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.     

Kinetic study of cytochrome P450 by capillary electrophoretically mediated microanalysis

An electrophoretically mediated microanalysis method for the determination of CYP3A4 activity using testosterone and nifedipine as substrates was developed. Initially, the enzymatic reaction was performed off-line and the samples were subsequently injected into the capillary by pressure. The CYP3A4 activity was determined by quantitation of the reactant cofactor, NADPH. To further optimize, speed-up and miniaturize the enzyme assay, the enzymatic reaction was performed directly in the capillary, prior to separation and quantitation of the product cofactor, NADP, employing the plug-plug mode of electrophoretically mediated microanalysis. 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 260 nm. This setup resulted in a fully automated assay, which can be carried out in less than 35 min. Using the Lineweaver-Burk equation, the Michaelis constants (K(m)) for the oxidation of testosterone and nifedipine by CYP3A4 were calculated to be 58.6+/-8.3 and 19.1+/-2.4 microM, respectively, which are consistent with off-line assay and previously reported values.     

毛细管胶束电动色谱法测定血管紧张素转化酶的活性

 建立了应用毛细管胶束电动色谱 (MECC)灵敏、快速的测定血管紧张素转化酶 (ACE)活性的方法。通过对电压、上样时间、电极缓冲液体系等影响因素的优化 ,探讨了方法的可行性 ,确立了最佳测定条件 (电压 :8 1kV ;上样时间 :1s;电极缓冲液 :2 0mmol/L硼酸盐缓冲液 (pH 9 0 ,含 5 0mmol/LSDS) ;检测波长 :2 2 8nm)。方法的最低ACE活性检测限为 5pmol/min(以 2倍的信噪比计 )。     

High temporal resolution monitoring of enzyme reaction and inhibition using optically gated vacancy capillary electrophoresis and immobilized enzyme

A novel method for monitoring of enzyme reaction and inhibition with high temporal resolution was developed by using optically gated vacancy capillary electrophoresis (OGVCE) with laser-induced fluorescence (LIF) detection and immobilized enzyme. Trypsin cleavage reaction and inhibition were investigated by the presented OGVCE-LIF assay, using carboxyfluorescein (FAM) end-labeled Angiotensin as the substrate and commercially available immobilized trypsin. The substrate and the product were continuously loaded into the capillary by the electroosmotic flow while the immobilized enzyme remained in the sample vial. Substrate consumption and product formation were monitored simultaneously at 5 s interval during the whole reaction time. The enzymatic reaction rates obtained from the substrate and the product were highly consistent. The enzyme activity and the Michaelis constants of trypsin cleavage reaction, as well as the inhibition constant (for reversible competitive inhibitor) and the inhibition fraction (for irreversible inhibitor), were obtained. It was showed that the reported OGVCE-LIF method can perform fast, accurate, sensitive and reproducible CE enzyme assay with high temporal resolution, thus has great potential in application of the enzyme-substrate systems with fast reaction rate and the fluorescent substrate and products.     

Improving the sensitivity of the determination of ceftiofur by capillary electrophoresis in environmental water samples: in-line solid phase extraction and sample stacking techniques

This paper describes two different approaches for increasing the sensitivity for the analysis of ceftiofur by capillary electrophoresis (CE). Two different techniques based on the introduction of an enlarged volume of sample, namely large volume sample stacking (LVSS) and in-line solid phase extraction (SPE) were studied and compared. LVSS allowed the on-column electrophoretic preconcentration of ceftiofur without modification of the separation capillary. In-line SPE-CE was developed by using a home-made microcartridge that was filled with a reversed-phase sorbent (C₁₈). The microcartridge was coupled in-line near the inlet of the separation capillary. LVSS and in-line SPE-CE allowed automated operation and improved sensitivity for the analysis of ceftiofur with respect to conventional CE. When environmental water samples were analyzed, an additional pretreatment step based on off-line SPE was necessary in both cases to further decrease the detection limits. In terms of sensitivity for the determination of ceftiofur in river water samples, the combination of off-line SPE with in-line SPE-CE was found the most sensitive with a detection limit of 10 ng L⁻¹, whereas the method based on the use of off-line SPE with LVSS presented a detection limit of 100 ng L⁻¹.     

Application of an integrated microchip system with capillary array electrophoresis to optimization of enzymatic reactions

In this work, a combination of complementary metal-oxide semiconductor (CMOS) microchip system with capillary array electrophoresis (CAE) is demonstrated as a system for optimizing conditions for enzymatic reaction. Dimethylacridinone (DDAO)-phosphate substrate and alkaline phosphatase conjugate were selected for the enzymatic reaction, which was applicable to the enzyme-linked immunosorbent assay (ELISA) technique. Laser-induced fluorometry with a miniature semiconductor laser was used to detect the enzymatic products. The speed of the enzymatic reaction between the DDAO-phosphate and the alkaline phosphatase conjugate was investigated as a function of reaction time. The microchip-CAE detection system could determine the pH condition and the concentration of enzyme that are suitable for rapid and low-cost analysis. This result shows the feasibility of using the microchip-CAE system for application to miniaturized screening systems.     

Determination of enzymatic activity and properties of secretory phospholipase A2 by capillary electrophoresis.

A capillary electrophoretic (CE) system coupled with a diode array UV detector was used for the assay of secretory phospholipase A2 (sPLA2) activity. This method is based on monitoring both the breakdown of substrates and the formation of products simultaneously using micellar electrokinetic chromatographic techniques. Under our developed separation conditions, we analyzed the substrates and products quantitatively, and investigated enzyme activity as a function of reaction time and presence of enzyme activator or inhibitor. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was also utilized to confirm the phosphatidylcholine, a substrate of sPLA2. In order to test the feasibility of the developed method for measurement of enzymatic activity, we compared it to the conventional radioactive assay method for sPLA2. On the basis of our results, the conventional method can be complemented, or even replaced, by this new CE method which possesses the advantages of short analysis time, use of non-radiolabeled and inexpensive substrates, simple measurement of enzymatic activity, and exact quantitation of substrate and product.     

Fast in-line bottom-up analysis of monoclonal antibodies: Toward an electrophoretic fingerprinting approach

For their characterization and quality control, monoclonal antibodies are frequently analyzed at the bottom-up level to generate specific fingerprints that can be used to tackle post-translational modifications or ensure production consistency between lots. To circumvent time-consuming and labor-intensive off-line sample preparation steps, the implementation of integrated methodologies from sample preparation to separation and detection is highly valuable. In this perspective, capillary zone electrophoresis appears as a choice technique since the capillary can subsequently be used as a vessel for sample preparation and electrophoretic discrimination/detection of the reaction products. Here, a fast in-line methodology for the routine quality control of mAbs at the bottom-up level is reported. Simultaneous denaturation and reduction (pretreatment step) were conducted with RapiGest® surfactant and dithiothreitol before in-line tryptic digestion. Reactant mixing was realized by transverse diffusion of laminar flow profile under controlled temperature. In-line digestion was carried out with a resistant trypsin to autolysis. The main parameters affecting the digestion efficiency (trypsin concentration and incubation conditions) were optimized to generate mAb electrophoretic profiles free from trypsin interferences. An acidic MS-compatible BGE was used to obtain high resolution separation of released peptides and in-line surfactant cleavage. The whole methodology was performed in less than two hours with good repeatability of migration times (RSD = 0.91%, n = 5) and corrected peak areas (RSD = 9.6%, n = 5). CE-fingerprints were successfully established for different mAbs and an antibody-drug conjugate.     

High-performance capillary electrophoretic analysis of chloramphenicol acetyl transferase activity.

This study highlights the potential utility of high-performance capillary electrophoresis (HPCE) for monitoring enzyme activity. Free-zone capillary electrophoresis is used to rapidly and reproducibly analyze the activity of the bacterial enzyme chloramphenicol acetyl transferase (CAT) which converts the substrates acetyl coenzyme A (CoA) and chloramphenicol to acetyl chloramphenicol and CoA. The results of this study indicate that HPCE may be an excellent tool for studying enzyme activities since it has several advantages over standard single parameters assays, most notably, the ability to monitor both loss of substrate and appearance of products simultaneously. Conditions have been identified for optimal separation of the substrate (chloramphenicol) from the products (acetylated derivatives). This presents a unique potential of HPCE for the analysis of enzymatic reactions that may be applied to areas of analytical research presently utilizing enzymatic reactions. One such analytical method is the CAT assay used for analysis of gene promoter activity. In this study, HPCE is shown to yield similar quantitative results with nonradiolabelled substrate in a fraction of the time. HPCE has several advantages over standard techniques including speed of analysis, no need for radiolabelled substrate, small sample volumes, high sensitivity/resolution and excellent quantitative capabilities.     

Capillary electrophoretic approach to screen for enzyme inhibitors in natural extracts

This paper demonstrates development of electrophoretically mediated micro analysis (EMMA), for screening protein tyrosine phosphatase (PTP) inhibitors in natural extracts. It is demonstrated that capillary electrophoresis (CE) separation of the substrate and the product allows for using the assay in an on-column format to monitor the reaction without typically used fluorogenic substrates. Michaelis-Menten kinetics parameters calculated based on the EMMA results (Km = 1.2-1.5 microM) were in a good agreement (Km = 1.0-1.5 microM) obtained using an off-line CE functional assay (CE FA). EMMA of PTP titrated with different concentrations of ligand demonstrated the peak-shift phenomenon normally seen in affinity capillary electrophoresis. This feature of EMMA gives an indication of the binding affinity of the ligand in addition to its functional activity, providing another dimension in characterization of the protein-inhibitor interaction. It was demonstrated that simultaneous screening of the primary PTP target and a secondary, counter target (PTP-C) using the EMMA format can be used to prioritize hits based on their specificity.     

Electrophoretically mediated microanalysis

This review describes the existing developments in the use of the capillary electrophoretic microanalytical technique for the in-line study of enzyme reaction, electrophoretically mediated microanalysis (EMMA). The article is divided into a number of parts. After an introduction, the different modes, basic principle, procedure, and some mathematical treatments of EMMA methodology are discussed and illustrated. The applications of EMMA for enzyme assay and for non-enzymatic determination are summarized into two tables. In addition to classical capillary electrophoresis (CE) instrument EMMA, special emphasis is given to a relatively new technique: EMMA on CE microchip. Finally, conclusions are drawn.     

基于DNA定向固定化技术构建毛细管固定化酶微反应器的研究进展

生物酶影响着物质代谢和质能转换等生命活动,生物体内某些酶的活性变化会导致疾病的发生。发展新型的酶分析方法对深刻理解生物代谢过程、疾病诊断和药物研发等具有重要意义。毛细管电泳（CE）具有分离效率高、分析速度快、操作简单和样品消耗少以及可与多种检测手段联用等优点,在酶分析研究中越来越受到关注。CE酶分析主要包括离线和在线两种模式,其中,固定化酶微反应器与毛细管电泳联用（CE-IMER）的在线酶分析已经成为主要的酶分析方法之一。CE-IMER充分结合了固定化酶和CE的优势,将游离酶固定在毛细管内,不仅可以显著提高酶的稳定性和重复使用性,而且可以实现纳升规模溶液的自动化酶分析,进而显著降低酶分析成本。目前已有大量方法制备IMER用于CE酶分析,然而如何构建性能良好、可再生使用、酶固载量大、自动化程度高的CE-IMER一直是该领域重点研究的问题。DNA定向固定化技术（DDI）可以充分利用DNA分子的碱基互补配对（A-T,C-G）,在温和的生理条件下特异性固定生物大分子。由于短链双螺旋DNA分子具有较强的机械刚性和物理化学稳定性,通过DDI将酶固定在载体表面,有利于降低传质阻力,提高酶与底物的接触能力,进而促进酶促分析过程。该文主要综述了利用DDI构建新型IMER在CE酶分析中的应用现状,并对其未来发展进行了展望。     

Measuring the activity of farnesyltransferase by capillary electrophoresis with laser-induced fluorescence detection

Enzymatic farnesylation of oncogenic forms of Ras proteins is the initial step in a series of posttranslational modifications essential for Ras activity. The modification is catalyzed by the enzyme, protein farnesyltransferase (PFTase), which transfers a farnesyl moiety from farnesyl diphosphate to the protein. We employed capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection to develop a rapid and sensitive method for the determination of PFTase activity in vitro. The limited substrate specificity of PFTase allowed us to use a fluorescently labeled pentapeptide instead of a Ras protein as a substrate for the enzyme; the product of the enzymatic reaction was the farnesylated pentapeptide. The product was separated from the substrate by CE and quantified with LIF detection. Under optimal conditions, the separation was achieved within 10 min with a resolution of 86. The mass and concentration limits of detection for the farnesylated product were 10(-19) mol and 0.28 nM, respectively. By measuring the rate of accumulation of the farnesylated product, we were able to determine the kinetic parameters of the enzymatic reaction. For yeast PFTase as an enzyme and difluorocarboxyfluorescein-labeled GCVIA peptide as a substrate, the values of k(cat) and K(M) were found to be (3.1 +/- 0.3)x10(-3) s(-1) and (12.0 +/- 1.2) nuM, respectively. Our results suggest that CE-LIF can be efficiently used for the determination of enzymatic activity of PFTase in vitro. After minor modifications, the developed method can be also applied to other reactions of enzymatic prenylation of proteins.     

Determination of catechol-O-methyltransferase activity in erythrocytes by high performance liquid chromatography with electrochemical detection

A rapid assay employing HPLC with electrochemical detection for catechol-O-methyltransferase (COMT) activity in red blood cells is described. Enzyme activity is determined from erythrocyte lysates using S-adenosyl-L-methionine as methyl donor and 3,4-dihydroxybenzoic acid as substrate. The 3-O- and 4-O-methylated reaction products are measured by high-performance liquid chromatography with electrochemical detection. Human erythrocyte soluble form of COMT had Km values of 6.1 microM and 26.0 microM for S-adenosyl-L-methionine and dihydroxybenzoic acid, respectively. The mean O-methylation ratio for the soluble form of COMT was 5.3. An O-methylation ratio of 15.5 was estimated in the membrane fraction of an erythrocyte pool from three samples. The activities of soluble COMT in erythrocytes of some animal species are also reported. The procedure is easily automated, and a large number of samples can be processed during one working day.     

Advances in Capillary Electrophoresis-Based Enzyme Assays

Capillary electrophoresis (CE) has become a flexible and accurate, high-efficiency analytical separation technique in many areas requiring only minute amounts of sample and chemicals. Thus, CE has also been recognized as a suitable technique to study enzymatic reactions including the determination of Michaelis–Menten kinetic data or the identification and characterization of inhibitors. The most often applied CE-based enzyme assay modes can be divided into two categories: (1) pre-capillary assays where incubations are performed offline followed by CE analysis of substrate(s) and/or product(s) and (2) in-capillary assays in which the enzymatic reaction and analyte separation are performed in the same capillary. In case of the in-capillary assays, the enzyme may be immobilized or in solution. The latter is also referred to as electrophoretically mediated microanalysis (EMMA), while in the case of immobilized enzyme the term immobilized enzyme reactor (IMER) is used. The present review summarizes the literature on CE-based enzyme assays published between January 2010 and April 2015. Immobilized enzyme reactors as well as microfluidic devices applied to the study of enzymatic activity will also be briefly addressed.

     

Micro enzymatic assay coupled to capillary electrophoresis via liquid junction

Summary A system for capillary electrophoresis combined with enzymatic assay has been evaluated for the two enzymes glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Instrumentation included a post-column reactor coupled to the separation capillary by a liquid junction. A technique for generating a substrate solution flow into the reactor by utilizing two high voltage supplies is proposed. This method offers a high degree of freedom in optimizing the separation and enzymatic reaction conditions individually. Possibilities for improving the enzymatic assay sensitivity were also examined.     

Electrophoretically mediated microanalysis for in-capillaryelectrical cell lysis and fast enzyme quantification by capillary electrophoresis

In this study, a novel capillary electrophoresis (CE)-based enzymatic assay was developed to evaluate enzymatic activity in whole cells. β-Galactosidase expression was used as an example, as it is a biomarker for assessing replicative senescence in mammalian cells. It catalyzes the hydrolysis of para-nitrophenyl-β-d-galactopyranoside (PNPG) into para-nitrophenol (PNP). The CE-based assay consisted of four main steps: (1) hydrodynamic injection of whole intact cells into the capillary, (2) in-capillary lysis of these cells by using pulses of electric field (electroporation), (3) in-capillary hydrolysis of PNPG by the β-galactosidase—released from the lysed cells—by the electrophoretically mediated microanalysis (EMMA) approach, and (4) on-line detection and quantification of the PNP formed. The developed method was applied to Escherichia coli as well as to human keratinocyte cells at different replicative stages. Results obtained by CE were in excellent agreement with those obtained from off-line cell lysates which proves the efficiency of the in-capillary approach developed. This work shows for the first time that cell membranes can be disrupted in-capillary by electroporation and that the released enzyme can be subsequently quantified in the same capillary. Enzyme quantification in cells after their in-capillary lysis has never been conducted by CE. The developed CE approach is automated, economic, eco-friendly, and simple to conduct. It has attractive applications in bacteria or human cells for early disease diagnostics or insights for development in biology.

A novel approach for a non competitive capillary electrophoresis immunoassay with laser-induced fluorescence detection for the determination of human serum albumin

A new non competitive capillary electrophoresis immunoassay format based on a separation into a capillary modified by analyte immobilisation is described. The injection of an excess of labelled antibody off-line preincubated with the analyte allows the surface capture of the free antibody and the immunocomplex detection. It was developed using the human serum albumin (HSA) as analyte, two FITC-labelled antibodies and a HSA covalently linked capillary. Two calibration curves with good run-to-run reproducibility and LOD--respectively 14.0 nM for the FITC-polyclonal antibody and 9.0 nM for the FITC-monoclonal antibody--were achieved. The assay was applied to HSA determination in spiked samples of human urine with acceptable recoveries.     

Development of an enzymatic microreactor based on microencapsulated laccase with off-line capillary electrophoresis for measurement of oxidation reactions

Microencapsulation is used here as a new technique to immobilize enzymes in a microreactor coupled off-line to capillary electrophoresis (CE), allowing the determination of enzymatic reaction products. The redox enzyme laccase was encapsulated using the method of interfacial cross-linking of poly(ethyleneimine) (PEI). The 50 μm diameter capsules were slurry packed from a suspension into a capillary-sized reactor made easily and quickly from a short length of 530 μm diameter fused-silica tubing. The volume of the bed of laccase microcapsules in the microreactor was in the order of 1.1 μL through which 50 μL of the substrate o-phenylenediamine (OPD) was flowed. The oxidation product 2,3-diaminophenazine (DAP) and the remaining OPD were quantified by CE in a pH 2.5 phosphate buffer. Peak migration time reproducibility was in the order of 0.4% RSD and peak area reproducibility was less than 1.7% RSD within the same day. Using the OPD peak area calibration curve, a conversion efficiency of 48% was achieved for a 2-min oxidation reaction in the microreactor.     

Comparison of off- and in-line solid-phase extraction for enhancing sensitivity in capillary electrophoresis using ochratoxin as a model compound

This paper proposes and compares two approaches based on off- and in-line solid-phase extraction (SPE), intended to enhance sensitivity in capillary electrophoresis with ultraviolet detection (CE-UV) using as a model the determination of ochratoxin A (OA) in river water samples. In the off-line SPE mode, the reversed-phase sorbent (octadecilsylane, C₁₈) selectively retains the target analyte (OA) and allows large volumes of the sample (70 mL) to be introduced and subsequently eluted in a small volume (0.1 mL) of an appropriate solution. In the in-line SPE mode, a custom-made microcartridge is inserted near the inlet of the capillary, which is filled with the same C₁₈ sorbent. This solid phase selectively retains OA present in a sample volume as low as approximately 640 μL for subsequent elution with ca. 135 nL of an appropriate eluent. The limit of detection (LOD) obtained with the in-line SPE method was 1 ng L^-1, which is 3 orders of magnitude lower than that obtained with CE-UV and roughly 1 order lower than that provided by the off-line SPE-CE-UV method.