Abstract
Heparin and heparan sulfate (HS) glycosaminoglycans have important roles in anticoagulation, human development, and human diseases. HS C5-epimerase, which catalyzes the epimerization of GlcA to IdoA, is a crucial enzyme involved in the biosynthesis of heparin-related biomolecules. Here, we describe a detailed method for measuring the total activity of HS C5-epimerase that involves the following steps: H/D exchange upon epimerization of the substrate with HS C5-epimerase, low-pH nitrous acid treatment of the substrate, the separation of low-pH nitrous acid-cleaved disaccharides using HPLC, and mass spectrometry analysis. This nonradioactive method is rapid and sensitive and, importantly, allows us to study the reversible nature of HS C5-epimerase.
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Acknowledgements
This work was supported in part by NIH grants (P01HL107152 and R01GM075168) to B.K. and by the NIH fellowship F31CA168198 to K.R. P.B. thanks Centre for Cellular and Molecular Platforms for support.
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Babu, P., Victor, X.V., Raman, K., Kuberan, B. (2015). A Rapid, Nonradioactive Assay for Measuring Heparan Sulfate C-5 Epimerase Activity Using Hydrogen/Deuterium Exchange-Mass Spectrometry. In: Balagurunathan, K., Nakato, H., Desai, U. (eds) Glycosaminoglycans. Methods in Molecular Biology, vol 1229. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1714-3_19
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DOI: https://doi.org/10.1007/978-1-4939-1714-3_19
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