Byrne, Dominic P., London, James A., Eyers, Patrick A. et al. (2 more authors) (2021) Mobility shift-based electrophoresis coupled with fluorescent detection enables real-time enzyme analysis of carbohydrate sulfatase activity. Biochemical journal. pp. 735-748. ISSN 1470-8728
Abstract
Sulfated carbohydrate metabolism is a fundamental process, which occurs in all domains of life. Carbohydrate sulfatases are enzymes that remove sulfate groups from carbohydrates and are essential to the depolymerisation of complex polysaccharides. Despite their biological importance, carbohydrate sulfatases are poorly studied and challenges remain in accurately assessing the enzymatic activity, specificity and kinetic parameters. Most notably, the separation of desulfated products from sulfated substrates is currently a time-consuming process. In this paper, we describe the development of rapid capillary electrophoresis coupled to substrate fluorescence detection as a high-throughput and facile means of analysing carbohydrate sulfatase activity. The approach has utility for the determination of both kinetic and inhibition parameters and is based on existing microfluidic technology coupled to a new synthetic fluorescent 6S-GlcNAc carbohydrate substrate. Furthermore, we compare this technique, in terms of both time and resources, to high-performance anion exchange chromatography and NMR-based methods, which are the two current 'gold standards' for enzymatic carbohydrate sulfation analysis. Our study clearly demonstrates the advantages of mobility shift assays for the quantification of near real-time carbohydrate desulfation by purified sulfatases, and will support the search for small molecule inhibitors of these disease-associated enzymes.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2021 The Author(s) |
Keywords: | Bacterial Proteins/analysis,Bacteroides thetaiotaomicron/enzymology,Boron Compounds/analysis,Carbohydrate Conformation,Chromatography, High Pressure Liquid,Chromatography, Ion Exchange,Computer Systems,Electrophoresis, Capillary/methods,Electrophoretic Mobility Shift Assay/methods,Fluorescent Dyes/analysis,Fluorometry/methods,Glycosaminoglycans/metabolism,High-Throughput Screening Assays/methods,Kinetics,Microfluidic Analytical Techniques/methods,Nuclear Magnetic Resonance, Biomolecular,Recombinant Proteins/analysis,Substrate Specificity,Sulfotransferases/analysis |
Dates: |
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Institution: | The University of York |
Academic Units: | The University of York > Faculty of Sciences (York) > Biology (York) |
Depositing User: | Pure (York) |
Date Deposited: | 19 Feb 2025 16:40 |
Last Modified: | 19 Feb 2025 17:10 |
Published Version: | https://doi.org/10.1042/BCJ20200952 |
Status: | Published |
Refereed: | Yes |
Identification Number: | 10.1042/BCJ20200952 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:223575 |
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Description: Mobility shift-based electrophoresis coupled with fluorescent detection enables real-time enzyme analysis of carbohydrate sulfatase activity
Licence: CC-BY 2.5