An Enzymatic Activity Assay for Heparanase That Is Useful for Evaluating Clinically Relevant Inhibitors and Studying Kinetics
© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature..
The enzyme heparanase cleaves heparan sulfate and is involved in a range of human diseases including cancer, inflammation, diabetes, and viral infection. There is a need for a simple and reliable enzymatic assay to allow for the screening of compounds to find inhibitors of heparanase. We have developed an assay that uses the heparinoid fondaparinux as enzyme substrate and detects one of the products of catalysis, which contains a newly formed reducing terminus, with the tetrazolium salt WST-1. Due to the homogenous substrate and single point of cleavage therein, this assay allows for more systematic kinetic analysis of heparanase inhibitors. Here, we provide a detailed method for conducting this assay and also provide information to assist researchers in evaluating whether the assay is performing properly in their laboratories.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2023 |
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| Erschienen: |
2023 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:2619 |
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| Enthalten in: |
Methods in molecular biology (Clifton, N.J.) - 2619(2023) vom: 02., Seite 227-238 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Hammond, Edward [VerfasserIn] |
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| Links: |
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| Themen: |
9050-30-0 |
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| Anmerkungen: |
Date Completed 24.01.2023 Date Revised 17.11.2023 published: Print Citation Status MEDLINE |
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| doi: |
10.1007/978-1-0716-2946-8_16 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| 520 | |a The enzyme heparanase cleaves heparan sulfate and is involved in a range of human diseases including cancer, inflammation, diabetes, and viral infection. There is a need for a simple and reliable enzymatic assay to allow for the screening of compounds to find inhibitors of heparanase. We have developed an assay that uses the heparinoid fondaparinux as enzyme substrate and detects one of the products of catalysis, which contains a newly formed reducing terminus, with the tetrazolium salt WST-1. Due to the homogenous substrate and single point of cleavage therein, this assay allows for more systematic kinetic analysis of heparanase inhibitors. Here, we provide a detailed method for conducting this assay and also provide information to assist researchers in evaluating whether the assay is performing properly in their laboratories | ||
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