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

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TitleA rapid, nonradioactive assay for measuring heparan sulfate C-5 epimerase activity using hydrogen/deuterium exchange-mass spectrometry.
Publication TypeJournal Article
Year of Publication2015
AuthorsBabu P, Victor XV, Raman K, Kuberan B
JournalMethods Mol Biol
Volume1229
Pagination209-19
Date Published2015
ISSN1940-6029
KeywordsAnimals, Biocatalysis, Carbohydrate Epimerases, Chromatography, Ion Exchange, Chromatography, Liquid, Deuterium Exchange Measurement, Disaccharides, Enzyme Assays, Glucuronic Acid, Heparitin Sulfate, Humans, Iduronic Acid, Mass Spectrometry, Sf9 Cells
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.

DOI10.1007/978-1-4939-1714-3_19
Alternate JournalMethods Mol. Biol.
PubMed ID25325956
Grant ListF31CA168198 / CA / NCI NIH HHS / United States
P01HL107152 / HL / NHLBI NIH HHS / United States
R01GM075168 / GM / NIGMS NIH HHS / United States