Determination of enzymatic activity by capillary electrophoresis

Glatz, Zdeněk

doi:10.1016/j.jchromb.2006.02.051

Cited by 77 publications

(63 citation statements)

References 155 publications

(36 reference statements)

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“…In the last two decades, CE has become a powerful technique, which can provide highly efficient separations; the evaluation of enzymatic reaction by CE has been under active investigation in recent years [28,29]. Compared to the other methods, the CE-based procedures are rapid, can be automated, and require only small sample amounts.…”

Section: Discussionmentioning

confidence: 99%

CE assay of methylmalonyl‐coenzyme‐A mutase activity

et al. 2007

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Methylmalonyl-coenzyme A mutase (MCM) is a 5'-deoxyadenosylcobalamin-linked mitochondrial enzyme that catalyzes the isomerization of L-methylmalonyl-coenzyme A to succinyl-coenzyme A. We described a method for methylmalonyl-CoA and succinyl-CoA separation by CE, suitable for the evaluation of MCM activity. The working conditions for optimal separation were obtained in order to achieve the best resolution in the shortest analysis time. The optimization of buffer composition together with other variables, such as injection time, separation voltage, migration time, and capillary temperature, resulted in a solution of 30 mM NaH2PO4 containing 15 mM SDS, pH 3.2. Separations were carried out in an uncoated fused-silica capillary (55 cm, 50 microm id) at -25 kV, reading at 254 nm. The method performance was evaluated by measuring total and holo-MCM activity in biological matrices such as rat liver and human peripheral blood lymphocytes (PBL). The mean MCM activity was expressed in nmol/h/mg protein of tissue/cell extract and was calculated from the amount of reaction product formed. The rapidity of analysis and utmost precision (repeatability and within-laboratory reproducibility) point out the potentialities of the proposed method for the differential diagnosis of methylmalonic acidemia, in relation to protein or coenzyme defects.

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Section: Discussionmentioning

confidence: 99%

CE assay of methylmalonyl‐coenzyme‐A mutase activity

et al. 2007

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“…(Yan, 1996). Because of this flexibility, CE has become increasingly popular in many bio-analytical fields, with uses ranging from enzyme kinetics, chiral separations to DNA sequencing (Anzenbacher and Hudecek, 2001;Dovichi and Zhang, 2000;Glatz, 2006;Ha et al, 2006;Portmann et al, 2010;Prost and Thormann, 2003;Ptolemy and Britz-McKibbin, 2008;Rabe et al, 2008;Suntornsuk).…”

Section: Enzymatic Kinetics By Cementioning

confidence: 99%

“…This is relevant in cases when products generated are stereoisomers (e.g., diastereomers, enantiomers) with similar spectroscopic properties that excludes conventional absorbance or fluorescence methods (Gavina et al, 2010). Many studies have used CE to examine enzymatic activity with off-line stopped reactions or on-line using the capillary as a microreactor in electrophoretically mediated microanalysis (EMMA; Glatz, 2006;Zhang et al, 2008a). The basic principle behind EMMA is the ability of the CE instrument to inject distinct plugs of reactant into the capillary and mix them together with pressure or voltage for an in-capillary reaction.…”

Section: Enzymatic Kinetics By Cementioning

confidence: 99%

Functional Screening of Cytochrome P450 Activity and Uncoupling by Capillary Electrophoresis

2011

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Abstract:Cytochrome P450s are a super-family of heme containing proteins that are found in all domains of life and are involved in the synthesis and breakdown of steroids, xenobiotics, and pharmaceuticals. Using five heterologously expressed zebrafish (Danio rerio) CYP1s, an assay was developed for CYP activity in order to monitor the consumption of the cofactor NADPH, providing a label-free screening tool to determine function of novel CYP genes. Using wellestablished fluorogenic substrates, NADPH and NADP + were separated by capillary electrophoresis (CE) from stopped CYP1 reactions and measured with UV absorbance detection as a surrogate to assess the rate of substrate metabolism. Product formation was confirmed by fluorometric detection of metabolites, giving rates of enzyme activity which could be compared to the rates of cofactor turn-over measured by CE. 17β-estradiol, four alkoxyresorufin and two coumarin based synthetic fluorogenic CYP substrates were screened for activity with recombinant zebrafish CYP1A, 1B1, 1C2, 1C2 and 1D1. Cofactor consumption was generally much larger than product formation for the majority of substrates and CYP1 isoforms, suggesting that the majority of metabolic events were uncoupled. Large uncoupling was seen in CYP1 when metabolizing estradiol, showing that endogenous compounds can also show severe uncoupling.Reactive oxygen species (ROS), a product of uncoupled events, were detected with 2,7-dichorofluorescein. Attempts for concomitant detection of ROS production and cofactor consumption with CE-UV detection were investigated, however, detection limits for 2,7-dichlorofluorescein were not adequate for detection of hydrogen peroxide production from CYP1 mediated reactions. Future work will be required to develop a single assay to quantitatively measure CYP activity by CE for functional determination of CYPs with unknown function.MSc Thesis -J.G. Harskamp McMaster University -Biology iv Acknowledgements:

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“…Capillary electrophoresis (CE) has emerged as a promising means to detect and monitor enzyme activity as an alternative to HPLC or radiochemistry [8][9][10] . Advantages of CE include low sample consumption, rapid analysis, high sensitivity, and efficient separation of product from substrate for detection and quantification.…”

Section: Introductionmentioning

confidence: 99%

Microfabricated Channel Array Electrophoresis for Characterization and Screening of Enzymes Using RGS−G Protein Interactions as a Model System

et al. 2008

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A microfluidic chip consisting of parallel channels designed for rapid electrophoretic enzyme assays was developed. Radial arrangement of channels and a common waste channel allowed chips with 16 and 36 electrophoresis units to be fabricated on a 7.62 × 7.62 cm glass substrate. Fluorescence detection was achieved using a Xe arc lamp source and commercial CCD camera to image migrating analyte zones in individual channels. Chip performance was evaluated by performing electrophoretic assays for G protein GTPase activity on chip using BODIPY-GTP as enzyme substrate. A 16-channel design proved to be useful in extracting kinetic information by allowing serial electrophoretic assays from 16 different enzyme reaction mixtures at 20 s intervals in parallel. This system was used to rapidly determine enzyme concentrations, optimal enzymatic reaction conditions, and MichaelisMenton constants. A chip with 36 channels was used for screening for modulators of the G protein: RGS protein interaction by assaying the amount of product formed in enzyme reaction mixtures that contained test compounds. 36 electrophoretic assays were performed in 30 s suggesting the potential throughput up to 4,320 assays per hour with appropriate sample handling procedures. Both designs showed excellent reproducibility of peak migration time and peak area. Relative standard deviations of normalized peak area of enzymatic product BODIPY-GDP were 5% and 11% respectively in the 16 and 36-channel designs.

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Determination of enzymatic activity by capillary electrophoresis

Cited by 77 publications

References 155 publications

CE assay of methylmalonyl‐coenzyme‐A mutase activity

CE assay of methylmalonyl‐coenzyme‐A mutase activity

Functional Screening of Cytochrome P450 Activity and Uncoupling by Capillary Electrophoresis

Microfabricated Channel Array Electrophoresis for Characterization and Screening of Enzymes Using RGS−G Protein Interactions as a Model System

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