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Updated: Aug 17, 2026
Analyzing Protein Dynamics Using Hydrogen Exchange Mass Spectrometry
Published on: November 29, 2013
A rapid, nonradioactive assay for measuring heparan sulfate C-5 epimerase activity using hydrogen/deuterium
Ponnusamy Babu1, Xylophone V Victor, Karthik Raman
1Glycomics and Glycoproteomics, Centre for Cellular and Molecular Platforms, NCBS-TIFR, GKVK Post, Bellary Road, Bangalore, 560065, India, babup@ncbs.res.in.
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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