A capillary electrophoresis method to assay catalytic activity of botulinum neurotoxin serotypes: implications for substrate specificity

The potent botulinum neurotoxin inhibits neurotransmitter release at cholinergic nerve terminals, causing a descending flaccid paralysis characteristic of the disease botulism. The currently expanding medical use of the neurotoxin to treat several disorders, as well as the potential misuse of the neurotoxin as an agent in biowarfare, has made understanding of the nature of the toxin's catalytic activity and development of inhibitors critical. To study the catalytic activity of botulinum neurotoxin more thoroughly and characterize potential inhibitors, we have developed a capillary electrophoresis method to measure catalytic activity of different serotypes of botulinum neurotoxin using peptides derived from the native substrates. This assay requires only a minute amount of sample (25 nl), is relatively rapid (15 min/sample), and allows the determination of enzyme kinetic constants for a more sophisticated characterization of inhibitors and neurotoxin catalytic activity. Using this method, we can measure activity of five of the seven serotypes of botulinum neurotoxin (A, B, E, F, and G) with two peptide substrates. Botulinum neurotoxin serotypes C and D did not cleave our peptides, lending insight into potential substrate requirements among the serotypes.     

Capillary electrophoresis for the investigation of prostate-specific antigen heterogeneity

Prostate-specific antigen (PSA) is a single-chain glycoprotein that is used as a biomarker for prostate-related diseases. PSA has one known posttranslational modification, a sialylated diantennary N-linked oligosaccharide attached to the asparagine residue N45. In this study capillary electrophoresis (CE) was employed to separate the isoforms of seven commercially available free PSA samples, two of which were specialized: enzymatically active PSA and noncomplexing PSA. The free PSA samples examined migrated as four to nine distinct, highly resolved peaks, indicating the presence of several isoforms differing in their oligosaccharide compositions. Overall, the use of CE provides a rapid, reproducible method for separation of PSA into its individual isoforms.     

Capillary electrophoresis with noncovalently bilayer-coated capillaries for stability study of allergenic proteins in simulated gastrointestinal fluids

A novel noncovalently bilayer-coated capillary using cationic polymer polybrene (PB) and anionic polymer (sodium 4-styrenesulfonate) (PSS) as coatings was prepared. This PB–PSS coating showed good migration-time reproducibility for proteins and high stability in the range of pH 2–10 and in the presence of 1 M NaOH, acetonitrile and methanol. Capillary electrophoresis with PB–PSS coated capillaries was successfully applied to quantitatively investigate the stability of bovine serum albumin, ovomucoid, β-lactoglobulin and lysozyme in simulated gastrointestinal fluids. β-lactoglobulin A and β-lactoglobulin B were both stable in simulated gastric fluid with degradation percentages of 34.3% and 17.2% after 60 min of incubation, respectively. Bovine serum albumin, ovomucoid and lysozyme were stable in simulated intestinal fluid with degradation percentages of 17.7%, 23.4% and 22.8% after 60 min of incubation, respectively. The superiority of the proposed method over sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and capillary electrophoresis with untreated fused silica capillaries was demonstrated and emphasized.     

Capillary affinity electrophoresis using lectins for the analysis of milk oligosaccharide structure and its application to bovine colostrum oligosaccharides

Animal colostrum and milk contain complex mixtures of oligosaccharides, which have species-specific profiles. Milk oligosaccharides have various types of structure related to the core structures of glycolipids and N- and O-glycans of glycoproteins and provide a good library to examine the binding of oligosaccharides to various lectins. Recently, we reported a capillary affinity electrophoresis (CAE) method for analyzing the interactions between lectins and complex mixtures of N-linked oligosaccharides prepared from serum glycoproteins. The present paper reports the interactions between 24 milk oligosaccharides and six lectins (PA-I, RCA(120), SBA, WGA, UEA-I, and AAL) analyzed using CAE. Based on the resulting data, we constructed a library that enables us to determine nonreducing terminal monosaccharides, such as Gal, GalNAc, GlcNAc, and Fuc, and to differentiate Gal- or Fuc-linked isomers, such as lacto-N-tetraose, lacto-N-neotetraose, and lacto-N-fucopentaose II and III. In addition, using the library, we show that a combination of the lectins can characterize the neutral oligosaccharides derived from bovine colostrum.     

Capillary electrophoresis for the quality control of chondroitin sulfates in raw materials and formulations

Exogenous administration of chondroitin sulfate (CS) is widely practiced for the treatment of osteoarthritis, although the efficacy of this treatment has not been completely established by clinical studies. A reason for the inconsistency of the results may be the quality of the CS preparations, which are commercially available as dietary supplements. In this article, we describe the development of a new method of capillary electrophoresis (CE) for the quantification of CS concentrations, screening for other glycosaminoglycan or DNA impurities and determination of hyaluronan impurities in CS raw materials, tablets, hard capsules, and liquid formulations. Analysis is performed within 12 min in bare fused silica capillaries using reversed polarity and an operating phosphate buffer of low pH. The method has high sensitivity (lower limit of quantitation [LLOQ] values of 30.0 microg/ml for CS and 5.0 microg/ml for hyaluronan), high precision, and accuracy. Analysis of 11 commercially available products showed the presence of hyaluronan impurities in most of them (up to 1.5%). CE analysis of the samples after treatment with chondroitinase ABC and ACII, which depolymerize the chains to unsaturated disaccharides, with a previously described method (Karamanos et al., J. Chromatogr. A 696 (1995) 295-305) confirmed the results of hyaluronan determination and showed that the structural characteristics (i.e., disaccharide composition) of CS are very different, showing the different species or tissue origin and possibly affecting the therapeutic outcome.     

A capillary electrophoretic method for monitoring the presence of alpha-tubulin in nuclear preparations

A sensitive capillary electrophoretic method was developed to detect the presence of alpha-tubulin, a microtubular cytoskeletal component, in isolated nuclear preparations. These preparations are treated with anti-alpha-tubulin primary mouse antibodies and then stained with a fluorescently labeled anti-mouse IgG antibody. The stained preparation is then analyzed by capillary electrophoresis with laser-induced fluorescence detection, a technique that allows for sensitive detection of fluorescently labeled species. Using this method, it is feasible to count individual subcellular aggregates containing alpha-tubulin (SATs), estimate the number of alpha-tubulin molecules per SAT, determine the cumulative intensity of all SATs as an estimate of the relative level of alpha-tubulin in a preparation, and obtain their apparent electrophoretic mobility distribution. The method was validated by comparing SATs from untreated cells with those from colchicine-treated cells. Since colchicine is a microtubule-disrupting agent, treatment reduced the number of SATs per cell as well as the cumulative intensity of all SATs in a preparation. In contrast, the apparent electrophoretic mobility distribution was not influenced by colchicine treatment, suggesting that this parameter is not strongly dependent on the alpha-tubulin content. Given the zeptomolar sensitivity of laser-induced fluorescence detection and the widespread availability of antibodies, the approach used here represents an improvement in the detection of cytoskeletal impurities in subcellular fractions.     

Modified method for the determination of capillary electrophoresis nitric oxide-correlated nitrate in tissue homogenates

A modified capillary electrophoretic method for the determination of nitric oxide correlated nitrate in several tissue homogenates is described in this study. The method was developed using a running buffer consisting of 200 mM lithium chloride and 10 mM borate buffer at pH 8.5, in a fused-silica column total 82 cm, effective 43 cm length and 75 μm I.D. The signal was measured at 214 nm and controlled current of 200 μA (equivalent to 12.7 kV) was applied in the reversed polarity direction. The sample was injected by vacuum pressure 50 ms (25 nl). In these conditions, bromide as internal standard and nitrate appeared at 7.2 and 8.9 min, respectively. Whole validation procedures were applied and satisfactory results were obtained. The nitrate levels of the tissue homogenates of control and -NAME applied (heart, brain, kidney, stomach, lung, testis and liver) were monitored by the present method and it was decided that the method is precise and accurate.     

Optimization by factorial design of a capillary zone electrophoresis method for the simultaneous separation of antihistamines

A 3(2) full factorial design was used to optimize the experimental conditions of a capillary zone electrophoresis method aimed at achieving simultaneous separation and quantification of the antihistamines brompheniramine, chlorpheniramine, cyproheptadine, diphenhydramine, doxylamine, hydroxyzine, and loratadine according to their therapeutic group. A statistical program, SPSS, was used to calculate the mathematical model with which to obtain the response surface. Critical parameters such as pH and applied voltage were studied to evaluate their effect on resolution and on efficiency. Optimum separation conditions were phosphate buffer pH 2.0, 5kV, and 2psis(-1) at 214nm. The analysis time was below 9min and the theoretical plates were between 6000 and 63,000N. Calibration curves were prepared for the antihistamines. The limits of detection were 4-14ngmL(-1), which allow their quantification in pharmaceuticals. The RSD% of each antihistamine was fairly good. Up to seven antihistamines belonging to the antihistaminic H(1)-receptor group were separated in the same electropherogram. The proposed method was then applied to the determination of antihistamines in pharmaceutical, urine, and serum samples with recoveries in agreement with the stated contents.     

A novel spectrofluorimetric method for the determination of calcitonin in ampules through derivatization with fluorescamine

In this study, a simple, sensitive and selective spectroflourimetric method has been developed for the determination of salmon calcitonin (sCT) in ampules. The method is based on the reaction between sCT and fluorescamine at pH 8.5 in borate buffer, resulting in a highly fluorescent derivative. Fluorescence of derivatized sCT solutions was measured by setting the excitation and emission monochromators and slit widths to 390, 484 and 10 nm, respectively. Sevaral derivatization parameters were optimized. A calibration graph was constructed using standard solutions of the derivatized calcitonin in the range 0.5–6.0 µg/mL. Limit of detection and limit of quantification values were determined to be 0.124 and 0.372 µg/mL, respectively. The proposed method was successfully applied for the determination of sCT in commercially available ampules. High recovery values (101.0–102.0 %), and a low relative standard deviation (RSD %) value (5.3–5.4) proved the accuracy and precision of the proposed method. An isocratic reversed‐phase high‐performance liquid chromatographic (HPLC) method, as a reference, was also developed for the determination of sCT. A reversed‐phase Nucleosil® C₁₈ column (250 mm × 4.6 mm i.d., 10 µm particle size, 120 Å pore size) was used and the detector was set at 210 nm. Statistical comparison of the results of the two methods showed clearly that there was no significant difference between them. Copyright © 2012 John Wiley & Sons, Ltd.     

A rapid capillary gel electrophoresis method for the quantitative determination of RuBisCo in spinach

A capillary gel electrophoretic (CGE) method for the quantitative analysis of RuBisCo in spinach leaves was developed. RuBisCo was resolved into large and small subunits in the presence of sodium dodecyl sulphate (SDS) by the CGE procedure which enabled the determination of the molecular weight of each unit accurately; the values so determined were in close agreement with those reported using other methods. Advantages of CGE over SDS-polyacrylamide gel electrophoresis and high-pressure gel filtration include decreased sample preparation and analysis time, superior resolution and greater sensitivity permitting reduced sample size and trace analysis. In addition, CGE provided precise quantification of RuBisCo and was demonstrated to be a viable alternative to other available methods of protein analysis.     

A colorimetric 96-well microtiter plate assay for the determination of urate oxidase activity and its kinetic parameters

Urate oxidase (E.C.1.7.3.3; uricase, urate oxygen oxidoreductase) is an enzyme of the purine breakdown pathway that catalyzes the oxidation of uric acid in the presence of oxygen to allantoin and hydrogen peroxide. A 96-well plate assay measurement of urate oxidase activity based on hydrogen peroxide quantitation was developed. The 96-well plate method included two steps: an incubation step for the urate oxidase reaction followed by a step in which the urate oxidase activity is stopped in the presence of 8-azaxanthine, a competitive inhibitor. Hydrogen peroxide is quantified during the second step by a horseradish peroxidase-dependent system. Under the defined conditions, uric acid, known as a radical scavenger, did not interfere with hydrogen peroxide quantification. The general advantages of such a colorimetric assay performed in microtiter plates, compared to other methods and in particular the classical UV method performed with cuvettes, are easy handling of large amounts of samples at the same time, the possibility of automation, and the need for less material. The method has been applied to the determination of the kinetic parameters of rasburicase, a recombinant therapeutic enzyme.     

Classification of the mode of inhibition of high-affinity choline uptake using capillary electrophoresis with electrochemical detection at an enzyme-modified microelectrode

A nonradiochemical in vitro assay using capillary electrophoresis with electrochemical detection at an enzyme-modified microelectrode has been developed to evaluate the inhibition of high-affinity choline transport in synaptosomes. Quantitative analysis of high-affinity choline transporter rates as a function of inhibitor and substrate concentrations allowed determination of the mode of inhibition for the quaternary ammonium-catechol-based inhibitors 3-[(trimethylammonio)methyl]catechol, N,N-dimethylepinephrine, and 6-hydroxy-N,N-dimethylepinephrine. The results are compared to the well-characterized inhibitor of choline transport, hemicholinium-3.     

A capillary electrophoresis method for the characterization of ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) and the analysis of inhibitors by in-capillary enzymatic microreaction

A capillary electrophoresis (CE) method for the characterization of recombinant NTPDases 1, 2, and 3, and for assaying NTPDase inhibitors has been developed performing the enzymatic reaction within the capillary. After hydrodynamic injection of plugs of substrate solution with or without inhibitor in reaction buffer, followed by a suspension of an enzyme-containing membrane preparation, and subsequent injection of another plug of substrate solution with or without inhibitor, the reaction took place close to the capillary inlet. After 5 min, the electrophoretic separation of the reaction products was initiated by applying a constant current of −60 μA. The method employing a polyacrylamide-coated capillary and reverse polarity mode provided baseline resolution of substrates and products within a short separation time of less than 7 min. A 50 mM phosphate buffer (pH 6.5) was used for the separations and the products were detected by their UV absorbance at 210 nm. The Michaelis–Menten constants (K_m) for the recombinant rat NTPDases 1, 2, and 3 obtained with this method were consistent with previously reported data. The inhibition studies revealed pronounced differences in the potency of reactive blue 2, pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), suramin, and N⁶-diethyl-β,γ-dibromomethylene-ATP (ARL67156) towards the NTPDase isoforms. Notably, ARL67156 does not inhibit all NTPDases, having only a minor inhibitory effect on NTPDase2. Dipyridamole is not an inhibitor of the NTPDase isoforms investigated. The new method is fast and accurate, it requires only tiny amounts of material (nanoliter scale), no sample pretreatment and can be fully automated; thus it is clearly superior to the current standard methods.     

Validation of a capillary electrophoresis method for the quantitative determination of free and total apigenin in extracts of Chamomilla recutita

A capillary electrophoretic method for the quantification of free and total apigenin in methanolic, ethanolic and glycolic extracts of Chamomilla recutita L. Rauschert (Asteraceae) is described. The method was validated for measurement of apigenin in the range 5.00-300 microg/mL (r2 = 0.993) and showed coefficients of intra-day (replicability) and inter-day (repeatability) variability of better than 2%. The limits of detection and quantification were 3.80 and 11.5 microg/mL, respectively, and the average recovery was 102.0 +/- 0.8% at three concentration levels of apigenin. Free and total apigenin contents in the extracts were, respectively, determined as 106 and 903 microg/g (methanolic extract), 77 and 817 microg/g (ethanolic extract) and 11.0 and 247 microg/g (glycolic extract).     

New method for the resolution of the enantiomers of 5,6-Dihydroxy-2-Methyl-Aminotetralin by selective derivatization and HPLC analysis: Application to biological fluids

A new chiral derivatization procedure for the HPLC resolution of chiral catecholamines and structurally related compounds is described. The homochiral reagent, (+)-(R)-1-phenylethyl isocyanate (RPEIC), was added to separate and quantitate the enantiomers of rac-5,6-dihydroxy-2-methyl-aminotetralin, the main metabolite of rac-5,6-diisobutyryl-2-methyl-aminotetralin, a potent dopamine agonist, by reversed-phase HLPC analysis. To avoid catecholamine degradation in the basic reaction medium and to obtain the selective and quantitative derivatization of the amino group of the compound, the reversible complex formation between diphenylborinic acid (DPBA) and the catechol group, in alkaline medium, was performed before homochiral isocyanate addition. The RPEIC derivatization was completed in 30 min and then the DPBA complex was dissociated by adding dilute acid. The structure of intermediates and urea derivatives was confirmed by mass spectrometry. The use of an electrochemical detector, operating in redox mode, allowed HPLC quantitation of enantiomers at the nanogram level in plasma and urine. The derivatization procedure is also suitable for other catecholamine-related compounds. © 1996 Wiley-Liss, Inc.     

A capillary zone electrophoresis method for the quantitative determination of hypoxoside in commercial formulations of African potato (Hypoxis hemerocallidea)

Hypoxoside is a norlignan diglucoside present in the corms of African potato, Hypoxis hemerocallidea, used as a popular African traditional medicine for its nutritional and immune boosting properties. A specific analytical method employing capillary zone electrophoresis has been developed and validated for the quantitative determination of this analyte. Sulfafurazole was used as internal standard, and electrophoretic separation of both analytes could be achieved within 12 min. Linearity of the method was established within the range 5-60 microg/mL and provided a high degree of accuracy (100 +/- 3%). The recovery of the method was found to be 100 +/- 5% and the RSDs of the intra- and inter-day precision were better than 5.19 and 2.52%, respectively. The limits of detection and quantification were calculated to be 0.5 and 2 microg/mL, respectively. The described method was used for the analysis and quality control of two commercially available products containing African potato. The method can also be used to determine product stability since it could separate the hypoxoside peak from its degraded products obtained from degradation studies.     

Spectrofluorimetric method for the determination of sulpiride in pharmaceutical preparations and human plasma through derivatization with 2‐cyanoacetamide

A sensitive and accurate spectrofluorimetric method has been developed for the determination of sulpiride in pharmaceutical preparations and human plasma. The developed method is based on the derivatization reaction of 2‐cyanoacetamide with sulpiride in 30% ammonical solution. The fluorescent derivatized reaction product exhibited maximum fluorescence intensity at 379 nm after excitation at 330 nm. The optimum conditions for derivatization reactions were studied and the fluorescence intensity versus concentration plot was found to be linear over the concentration range 0.2–20.0 µg/mL with a correlation coefficient of 0.9985. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.82 and 2.73 ng/mL, respectively. The proposed method was validated according to ICH guidelines. The effects of common excipients and co‐administered drugs were also studied. The accuracy of the method was checked using the standard addition method and percent recoveries were found to be in the range of 99.00–101.25% for pharmaceutical preparations and 97.00–97.80% for spiked human plasma. The method was successfully applied to commercial formulations and the results obtained for the proposed method were compared with a high‐performance liquid chromatography reference method and statistically evaluated using the Student's t‐test for accuracy and the variance ratio F‐test for precision. A reaction pathway was also proposed. Copyright © 2012 John Wiley & Sons, Ltd.     

Capillary electrophoresis-based nanoscale assays for monitoring ecto-5'-nucleotidase activity and inhibition in preparations of recombinant enzyme and melanoma cell membranes

Powerful capillary electrophoresis (CE) methods were developed for monitoring the reaction of ecto-5'-nucleotidase (ecto-5'-NT, CD73), a (patho)biochemically important enzyme that hydrolyzes nucleoside-5'-monophosphates to the corresponding nucleosides. The enzymatic reaction was performed either before injection into the capillary (method A) or directly within the capillary (method B). In method A, separation of substrates and products was achieved within 8 min using an eCAP fused-silica capillary (20 cm effective length, 75 microM i.d., UV detection at 260 nm), 40 mM sodium borate buffer (pH 9.1), normal polarity, and a constant voltage of 15 kV. In method B, the sandwich technique was applied; substrate dissolved in reaction buffer (10mM Hepes [pH 7.4], 2mM MgCl2, and 1mM CaCl2) was hydrodynamically injected into a fused-silica capillary (30 cm, 75 microM i.d.), followed by enzyme (recombinant rat ecto-5'-NT) and subsequent injection of substrate solution. The reaction was initiated by the application of 1 kV voltage for 1 min. The voltage was turned off for 1 min and again turned on at a constant voltage of 15 kV to elute products (nucleosides) within 4 min using borate buffer (40 mM, pH 9.1). Thus, assays could be performed within 6 min, including enzymatic reaction, separation, and quantification of the formed nucleoside. The CE methods were used for measuring enzyme kinetics and for assaying inhibitors and substrates. In addition, the online assay was successfully applied to melanoma cell membrane preparations natively expressing the human ecto-5'-NT.     

Effective pH pretreatment and cell disruption method for real-time intracellular enzyme activity assay of a marine fungus covered with pigments

Filamentous fungi are capable producers of many bioactive compounds, and real-time intracellular enzyme activity assay is an essential guidance for their bioprocess developments. However, there are many difficulties in preparing homogenate for enzyme activity assay, such as disrupting fungal cell with complicated cellular structure and solid cell wall, removing abundant extracellular metabolites accumulating on mycelia, and so on. Halorosellinia sp. (No. 1403) was a marine-derived filamentous fungus producing a potential antitumor compound 1403C, and the deep red pigments (with main component of 1403C) covering on its mycelia showed strong absorption in a wide range, which critically affected the measurement of many enzyme activities. In this study, we developed an effective pH pretreatment and cell disruption method to prepare homogenate for enzyme activity assay. When mycelia were washed by the solution with pH 5.0 for 3?min, most pigments could be removed without severe loss on enzyme activities. Afterward, grinding with mini bead for 15?min with alternating cooling could effectively disrupt both cell wall and mitochondrial membrane. These methods have been successfully applied on real-time intracellular enzyme activity assay of Halorosellinia sp. (No. 1403) and can offer enlightenment for other filamentous fungi with similar problems.     

A method for activity staining after native polyacrylamide gel electrophoresis using a coupled enzyme assay and fluorescence detection: application to the analysis of several glycolytic enzymes.

We describe a method for the detection of isoforms of several glycolytic enzymes by activity staining after native PAGE. The staining is based on coupled enzyme assays carried out on the gel after electrophoresis and is linked to the disappearance of NADH, which is visualized by fluorescence. This method offers reliable and sensitive detection for phosphoenolpyruvate carboxylase, PPi-dependent phosphofructokinase, and pyruvate kinase from plant tissues. It can be applied to the detection of all enzymes which are normally detected spectrophotometrically using coupled enzyme assays consuming NAD(P)H.