Advances in Capillary Electrophoresis-Based Enzyme Assays

Abstract

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...

Capillary electrophoresis‐based enzyme assays for β‐lactamase enzymes

“…In the case of β‐lactamases, various formats have been employed including photometric, biochemical, mass spectrometric or polymerase chain reaction‐based and other sequencing assays as summarized, for example, in . Due to the high efficiency and minute sample requirements, CE has been shown to be a powerful microanalytical technique for the determination of the activity of enzymes including the characterization of substrates and inhibitors . With regard to penicillinases, CE–MS has been used to identify β‐lactamase‐derived proteins .…”

Fast and accurate enzyme activity measurements using a chip-based microfluidic calorimeter

“…Therefore various label-free methods for enzyme activity measurements have been developed, based on different techniques, such as liquid chromatography-mass spectrometry (LC-MS) [3], capillary electrophoresis [4,5], a method based on macrocycle-fluorescent dye complex formation [6], and methods based on heat measurements with isothermal titration calorimetry (ITC) [7]. Recently, a method for timeresolved initial rate measurements using isothermal titration calorimetry (ITC) has been developed as a generic assay for the activity of enzymes.…”

Enzyme and inhibition assay of urease by continuous monitoring of the ammonium formation based on capillary electrophoresis

“…On the other hand, for on‐line enzyme assays, all the necessary procedures for the assay, including the load of the substrate and enzyme/inhibitor, the trigger and termination of the reaction, the determination and measurement of the product are performed in one analytical instrument. During the past two decades, on‐line assays have been developed rapidly for a variety of enzymes, which have shown significant advantages over off‐line assays for rapid and accurate determination of various enzyme activity and inhibition .…”

“…As a high performance liquid separation technique, CE offers several advantages, such as high efficiency, sensitivity, fast analysis, low sample volume requirement and so on. Over the past few decades, CE has been applied not only as a separation tool with high performance but also as a versatile platform for on‐line enzyme studies . Several approaches have been developed based on CE for on‐line assay of various kinds of enzymes.…”

“…In one approach, namely electrophoretically mediated microanalysis technique , the injected enzyme and substrate plugs inside the capillary are mixed to initiate the reaction due to their different electrophoretic mobilities, and components of the reaction mixture are then separated for CE analysis. Another commonly used approach is immobilized enzyme reactor , where enzyme is immobilized on the capillary inner surface or a suitable carrier attached to the capillary. After injected into capillary, reactant (substrate(s), coenzyme) flow first passes through the part of the capillary containing an immobilized enzyme reactor to initiate enzyme reaction.…”