Development of both colorimetric and fluorescence heparinase activity assays using fondaparinux as substrate

Analytical Biochemistry
Simone SchiemannSusanne Alban

Abstract

Because tumors and other diseases are characterized by increased heparanase levels, human heparanase is a promising drug target and diagnostic marker. Therefore, methods are needed to determine heparanase activity and to examine potential inhibitors. Because of substrate comparability, we used the bacterial enzyme heparinase II (heparinase) for the assay development. Usually the substrate of heparanase assays is heparan sulfate, which has several disadvantages. Because of that, we used fondaparinux, which is being cleaved by both heparanase and heparinase. Two concepts to detect its degradation were examined: measurement of anti-factor Xa activity of fondaparinux and its direct quantification with the fluorescent sensor polymer-H. Using fondaparinux as substrate, the anti-factor Xa assay was shsown to be appropriate to determine heparinase activity. The detection with polymer-H was easier and even faster to perform. Linearity was given with fondaparinux as well as heparan sulfate, and heparin as substrates, but fondaparinux turned out to be most suitable. By modifications (incubation time, fondaparinux concentration, and polymer-H concentration), the limit of quantification and the linear range can be adapted to the respective ...Continue Reading

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Citations

Mar 29, 2014·Journal of Pharmaceutical and Biomedical Analysis·Ann-Kathrin SchoenfeldSusanne Alban
Apr 9, 2016·Future Medicinal Chemistry·Silvia RivaraGiuseppe Giannini
Nov 18, 2018·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Mohit Chhabra, Vito Ferro
Sep 5, 2019·Bio-protocol·Jyothi C Sistla, Umesh R Desai
Jul 17, 2018·Journal of Medicinal Chemistry·Valentina Noemi MadiaGiuseppe Giannini
Sep 5, 2019··JC SistlaUmesh Desai

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