Mobility shift-based electrophoresis coupled with fluorescent detection enables real-time enzyme analysis of carbohydrate sulfatase activity

Byrne, Dominic P, London, James A ORCID: 0000-0002-5823-7057, Eyers, Patrick A ORCID: 0000-0002-9220-2966, Yates, Edwin A ORCID: 0000-0001-9365-5433 and Cartmell, Alan ORCID: 0000-0002-5512-249X (2021) Mobility shift-based electrophoresis coupled with fluorescent detection enables real-time enzyme analysis of carbohydrate sulfatase activity. BIOCHEMICAL JOURNAL, 478 (4). pp. 735-748.

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Official URL: http://dx.doi.org/10.1042/bcj20200952

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.

Item Type:	Article
Uncontrolled Keywords:	Boron Compounds, Sulfotransferases, Glycosaminoglycans, Bacterial Proteins, Recombinant Proteins, Fluorescent Dyes, Chromatography, High Pressure Liquid, Chromatography, Ion Exchange, Electrophoresis, Capillary, Electrophoretic Mobility Shift Assay, Microfluidic Analytical Techniques, Fluorometry, Nuclear Magnetic Resonance, Biomolecular, Carbohydrate Conformation, Substrate Specificity, Kinetics, Computer Systems, High-Throughput Screening Assays, Bacteroides thetaiotaomicron
Divisions:	Faculty of Health and Life Sciences Faculty of Health and Life Sciences > Institute of Systems, Molecular and Integrative Biology
Depositing User:	Symplectic Admin
Date Deposited:	07 Apr 2021 10:05
Last Modified:	18 Jan 2023 22:54
DOI:	10.1042/BCJ20200952
Related URLs:	Author Publisher
URI:	https://livrepository.liverpool.ac.uk/id/eprint/3118205