New development in in-capillary electrophoresis techniques for kinetic and inhibition study of enzymes

Nehmé, Hala; Nehmé, Reine; Lafite, Pierre; Routier, Sylvain; Morin, Pascal

doi:10.1016/j.aca.2012.02.003

Cited by 50 publications

(30 citation statements)

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“…However, mixing more than two reactants by EMMA is difficult [8,25,26,28]. In this work, even when combining EMMA to a zero potential amplification step [8,9,26,27] the ADP produced was below the LOQ (about 0.20 M).…”

Section: Preliminary Study Of In-capillary Mixingmentioning

confidence: 69%

“…Electrophoretically mediated microanalysis (EMMA) is the most widely used in-capillary mixing technique for enzymatic studies [24][25][26][27]. However, mixing more than two reactants by EMMA is difficult [8,25,26,28].…”

Section: Preliminary Study Of In-capillary Mixingmentioning

confidence: 99%

“…Moreover, in the case of CDK1/cyclin B and CDK5/p25, applying an electric field during the enzymatic reaction may have also engendered a dissociation of the enzyme CDK1 or CDK5 from its activator altering the enzymatic activity. At-inlet reaction was also performed [25]. However, even after incubating for 30 min, the ADP formed was not quantifiable.…”

Section: Preliminary Study Of In-capillary Mixingmentioning

confidence: 99%

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Human protein kinase inhibitor screening by capillary electrophoresis using transverse diffusion of laminar flow profiles for reactant mixing

Nehmé

Lafite

et al. 2013

Journal of Chromatography A

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Section: Preliminary Study Of In-capillary Mixingmentioning

confidence: 69%

Section: Preliminary Study Of In-capillary Mixingmentioning

confidence: 99%

Section: Preliminary Study Of In-capillary Mixingmentioning

confidence: 99%

See 1 more Smart Citation

Human protein kinase inhibitor screening by capillary electrophoresis using transverse diffusion of laminar flow profiles for reactant mixing

Nehmé

Lafite

et al. 2013

Journal of Chromatography A

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“…Nehme and colleagues compared pre-capillary assays with EMMA as well as a pressure-mediated microanalysis (PMMA) mode in addition to a spectrophotometric assay [49]. In PMMA, the reactant plugs are injected and mixing is achieved by diffusion.…”

Section: In-capillary Enzyme Assaysmentioning

confidence: 99%

“…Enzyme kinetic data, enzyme inhibition [49] Lysosomal β-glucocerebrosidase 4-Methylumbelliferyl-β-d-glucopyranoside 150 mM sodium borate, pH 9.5…”

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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Enzymatic Kinetic Determinations

Hüttl

Frank

2000

Encyclopedia of Analytical Chemistry

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Kinetic methods for analytical applications can be divided into noncatalytic and catalytic methods. Enzyme kinetic determinations are a special type of catalytic methods. Enzymatic reactions are used analytically to determine enzyme activities – for instance, in the diagnosis of diseases, substrate concentrations in the food industry or medicine, and concentrations of effectors – for example, in trace analysis. Owing to the high selectivity of enzymatic analysis, the importance of this method in various fields is growing rapidly. The widest use is observed in clinical chemistry and food chemistry. This article gives only a general survey of the high potential of enzymatic analysis, with references to more detailed literature. Enzymatic reactions are characterized by some special features, explained after the introduction. The theoretical background for kinetic determination is the comprehension of the principles on enzymatic kinetics. In Section 3, the simplest mechanism, outlined by Michaelis and Menten, and several transformations of the equation are described. In this section, two kinetic parameters, enzyme activity and the Michaelis constant, are introduced. Section 4 deals with substances, the so‐called effectors , influencing the rate of an enzymatic reaction. Simple mechanisms of these effectors are shown. Different methods for determination of substrates, enzymes, and effectors, illustrated by many examples, are represented in Section 5. While substrate concentrations can be determined using both equilibrium or kinetic methods, the determination of enzymes and effectors can be performed only by kinetic approaches. In the last two sections, the application of enzymes in flow systems and biosensor development are shown.

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New development in in-capillary electrophoresis techniques for kinetic and inhibition study of enzymes

Cited by 50 publications

References 43 publications

Human protein kinase inhibitor screening by capillary electrophoresis using transverse diffusion of laminar flow profiles for reactant mixing

Human protein kinase inhibitor screening by capillary electrophoresis using transverse diffusion of laminar flow profiles for reactant mixing

Advances in Capillary Electrophoresis-Based Enzyme Assays

Enzymatic Kinetic Determinations

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