Study of enzymatic reaction by electrophoretically mediated microanalysis in a partially filled capillary with indirect or direct detection

Electrophoretically mediated microanalysis (EMMA), in combination with a partial filling technique and indirect or direct detection, is described for the study of enzymes reacting with the high mobility inorganic or organic anions as substrates or products. Part of the capillary is filled with a buffer optimized for the enzymatic reaction, the rest of the capillary with the background electrolyte being optimal for the separation of substrates and products. With haloalkane dehalogenase, chosen as a model enzyme, the enzymatic reaction was performed in a 20 mM glycine buffer (pH 8.6). Because of the wide substrate specificity of this enzyme, utilizing chlorinated as well as brominated substrates and producing either nonabsorbing chloride or absorbing bromide ions, two different background electrolytes and detection approaches were adopted. A 10 mM chromate-0.1 mM cetyltrimethylammonium bromide background electrolyte (pH 9.2) was used in combination with indirect detection and 20 mM beta-alanine-hydrochloric acid (pH 3.5) in combination with direct detection. The Michaelis constant (K(m)) of haloalkane dehalogenase for 1-bromobutane was determined. The K(m) values 0.59 mM estimated by means of indirect detection method and 0.17 mM by means of direct detection method were comparable with the value 0.13 mM estimated previously by gas chromatography.     

Study of substrate inhibition by electrophoretically mediated microanalysis in partially filled capillary

Substrate inhibition is a common phenomenon in enzyme kinetics. We report here for the first time its study by a combination of the electrophoretically mediated microanalysis (EMMA) methodology with a partial filling technique. In this setup, the part of capillary is filled with the buffer best for the enzymatic reaction whereas, the rest of the capillary is filled with the background electrolyte optimal for separation of substrates and products. In the case of haloalkane dehalogenase, a model enzyme selected for this study, the enzymatic reaction was performed in 20 mM glycine buffer (pH 8.6) whereas 20 mM beta-alanine-hydrochloric acid buffer (pH 3.5) was used as a background electrolyte in combination with direct detection at 200 nm. The whole study was performed on poorly soluble brominated substrate--1,2-dibromoethane. As a result it was first necessary to find the compromise between the concentrations of the enzyme and the substrate preserving both the adequate sensitivity of the assay and at the same time the attainable substrate solubility. By means of the developed EMMA methodology we were able to determine the Michaelis constant (K(M)) as well as the substrate inhibition constant (K(SI)). The value of K(M) and K(SI) obtained were 7.7+/-2.5 mM and 1.1+/-0.4 mM, respectively. Observation of the substrate inhibition of haloalkane dehalogenase by 1,2-dibromoethane is in accordance with previous literature data.     

Development of off-line and on-line capillary electrophoresis methods for the screening and characterization of adenosine kinase inhibitors and substrates

Fast and convenient CE assays were developed for the screening of adenosine kinase (AK) inhibitors and substrates. In the first method, the enzymatic reaction was performed in a test tube and the samples were subsequently injected into the capillary by pressure and detected by their UV absorbance at 260 nm. An MEKC method using borate buffer (pH 9.5) containing 100 mM SDS (method A) was suitable for separating alternative substrates (nucleosides). For the CE determination of AMP formed as a product of the AK reaction, a phosphate buffer (pH 7.5 or 8.5) was used and a constant current (95 microA) was applied (method B). The methods employing a fused-silica capillary and normal polarity mode provided good resolution of substrates and products of the enzymatic reaction and a short analysis time of less than 10 min. To further optimize and miniaturize the AK assays, the enzymatic reaction was performed directly in the capillary, prior to separation and quantitation of the product employing electrophoretically mediated microanalysis (EMMA, method C). After hydrodynamic injection of a plug of reaction buffer (20 mM Tris-HCl, 0.2 mM MgCl2, pH 7.4), followed by a plug containing the enzyme, and subsequent injection of a plug of reaction buffer containing 1 mM ATP, 100 microM adenosine, and 20 microM UMP as an internal standard (I.S.), as well as various concentrations of an inhibitor, the reaction was initiated by the application of 5 kV separation voltage (negative polarity) for 0.20 min to let the plugs interpenetrate. The voltage was turned off for 5 min (zero-potential amplification) and again turned on at a constant current of -60 microA to elute the products within 7 min. The method employing a polyacrylamide-coated capillary of 20 cm effective length and reverse polarity mode provided good resolution of substrates and products. Dose-response curves and calculated K(i) values for standard antagonists obtained by CE were in excellent agreement with data obtained by the standard radioactive assay.     

Electrophoretically mediated microanalysis with partial filling technique and indirect or direct detection as a tool for inhibition studies of enzymatic reaction

The inhibition of the model enzyme, haloalkane dehalogenase from Sphingomonas paucimobilis, was investigated by a combination of electrophoretically mediated microanalysis with a partial filling technique, followed by indirect or direct detection. In this setup, part of the capillary is filled with a buffer suitable for the enzymatic reaction (20 mM glycine buffer, pH 8.6) whereas the rest of the capillary is filled with the background electrolyte optimal for separation of substrates and products. Two different background electrolytes and corresponding detection approaches were used to show the versatility of the developed method. The inhibition effect of 1,2-dichloroethane on the dehalogenation of brominated substrate 1-bromobutane was studied by means of 10 mM chromate - 0.1 mM cetyltrimethylammonium bromide (pH 9.2) in combination with indirect detection or 20 mM beta-alanine - hydrochloric acid (pH 3.5) in combination with direct detection. The method was used to estimate the inhibition constant K(I) (0.44 mM by indirect detection and 0.63 mM by of direct detection) and to determine the inhibition type. Compared to spectrophotometric and other discontinuous assays, the method is rapid, can be automated, and requires only small amount of reagents that is especially important in the case of enzymes and inhibitors.     

Determination of organic peroxides by liquid chromatography with on-line post-column ultraviolet irradiation and peroxyoxalate chemiluminescence detection

A combination of the electrophoretically mediated microanalysis methodology with a partial filling technique was applied for the inhibition study of bovine liver rhodanese by 2-oxoglutarate. In this set-up, part of the capillary is filled with the best buffer for the enzymatic reaction, while the rest of the capillary is filled with the optimal background electrolyte for separation of substrates and products. The estimated value of K_I for 2-oxoglutarate was 3.62·10⁻⁴±1.43·10⁻⁴ M with respect to cyanide and 1.40·10⁻³±1.60·10⁻⁴ M with respect to thiosulfate. In addition, the type of inhibition was also evaluated. The findings of 2-oxoglutarate as the competitive inhibitor with respect to cyanide and as the uncompetitive inhibitor with respect to thiosulfate are in accordance with previous literature data.     

Michaelis-Menten analysis of bovine plasma amine oxidase by capillary electrophoresis using electrophoretically mediated microanalysis in a partially filled capillary

A method for determining bovine plasma amine oxidase (PAO; EC 1.4.3.6) activity with benzylamine (Bz) as substrate is described. Electrophoretically mediated microanalysis (EMMA) combined with micellar electrokinetic capillary chromatography (MEKC) was used to perform an on-capillary enzymatic reaction and to separate the generated benzaldehyde from the other reaction products. The capillary was only partially filled with the separation solution, since the enzyme was unstable in the presence of the applied surfactant. The initial reaction velocity of the enzyme-catalyzed reaction was estimated from the peak area of the enzyme product, benzaldehyde. An amplification step was introduced by means of an on-capillary incubation of 15 min, in order to accumulate enough reaction product to detect spectrophotometrically at 254 nm. This set-up resulted in a fully automated assay, which can be carried out in less then 35 min. Using the Lineweaver-Burk equation, an average Michaelis constant (K(M)) for PAO was calculated to be 0.74 mM +/- 0.05 mM, which is consistent with previously reported values.     

Development of electrophoretically mediated microanalysis method for the kinetics study of flavin-containing monooxygenase in a partially filled capillary

An electrophoretically mediated microanalysis method with a partial filling technique was developed for flavin-containing monooxygenase, form 3 (FMO3). The in-line enzymatic reaction was performed in 100 mM phosphate reaction buffer (pH 7.4) whereas 150 mM phosphate buffer (pH 3.3) was used as a background electrolyte. A long plug of cofactor NADPH dissolved in reaction buffer was hydrodynamically injected into a fused-silica capillary, followed by enzyme and substrate solution. The reaction was initiated at 37 degrees C in the thermostated part of the cartridge by the application of 9 kV for 0.9 min. The voltage was turned off to increase the product amount (zero-potential amplification) and again turned on at a constant voltage of 10 kV to elute all the components. Direct detection was performed at 191 nm. The developed electrophoretically mediated microanalysis method was applied for the kinetics study of FMO3 using clozapine as a substrate probe. A Michaelis-Menten constant (K(m)) of 410.3 microM was estimated from the corrected peak area of the product, clozapine N-oxide. The calculated value of the maximum reaction velocity (V(max)) was found to be 1.86 nmol/nmol enzyme/min. The acquired FMO3 kinetic parameters are in accordance with the published literature data.     

Electrophoretically mediated microanalysis assay for sirtuin enzymes

An electrophoretically mediated microanalysis (EMMA) assay for the human sirtuin SIRT1 has been developed using 9-fluorenylmethoxycarbonyl (Fmoc)-labeled peptides, i.e. Fmoc-KK(Ac)-NH(2), Fmoc-KK(Ac)L-NH(2) and Fmoc-RHKK(Ac)-NH(2), as substrates. The partial filling mode was applied due to the incompatibility between the incubation buffer, pH 8.0, and the BGE that had a pH of 2.7 or 2.3 depending on the analytes. Incubation and subsequent analyte separation were carried out in a 37/30 cm, 50 μm id fused-silica capillary at 37°C. An injection sequence of incubation buffer, enzyme, substrate, enzyme and incubation buffer was selected because the electrophoretic mobility of SIRT1 was not known. The assay was optimized with regard to the length of the injected plugs, the mixing voltage and mixing time as well as the activity (concentration) of SIRT1. The EMMA assay was subsequently applied to the determination of the Michaelis-Menten constants, K(m), and the maximum velocity, V(max), as well as the determination of the inhibitory constants, IC(50), of inhibitors. Data obtained with the in-capillary assay were in accordance with the literature data or an offline SIRT1 assay.     

Kinetic study of gamma-glutamyltransferase activity by electrophoretically mediated microanalysis combined with micellar electrokinetic capillary chromatography

The use of capillary electrophoresis for the determination of gamma-glutamyltransferase (GGT) activity with gamma-glutamyl-p-nitroanilide (Glu-p-NA) as a substrate was investigated. The reaction velocity was quantified spectrophotometrically by the corrected peak area of the product p-nitroaniline (pNA) at 380 nm. Micelles composed of sodium deoxycholic acid were used in the background electrolyte in order to obtain a baseline separation between the substrate and the product. The presence of the micelles did not influence the enzymatic reaction. The electrophoretic system was used, not only for the separation and quantitation of the different reaction compounds but also for the in-capillary mixing of the enzyme and substrate plugs. This methodology is known as electrophoretically mediated microanalysis (EMMA). With the developed in-capillary activity assay an average Michaelis constant (K(M)) for GGT was calculated to be 2.09 mM (RSD = 7.3%, n = 3), a value consistent with previously reported values.     

Enantiomeric separation of citalopram and its metabolites by capillary electrophoresis

A simple and fast capillary electrophoretic method has been developed for the enantioselective separation of citalopram and its main metabolites, namely N-desmethylcitalopram and N,N-didesmethylcitalopram, using beta-cyclodextrin (beta-CD) sulfate as the chiral selector. For method optimisation several parameters were investigated, such as CD and buffer concentration, buffer pH, and capillary temperature. Baseline enantioseparation of the racemic compounds was achieved in less than 6 min using a fused-silica capillary, filled with a background electrolyte consisting of a 35 mM phosphate buffer at pH 2.5 supplemented with 1% w/v beta-CD sulfate and 0.05% w/v beta-CD at 25 degrees C and applying a voltage of -20 kV. A fast separation method for citalopram was also optimized and applied to the analysis of pharmaceutical formulations. Racemic citalopram was resolved in its enantiomers in less than 1.5 min using short-end injection (8.5 cm, effective length) running the experiments in a background electrolyte composed of a 25 mM citrate buffer at pH 5.5 and 0.04% w/v beta-CD sulfate at a temperature of 10 degrees C.     

Determination of haloalkane dehalogenase activity by capillary zone electrophoresis

A new sensitive method has been developed for the determination of haloalkane dehalogenase activity. The enzymatic reactions were carried out directly in thermostatted autosampler vials and the formation of product - bromide or chloride ions - was monitored by sequential capillary zone electrophoresis runs. The determinations were performed in a 75 microm fused-silica capillary using 5 mM chromate, 0.5 mM tetradecyltrimethylammonium bromide (pH 8.4) as a background electrolyte, separation voltage 15 kV (negative polarity) and indirect detection at sample wavelength 315 nm, reference wavelength 375 nm for brominated and chlorinated substrates, respectively 0.1 M beta-alanine-HCl (pH 3.50) as a background electrolyte, separation voltage 18 kV (negative polarity) and direct detection at 200 nm for brominated substrates. The temperature of capillary was in both cases 25 degrees C. The method is rapid, can be automated, and requires only small amount of enzyme preparation and substrate.     

Determination of iodide in samples with complex matrices by hyphenation of capillary isotachophoresis and zone electrophoresis

A method has been developed for the determination of iodide in mineral water, seawater, cooking salt, serum, and urine based on hyphenation of capillary ITP and zone electrophoresis. A commercially available instrumentation for capillary ITP with column-switching system was used. ITP served for removal of chloride present in the analyzed samples in a ratio of 10(6)-10(7):1 to iodide, zone electrophoresis was used for evaluation. Isotachophoretic separation proceeded in a capillary made of fluorinated ethylene-propylene copolymer of 0.8 mm id and 90 mm total length to the bifurcation point filled with a leading electrolyte (LE) composed of 8 mM HCl + 16 mM beta-alanine (beta-Ala) + 10% PVP + 2.86 mM N(2)H(4)x2HCl, pH 3.2; and a terminating electrolyte composed of 8 mM H(3)PO(4) + 16 mM beta-Ala + 10% PVP + 5 mM N(2)H(4), pH 3.85 for all the matrices except seawater. For ITP of seawater the LE consisted of 50 mM HCl + 100 mM beta-Ala + 10% PVP + 2.86 mM N(2)H(4)x2HCl, pH 3.52. Distance of conductivity detector from the injection point and bifurcation point was 52 and 38 mm, respectively. Zone electrophoresis was performed in a capillary made of fused silica of 0.3 mm id and 160 mm total length filled with LE from isotachophoretic step. LODs reached for all matrices were 2-3x10(-8) M concentration (2.5-4 microg/L) enabled monitoring of iodide in all analyzed samples with RSD 0.4-9.3%. Estimated concentrations of iodide in individual matrices were 10(-6)-10(-8) M.     

Comparison of the separation of large DNA fragments in the presence and absence of electroosmotic flow at high pH

This paper describes the analysis of large DNA fragments at pH > 10.0 by capillary electrophoresis (CE) in the presence of electroosmotic flow (EOF) using hydroxyethylcellulose (HEC) solution. HEC solution in the anodic reservoir enters the capillaries filled with high-pH buffer by EOF after sample injection. With respect to resolution, sensitivity, and speed, separation conducted under discontinuous conditions (different pH values of HEC solutions and buffer filling the capillary) is appropriate. Using HEC solution at concentrations higher than its entanglement threshold ensures a good separation of large DNA fragments in the presence of EOF at high pH. In addition to pH and HEC, the electrolyte species, dimethylamine, methylamine, and piperidine, play different roles in determining the resolution. The separation of DNA fragments ranging in size from 5 to 40 kilo base pairs was completed in 6 min using 1.5% HEC prepared in 20 mM methylamine-borate, pH 12.0, and the capillary filled with 40 mM dimethylamine-borate, pH 10.0. In comparison, this method allows faster separations of large DNA fragments compared with that conducted in the absence of EOF using dilute HEC solutions.     

Capillary electrochromatographic separation of non-steroidal anti-inflammatory drugs with a histidine bonded phase

An open tubular wall-coated capillary column containing histidine functional groups was prepared and employed for the capillary electrochromatographic separation of non-steroidal anti-inflammatory drugs. The anion exchange along with the hydrogen bonding and hydrophobic properties of the surface coating allowed the separation of analytes with very similar ionic mobility. Selectivity and resolution were studied by changing the pH over the range from 3.5 to 5.0 and the concentration of the buffer from 10 to 25 mM, as well as variation of the organic modifier, such as methanol, ethanol and 1-propanol over the range 7.5 to 20%. The optimum experimental conditions for the separation of a drug mixture, which consisted of indoprofen, ketoprofen, suprofen, naproxen, flurbiprofen, fenoprofen and ibuprofen were using a mixture of acetate buffer (20 mM, pH 5.0)-ethanol (1:5, v/v) as background electrolyte and an applied voltage of -20 kV with UV detection at 220 nm. The separation of these drugs could be achieved with an average plate number of 1.0 x 10(5) m(-1).     

Microchip separations of transition metal ions via LED absorbance detection of their PAR complexes

Micellar electrokinetic chromatography was utilized in the electrophoretic separation of seven transition metal ions, colorimetrically complexed by 4-(2-pyridylazo)resorcinol (PAR) on a glass capillary electrophoresis microchip. Detection of the PAR metal chelates was demonstrated using a green light emitting diode (540 nm) and a miniature photomultiplier tube. Parameters investigated included the effect of buffer type, pH and surfactant concentration (sodium dodecyl sulfate, SDS) on the separation efficiency. The optimally determined background electrolyte contained 10 mM ammonium phosphate buffer (pH 7.5), 1 mM PAR to prevent kinetic lability problems and 75 mM SDS for enhanced resolution. The separation of seven transition metal ions, Co2+, V3+, Ni2+, Cu2+, Fe2+, Mn2+ and Cd2+, was achievable in under 65 s, with the resolution of each metal ion in excess of 1.60. Detection limits obtained ranged from 400 ppb for Ni2+ to 1.2 ppm for Mn2+.     

Non-aqueous capillary electrophoresis of the positional isomers of a sulfated monosaccharide

A non-aqueous capillary electrophoresis (NACE) method coupled to indirect absorbance detection has been developed for the separation of the three positional isomers of monosulfated fucose. The optimized electrolyte was composed of 12 mM ethanolamine, 2 mM trimesic acid buffer in a methanol-ethanol (1:1, v/v) mixture. As the retained electrolyte entails no separating agent other than the pH buffer, the NACE separation of the positional isomers has been ascribed mainly to selective ion-pairing with the electrolyte counter-ion and the possibility of a selective solvation effect in the alcohol mixture. In the absence of pure isomeric standards, peak identification was completed by MS and NMR spectroscopy and selective enzymatic desulfation. This method should be of interest for the structure elucidation of monosulfated fucose-based polysaccharides and for the screening of sulfoesterase of unknown activity.     

Separation of 2-aminobenzoic acid-derivatized glycosaminoglycans and asparagine-linked glycans by capillary electrophoresis.

A capillary electrophoresis method was developed for the analysis of oligosaccharides combined with derivatization with 2-aminobenzoic acid. Glycosaminoglycan delta-disaccharides were effectively resolved on a fused-silica capillary tube using 150 mM borate, pH 8.5, as a running electrolyte solution. This analytical method was applied to the identification of glycosaminoglycan in combination with enzymatic digestion. The separation of N-glycans or glucose-oligomers was performed with a phosphate buffer containing polyethylene glycol or borate as an electrolyte solution. This method is expected to be useful in the determination of oligosaccharide structures in a glycoprotein.     

Preliminary study on the monitoring of glutathione S-tranferase activity toward styrene oxide by electromigration methods

A new method has been developed for the monitoring of glutathione S-tranferase (GST) detoxification activity toward styrene oxide (SO). The enzymatic reaction was carried out directly in a thermostatted autosampler vial and the formation of conjugates between glutathione (GSH) and SO was monitored by sequential MEKC runs. The determinations were performed in a 50-microm fused silica capillary using 50 mM SDS in 20 mM phosphate 20 mM tetraborate buffer (pH 8.3) as a background electrolyte; separation voltage 28 kV (positive polarity), temperature of capillary 25 degrees C, and detection at 200 nm. The method is rapid, amenable to automation, and requires only small amounts of samples, which is especially important in the case of GST isoenzyme analyses.     

Determination of rhodanese enzyme activity by capillary zone electrophoresis.

A new sensitive method has been developed for the determination of rhodanese activity. The enzymatic reactions were carried out directly in thermostatted autosampler vials and the formation of SCN- was monitored by sequential capillary zone electrophoretic runs. The determinations were performed in a 75-micron fused-silica capillary using 0.1 M beta-alanine-HCl (pH 3.50) as a background electrolyte, a separation voltage of 18 kV (negative polarity), a capillary temperature of 25 degrees C and direct detection at 200 nm. Short-end injection or long-end injection procedures were used for sample application. The method is rapid, able to be automated and requires only small amounts of sample and substrates, which is especially important in the case of highly toxic cyanide. The developed capillary electrophoretic method also has great potential for thiocyanate determinations in other applications.     

Rapid screening of aminopeptidase N inhibitors by capillary electrophoresis with electrophoretically mediated microanalysis

In this work, an electrophoretically mediated microanalysis (EMMA) method with a partial‐filling technique was setup to evaluate the inhibitory potency of novel compounds toward aminopeptidase N (APN). It was necessary to optimize the electrophoretic conditions with respect to the kinetic constraints and for attaining high sensitivity. In our setup, a part of the capillary was filled with the incubation buffer for the enzyme reaction, whereas the rest was filled with a suitable BGE for the separation of substrates and products. To monitor the performance of the newly developed method, the kinetic constants (K_m and V_max) for the catalyzed dissociation of l ‐Leucine‐p‐nitroanilide in the presence of APN as well as the inhibition constant (IC₅₀) of a known competitive inhibitor, that is bestatin, were determined and these results were compared with those obtained by a classical spectrophotometric assay. The developed EMMA method was subsequently applied to the screening of 30 APN inhibitors. Whereas the inhibition potency of these inhibitors (expressed in IC₅₀ values) were significantly underestimated by the EMMA method, the order of the inhibitory potential of these various compounds was found in agreement with the literature.