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Development of a solid phase array assay for the screening of galactose oxidase activity and for Fast identification of inhibitors

Development of a solid phase array assay for the screening of galactose oxidase activity and for Fast identification of inhibitors
Development of a solid phase array assay for the screening of galactose oxidase activity and for Fast identification of inhibitors
Background: Galactose oxidase (GOase) catalyses the highly selective oxidation of terminal galactosides on a wide range of natural glycoconjugates and has found wide applications in biotechnology – particularly in biocatalysis. GOase is copper dependent and uses oxygen to oxidise the C6-primary alcohol of galactose and produces hydrogen peroxide. The enzyme activity can be conveniently assessed by a colorimetric assay. Objectives: The objective of the present study was to develop an assay system, which is independent of the hydrogen peroxide formation to identify possible fluorinated GOase inhibitors. In case that the inhibitor bears a primary or secondary alcohol, it could also be oxidised by the enzyme. In such case, the colorimetric assay is not able to distinguish between substrate and inhibitor, since oxidation of both molecules would result in the formation of hydrogen peroxide. Methods: D-galactose (D-Gal) was immobilised onto a gold surface functionalised by selfassembled monolayers (SAMs,). A GOase solution was then added to the surface in a droplet for a certain period of time and thereafter washed away. The activity of GOase on the immobilised D-Gal can then be quantified by MALDI-ToF MS. Results: For inhibition studies, GOase was incubated together with 62.5 mM of deoxy-fluorinated monosaccharides on the D-Gal displaying platform. Five deoxy-fluorinated D-Gal showed a >50% inhibition of its activity. The array system has been moreover utilised to determine the apparent IC50 value of 3-F-Gal 15 as a proof of principle. Conclusion: The developed array platform allows the fast identification of GOase substrates and inhibitors from a library of deoxy-fluorinated sugars using MALDI-ToF MS as a label–free readout method. In addition, the enzymatic reaction enables for the in situ activation of sugar-coated surfaces to bioorthogonal aldehydes, which can be utilised for subsequent chemical modifications.
0929-8665
742-746
Weissenborn, Martin J.
f5f3ee0d-411e-49aa-a04e-87c419671d20
Debecker, Damien P.
acf3ce9c-bdc1-4a28-9f74-9d95cf411f40
Golten, Samuel
214a0aed-950c-41b0-8b3f-b71614ce9d5e
Linclau, Bruno
19b9cacd-b8e8-4c65-af36-6352cade84ba
Turner, Nicolas J.
a0f6562a-e317-4f29-a3a4-8ee09add8f31
Flitsch, Sabine L.
74eec2dd-f329-4f0f-999d-73edbcf34e96
Weissenborn, Martin J.
f5f3ee0d-411e-49aa-a04e-87c419671d20
Debecker, Damien P.
acf3ce9c-bdc1-4a28-9f74-9d95cf411f40
Golten, Samuel
214a0aed-950c-41b0-8b3f-b71614ce9d5e
Linclau, Bruno
19b9cacd-b8e8-4c65-af36-6352cade84ba
Turner, Nicolas J.
a0f6562a-e317-4f29-a3a4-8ee09add8f31
Flitsch, Sabine L.
74eec2dd-f329-4f0f-999d-73edbcf34e96

Weissenborn, Martin J., Debecker, Damien P., Golten, Samuel, Linclau, Bruno, Turner, Nicolas J. and Flitsch, Sabine L. (2017) Development of a solid phase array assay for the screening of galactose oxidase activity and for Fast identification of inhibitors. Proteins & Peptide Letter, 24 (8), 742-746. (doi:10.2174/0929866524666170724114348).

Record type: Article

Abstract

Background: Galactose oxidase (GOase) catalyses the highly selective oxidation of terminal galactosides on a wide range of natural glycoconjugates and has found wide applications in biotechnology – particularly in biocatalysis. GOase is copper dependent and uses oxygen to oxidise the C6-primary alcohol of galactose and produces hydrogen peroxide. The enzyme activity can be conveniently assessed by a colorimetric assay. Objectives: The objective of the present study was to develop an assay system, which is independent of the hydrogen peroxide formation to identify possible fluorinated GOase inhibitors. In case that the inhibitor bears a primary or secondary alcohol, it could also be oxidised by the enzyme. In such case, the colorimetric assay is not able to distinguish between substrate and inhibitor, since oxidation of both molecules would result in the formation of hydrogen peroxide. Methods: D-galactose (D-Gal) was immobilised onto a gold surface functionalised by selfassembled monolayers (SAMs,). A GOase solution was then added to the surface in a droplet for a certain period of time and thereafter washed away. The activity of GOase on the immobilised D-Gal can then be quantified by MALDI-ToF MS. Results: For inhibition studies, GOase was incubated together with 62.5 mM of deoxy-fluorinated monosaccharides on the D-Gal displaying platform. Five deoxy-fluorinated D-Gal showed a >50% inhibition of its activity. The array system has been moreover utilised to determine the apparent IC50 value of 3-F-Gal 15 as a proof of principle. Conclusion: The developed array platform allows the fast identification of GOase substrates and inhibitors from a library of deoxy-fluorinated sugars using MALDI-ToF MS as a label–free readout method. In addition, the enzymatic reaction enables for the in situ activation of sugar-coated surfaces to bioorthogonal aldehydes, which can be utilised for subsequent chemical modifications.

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e-pub ahead of print date: 31 July 2017
Published date: 24 October 2017

Identifiers

Local EPrints ID: 417286
URI: http://eprints.soton.ac.uk/id/eprint/417286
ISSN: 0929-8665
PURE UUID: 0458071b-ff00-49d6-9219-6a0ae3848f90
ORCID for Bruno Linclau: ORCID iD orcid.org/0000-0001-8762-0170

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Date deposited: 26 Jan 2018 17:31
Last modified: 19 Feb 2021 02:36

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Contributors

Author: Martin J. Weissenborn
Author: Damien P. Debecker
Author: Samuel Golten
Author: Bruno Linclau ORCID iD
Author: Nicolas J. Turner
Author: Sabine L. Flitsch

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