Advances in enzyme substrate analysis with capillary electrophoresis

Abstract

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 K_M 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 K_I 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.

Keywords: Capillary electrophoresis; Enzyme; Inhibitor; Michaelis-Menten constant.

Fig. 1

Schematic of capillary electrophoresis setup. It consists of capillary, buffer reservoir containing background electrolyte and a detector. Analytes are separated in capillary under the influence of the electric field supplied by high voltage power supply. A color version of this figure is available on-line.

Fig. 2

Conceptual electropherogram showing the separation of a hypothetical mixture of five analytes using capillary electrophoresis. The mixture is separated by the charge-to-size ratio of the analytes. The charge and size of the analytes depend on the molecular pK_a value and the hydrodynamic radius, respectively. The molecular weights (MW) of the analytes are proportional to the hydrodynamic volumes. The vectors represent the electroosmotic flow (EOF), and the electrophoretic (EPH) and net velocities of the analytes. In this conceptual depiction the pH of the background electrolyte maintains positive charge of cation 1 and cation 2. Because it is larger, cation 2 has a slower migration time. Although anion 1 and anion 2 are both negatively charged, anion 2 has a slower migration because it is smaller and migrates against the bulk EOF. A color version of this figure is available on-line.

Fig. 3

Conceptual diagrams demonstrating K_M analysis using capillary electrophoresis. Electropherograms in inset show five different substrate concentrations and the products generated after the enzyme reactions. The generated products were zoomed to emphasize the product area increases as the initial substrate concentration increases. The curve on the right depicts the Michaelis-Menten curve is generated by plotting the rate of product formation versus the substrate concentration. A color version of this figure is available on-line.

Fig. 4

Electropherogram of standards relevant to the deacetylation of the substrate, (2S)-6-(acetylamino)-2-[[(7-methoxy-2-oxo-2H-1-benzopyran-4-yl) methyl]amino]-hexanamide, (peak 4) with human SIRT1 enzyme. The product, (2S)-6-amino-2-[[(7-methoxy-2-oxo-2H-1-benzopyran-4-yl)methyl]amino]-hexanamide, (peak 1), migrates faster than the substrate. Peaks 2 and 3 are the co-product nicotinamide, and the internal standard 4-(aminomethyl)benzoic acid, respectively. From H. Abromeit, S. Kannan, W. Sippl, G.K.E. Scriba, A new nonpeptide substrate of human sirtuin in a capillary electrophoresis-based assay. Investigation of the binding mode by docking experiments, Electrophoresis 33(11) (2012) 1652–1659. Copyright © 2012 by John Wiley Sons, Inc. Reprinted by permission of John Wiley & Sons, Inc.

Fig. 5

Conceptual diagram of electrophoretic mixing in the fixed enzyme zone. The mixing duration was determined by the total number of times the substrate passes through the fixed enzyme zone and the formation of product increases with the mixing time. The results of the electrophoretic mixing are shown in the adjacent electropherogram where the rate of product formation are obtained. Reprinted with permission from S. Gattu, C.L. Crihfield, L.A. Holland, Microscale Measurements of Michaelis–Menten Constants of Neuraminidase with Nanogel Capillary Electrophoresis for the Determination of the Sialic Acid Linkage, Analytical Chemistry 89(1) (2017) 929–936. Copyright 2017 American Chemical Society. Further permissions related to the material excerpted should be directed to the ACS. <http://pubs.acs.org/doi/abs/10.1021/acs.analchem.6b04074>.

Fig. 6

Conceptual diagrams demonstrating K_I analysis using capillary electrophoresis. Traces in A depict electropherograms generated in the absence and presence of inhibitor showing decrease in product area when inhibitor was present. Traces in B depict the product generated in the absence and presence of inhibitor at various substrate concentrations with the concentration of inhibitor being same. The traces in grey with inhibitor shows the decrease in product area and were offset in time for clearer representation. The graph in C is a hypothetical Michaelis-Menten curve generated by plotting the substrate concentration versus the rate of product formation in the absence (− inhibitor) and the presence (+ inhibitor) of the inhibitors. A color version of this figure is available on-line.

Fig. 7

Scanning electron microscopic image of silica monolith. The image shows a rigid skeleton and even distribution of macropores in synthesized porus silica monolith within the capillary bioreactor. Reprinted with permission from C. Zhao, R. Yin, J. Yin, D. Zhang, H. Wang, Capillary Monolithic Bioreactor of Immobilized Snake Venom Phosphodiesterase for Mass Spectrometry Based Oligodeoxynucleotide Sequencing, Anal. Chem. 84(2) (2012) 1157–1164. Copyright 2012 American Chemical Society.

Fig. 8

Schematic of capillary zone electrophoresis coupled with mass spectrometry showing on-line protein digests. The schematic in A is of strong cation exchange based monolith based microreactor coupled with capillary zone electrophoresis-mass spectrometry. The mass spectrum in B is of an on-line analysis of Xenopus laevis protein digests achieved by the set up used in A. Reprinted with permission from Z. Zhang, L. Sun, G. Zhu, O.F. Cox, P.W. Huber, N.J. Dovichi, Nearly 1000 Protein Identifications from 50 ng of Xenopus laevis Zygote Homogenate Using Online Sample Preparation on a Strong Cation Exchange Monolith Based Microreactor Coupled with Capillary Zone Electrophoresis, Analytical Chemistry 88(1) (2016) 877–882. Copyright 2016 American Chemical Society.