A rapid, nonradioactive assay for measuring heparan sulfate C-5 epimerase activity using hydrogen/deuterium exchange-mass spectrometry

Babu P, Victor XV, Raman K, Kuberan B. Methods Mol Biol. 2015;1229:209-19. doi: 10.1007/978-1-4939-1714-3_19. PMID: 25325956

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.