A highly sensitive capillary electrophoresis method using p-nitrophenyl 5′-thymidine monophosphate as a substrate for the monitoring of nucleotide pyrophosphatase/phosphodiesterase activities

Lee, Sang-Yong; Lévesque, Sébastien A.; Sévigny, Jean; Müller, Christian

doi:10.1016/j.jchromb.2012.10.044

Cited by 17 publications

(18 citation statements)

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“…Our group has long-standing experience in the quantification of nucleotides by CE, and especially in enzyme assays involving the formation or transformation of nucleotides [15][16][17][18][19][20][21][22]. Thus we decided to quantify the nucleotide PAP which is formed from the co-substrate PAPS during the CST reaction.…”

Section: Development and Optimization Of Ce Methods For Monitoring Cstmentioning

confidence: 99%

A capillary electrophoresis method with dynamic pH junction stacking for the monitoring of cerebroside sulfotransferase

Zech

Gieselmann

et al. 2015

Journal of Chromatography A

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Section: Development and Optimization Of Ce Methods For Monitoring Cstmentioning

confidence: 99%

A capillary electrophoresis method with dynamic pH junction stacking for the monitoring of cerebroside sulfotransferase

Zech

Gieselmann

et al. 2015

Journal of Chromatography A

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“…An off-line enzyme incubation is preferred if the working pH range of the enzyme reaction does not support the separation of product from substrate. These reactions typically use reaction volumes of 100 μL or greater [29,38,39,49,53–55], although smaller volumes have been reported [36,37]. Off-line enzyme reactions can be quenched to allow more flexibility in the separation of each sample; for example, when reactions are run in parallel.…”

Section: Adapting the Separation To Determine Km Valuesmentioning

confidence: 99%

“…Most of the on-line incubations described in the paper either use the same pH [13,18,23,24,34,43,56–62], or a pH close to enzyme reaction [26,32,33,41,55]. In some instances a wide difference of pH existed between the enzyme incubation buffer and the separation buffer [31,63].…”

Section: Adapting the Separation To Determine Km Valuesmentioning

confidence: 99%

“…Off-line analysis for enzymatic inhibition studies are performed by incubation of enzyme and substrate before the capillary electrophoresis analysis. The enzymatic reactions are then quenched and injected into the capillary for separation [14,15,20,21,28,29,38,40,55,65,71,72]. The substrates and products are separated and quantified for kinetic analysis of enzyme reaction and elucidation of the inhibitor mechanism and effectiveness.…”

Section: Evaluating Enzyme Inhibitors Using Capillary Electrophoresismentioning

confidence: 99%

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Advances in enzyme substrate analysis with capillary electrophoresis

Gattu

Crihfield

et al. 2018

Methods

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Capillary electrophoresis provides a rapid, cost-effective platform for enzyme and substrate characterization. The high resolution achievable by capillary electrophoresis enables the analysis of substrates and products that are indistinguishable by spectroscopic techniques alone, while the small volume requirement enables analysis of enzymes or substrates in limited supply. Furthermore, the compatibility of capillary electrophoresis with various detectors makes it suitable for KM determinations ranging from nanomolar to millimolar concentrations. Capillary electrophoresis fundamentals are discussed with an emphasis on the separation mechanisms relevant to evaluate sets of substrate and product that are charged, neutral, and even chiral. The basic principles of Michaelis-Menten determinations are reviewed and the process of translating capillary electrophoresis electropherograms into a Michaelis-Menten curve is outlined. The conditions that must be optimized in order to couple off-line and on-line enzyme reactions with capillary electrophoresis separations, such as incubation time, buffer pH and ionic strength, and temperature, are examined to provide insight into how the techniques can be best utilized. The application of capillary electrophoresis to quantify enzyme inhibition, in the form of KI or IC50 is detailed. The concept and implementation of the immobilized enzyme reactor is described as a means to increase enzyme stability and reusability, as well as a powerful tool for screening enzyme substrates and inhibitors. Emerging techniques focused on applying capillary electrophoresis as a rapid assay to obtain structural identification or sequence information about a substrate and in-line digestions of peptides and proteins coupled to mass spectrometry analyses are highlighted.

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“…This can be overcome by the application of stacking techniques such as online preconcentration step which have been summarized in recent general reviews on the topic [14][15][16][17]. For example, sweeping was employed to achieve the appropriate sensitivity when monitoring the activity of nucleotide pyrophosphatases/phosphodiesterases [18] as well as for a MEKC assay of sirtuin enzymes [19]. Huang and Tzeng used polyethylene glycol 4000 in the BGE as the EOF suppressor for sample stacking by transient isotachophoresis when analyzing the activity of ribonuclease reductase [20].…”

Section: Pre-capillary Enzyme Assaysmentioning

confidence: 99%

Advances in Capillary Electrophoresis-Based Enzyme Assays

Scriba

Belal

2015

Chromatographia

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IntroductionEnzymes catalyze metabolic reactions in cells regulating homeostasis, growth and reproduction of living organisms. Enzymes are also often involved in human disease. The analysis of the human genome has revealed that within the so-called druggable genome a significant portion (in some analyses more than half) of the potential drug targets are enzymes [1][2][3]. Consequently, enzymes are important targets in the development of new drugs. Moreover, enzymes play a role in the clinical diagnosis of diseases. Therefore, effective methods for characterization of enzymes as well as for the determination of their activity are important, for example, for the understanding of enzyme-mediated metabolic reactions or the identification of substrates and inhibitors for the treatment of human diseases.Capillary electrophoresis (CE) has been applied to enzyme analysis for more than 20 years since the first report by Banke et al. on an assay of alkaline protease [4]. Since then, all aspects of enzyme-related analysis have been studied by CE methods including the evaluation of enzymatic activity, enzyme kinetics, identification and characterization of enzyme inhibitors and activators as well as metabolic pathways as, for example, summarized in [5][6][7][8][9][10][11][12].Generally, CE-based enzyme assays can be divided in three groups, pre-capillary (offline) assays, in-capillary (online) assays and post-capillary assays. In the pre-capillary assays, all required components including enzyme, substrate, co-factors, etc., are mixed in a vial and incubated for an intended period of time. Subsequently, the reaction is quenched and an aliquot is subjected to CE analysis for the determination of substrate(s) and/or co-substrate(s) and/ or product(s). Multiple sampling or repeated sampling in combination with sequential runs for the determination of Abstract 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 CEbased enzyme assays published between January 2010 and April 2015. Immobilized enzyme reactors as well as microfluidic devices applied to the study of enzymatic a...

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A highly sensitive capillary electrophoresis method using p-nitrophenyl 5′-thymidine monophosphate as a substrate for the monitoring of nucleotide pyrophosphatase/phosphodiesterase activities

Cited by 17 publications

References 50 publications

A capillary electrophoresis method with dynamic pH junction stacking for the monitoring of cerebroside sulfotransferase

A capillary electrophoresis method with dynamic pH junction stacking for the monitoring of cerebroside sulfotransferase

Advances in enzyme substrate analysis with capillary electrophoresis

Advances in Capillary Electrophoresis-Based Enzyme Assays

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