that the docking of stems I and II occurs at low Mg2+ concentrations ([Mg2+]1/2,dock = 0.7 mM, 0.1 M NaCl), but a much weaker Mg2+ interaction ([Mg2+]1/2,cat approximately 90 mM) increases activity to very high maximum rates of approximately 1 s-1 at 0.1 M Na+ and pH 7.0.     

NMR evidence for Mg(II) binding to N1 of ATP

The conformation of the complex of [ATP-Mg]2+ is studied by 1H, 15N and 31P NMR on ATP in the absence and presence of MgCl2 in a wide pH range from 1 to 10. 1H-15N HMBC experiments show a large change in the 15N chemical shift of N1 up to 10 ppm around pH 3.7, suggesting that there is a strong interaction between Mg2+ and N1 of ATP at this pH. 31P NMR indicates that at pH 3.7 the phosphate chain also binds Mg2+. 1H diffusion measurements imply that the [ATP-Mg]2+ complex involves only one ligand and one metal ion.     

Purification of clathrin assembly protein from rat liver

Recently, the gene encoding clathrin assembly protein of lymphoid myeloid leukemia (CALM), which is homologous to the AP180, was cloned from rat brain, and its expression differential to AP180 was reported (Kim and Lee, 1999). This gene product promotes the polymerization of clathrin into clathrin cage and found to be a regulator in membrane trafficking between intracellular compartments in eukaryotic cells (Kim et al., 2000). In this study, we have purified the CALM protein from clathrin-coated vesicles of rat liver using the monoclonal antibody against the recombinant N-terminal region of the CALM. The coated proteins extracted from the coated vesicle fraction was further purified by multi-step procedures involving gel-filtration and ion-exchange chromatography and SDS-PAGE. The purified protein with an apparent molecular weight of 100 kD promoted the assembly of clathrin triskelia into clathrin cage. In this respect the CALM protein bears a functional resemblance to the AP180 that has been previously described.     

Adsorption of alpha-chymotrypsin onto mica in laminar flow conditions. Adsorption kinetic constant as a function of tris buffer concentration at pH 8.6

We examined the adsorption kinetics of alpha-chymotrypsin (pH 8.6, 10(-2) to 0.5 M Tris buffer) on muscovite mica in conditions of laminar flow through a slit. The range of buffer concentrations is between two limits: (i) no adsorption in 1 M Tris and (ii) no desorption in 10(-3) M Tris. Studying the dependence of adsorption kinetics on the wall shear rate leads to the determination of the interfacial adsorption kinetic constant ka and the diffusion coefficient. The obtained value for the diffusion coefficient is close to the one expected from the molecular size of alpha-chymotrypsin. The interfacial adsorption kinetic constant of alpha-chymotrypsin decreases when ionic strength increases, while the initial desorption constant (over a part of all the adsorbed population) shows the contrary. Although alpha-chymotrypsin is almost at its isoelectric point, the effect of ionic strength on the adsorption kinetics suggests the importance of electrostatic interactions between the protein and mica. We observed an increase in the adsorption rate, at a surface coverage near 0.14 microg cm(-2), for adsorption in 10(-2) M Tris and the low wall shear rates (<300 s(-1)). This change in the adsorption rate suggests a structural transition, that we assume again to be due to electrostatic interactions, but between proteins. The large dipole moment of the protein may induce this transition, illustrated here by the ferroelectric/antiferroelectric pattern. The variation of the zeta potential with interfacial concentration seems to be in agreement with such a model.     

Isolation of membrane vesicles with inverted topology by osmotic lysis of Azotobacter vinelandii spheroplasts.

Membrane vesicles were prepared from Azotobacter vinelandii spheroplasts by lysis in either potassium phosphate (pH 7.0) or Tris1-acetate (pH 7.8) buffers. These 2 types of preparations differ considerably in their properties: 1) Examination by scanning electron microscopy reveals that the Pi vesicles consist primarily of closed structures 0.6-0.8 micrometer in diameter with a rough or particulate surface similar to that of spheroplasts. The Tris vesicles are significantly smaller, 0.1-0.3 micrometer in diameter, and have a much smoother surface structure. 2) Antisera from rabbits immunized with A. vinelandii lipopolysaccharide antigen will agglutinate Pi vesicles but not Tris vesicles. 3) Tris vesicles have a fourfold higher specific activity of latent H+-ATPase than Pi vesicles. After exposure to Triton X-100 similar ATPase activities are observed for both types of vesicles. 4) Pi vesicles transport calcium in the presence of ATP or lactate at less than 30% of the rats observed for Tris vesicles. 5) Tris vesicles have less than 22% of the transport capacity of Pi vesicles for accumulation of labeled sucrose and less than 3% of the capacity for valinomycin-induced uptake of rubidium observed during respiration. 6) Quinacrine fluorescence intensity is reduced by 30% during lactate oxidation and 20% during ATP hydrolysis by Tris vesicles. Under similar conditions, fluorescence in Pi vesicles is quenched by only 7% and less than 2%, respectively. These findings suggest that Pi vesicles have the normal orientation of the intact cell whereas Tris vesicles have an inverted topology.     

Application of a multivariate approach for analyte focusing by micelle collapse‐micellar electrokinetic chromatography for analyzing sunscreen agents in cosmetics

The operating parameters that affect the performance of the online preconcentration technique “analyte focusing by micelle collapse‐MEKC (AFMC‐MEKC)” were examined using a multivariate approach involving experimental design to determine the sunscreen agents in cosmetics. Compared to the single‐variable approach, the advantage of the multivariate approach was that many factors could be investigated simultaneously to obtain the best separation condition. A fractional factorial design was used to identify the fewest significant factors in the central composite design (cCD). The cCD was adopted for evaluating the location of the minimum or maximum response in this study. The influences of the experimental variables on the response were investigated by applying a chromatographic exponential function. The optimized condition and the relationship between the experimental variables were acquired using the JMP software. The ANOVA analysis indicated that the Tris pH value, SDS concentration, and ethanol percentage influenced the separation quality and significantly contributed to the model. The optimized condition of the running buffer was 10 mM Tris buffer (pH 9.5) containing 60 mM SDS, 7 mM γ‐CD, and 20% v/v ethanol. The sample was prepared in 100 mM Tris buffer (pH 9.0) containing 7.5 mM SDS and 20% v/v ethanol. The SDS concentration in the sample matrix was slightly greater than the CMC value that makes the micelle be easily collapsed and the analytes be accumulated in the capillary. In addition, sunscreen agents in cosmetics after 1000‐fold dilution were successfully determined by AFMC‐MEKC.     

Evaluation of the arsenic binding capacity of plant proteins under conditions of protein extraction for gel electrophoretic analysis

As prerequisite for the investigation of arsenic-binding proteins in plants, the general influence of different extraction parameters on the binding behaviour of arsenic to the plant protein pool was investigated. The concentration of the extraction buffer affected the extraction yield both for proteins and for arsenic revealing an optimal buffer concentration of 5 mM Tris/HCl, pH 8. The addition of 1 or 2% (w/v) SDS to the extraction buffer produced a two- to threefold enhancement of the total protein extraction yield but strongly suppressed the simultaneous extraction of arsenic from 80 ± 8% extraction yield obtained without SDS to 48 ± 2% in presence of 2% (w/v) SDS. The arsenic binding capacity of the protein fraction obtained after extraction with Tris buffer and protein precipitation by trichloroacetic acid in acetone was estimated to be 1.4 ± 0.6% independently on the original spiking concentration of arsenic provided in the form of monomethylarsonate to the extracts. Due to the low total protein concentrations of the plant extracts that varied in the range from 75 to 412 μg mL⁻¹ depending on the extraction parameters, high arsenic concentrations of 263-1001 mg (kg protein mass)⁻¹ resulted for spiking concentrations of 10 mg As L⁻¹. The optimized protein isolation procedure was applied to plants grown under arsenic exposure and revealed a similar arsenic binding capacity as for the spiked protein extracts.     

Dynamic modification of the capillary wall for electrophoretic separations of small ions

Separations of small ions were carried out under nonequilibrated conditions using capillaries treated with NaOH, HCl, or tris(hydroxymethyl)aminomethane (Tris) prior to analysis. For separations of benzoic acid isomers or acids and amines under weakly acidic conditions, capillaries flushed with 0.1 M NaOH and subsequently with running buffers prior to analysis were used. Separations of six benzoic acid isomers were accomplished in 4 min in 1 mM phosphate buffers, pH 4.01, containing 2.5 mM hydroxypropyl-beta-cyclodextrin. Without additives, the separation of biological amines and acids were also achieved in 10 min at pH 4.01. Capillaries treated with 0.1 M HCl prior to analysis were tested in separations of six phenols in 5 mM Tris solutions at pH 7.0. As a result of small electrophoretic mobilities of phenols against a small electroosmotic flow, resolution was optimized. We also found that reproducibility was improved using capillaries treated with HCl. The relative standard deviations of migration mobility of phenols were less than 1%, which were smaller than those obtained using capillaries treated with 0.1 M NaOH or Tris.