title: A rapid, nonradioactive assay for measuring heparan sulfate C-5 epimerase activity using hydrogen/deuterium exchange-mass spectrometry.
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.
authors.affiliation: Glycomics and Glycoproteomics, Centre for Cellular and Molecular Platforms, NCBS-TIFR, GKVK Post, Bellary Road, Bangalore, 560065, India, babup@ncbs.res.in.
authors.forename: Ponnusamy
authors.initials: P
authors.lastname: Babu
authors.forename: Xylophone V
authors.initials: XV
authors.lastname: Victor
authors.forename: Karthik
authors.initials: K
authors.lastname: Raman
authors.forename: Balagurunathan
authors.initials: B
authors.lastname: Kuberan
mesh_terms: D000818:Animals; D055162:Biocatalysis; D002238:Carbohydrate Epimerases / Q000302:isolation & purification / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D041961:Deuterium Exchange Measurement / Q000379:methods*; D004187:Disaccharides / Q000378:metabolism; D057075:Enzyme Assays / Q000379:methods*; D020723:Glucuronic Acid / Q000737:chemistry / Q000378:metabolism; D006497:Heparan Sulfate / Q000378:metabolism*; D006801:Humans; D007067:Iduronic Acid / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D061987:Sf9 Cells
chemical_list: D002238:Carbohydrate Epimerases; D004187:Disaccharides; D020723:Glucuronic Acid; D006497:Heparan Sulfate; D007067:Iduronic Acid; C560363:heparosan