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

Methods Mol Biol. 2015;1229:209-19. doi: 10.1007/978-1-4939-1714-3_19.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biocatalysis
  • Carbohydrate Epimerases / isolation & purification
  • Carbohydrate Epimerases / metabolism*
  • Chromatography, Ion Exchange
  • Chromatography, Liquid
  • Deuterium Exchange Measurement / methods*
  • Disaccharides / metabolism
  • Enzyme Assays / methods*
  • Glucuronic Acid / chemistry
  • Glucuronic Acid / metabolism
  • Heparitin Sulfate / metabolism*
  • Humans
  • Iduronic Acid / chemistry
  • Iduronic Acid / metabolism
  • Mass Spectrometry / methods*
  • Sf9 Cells

Substances

  • Disaccharides
  • heparosan
  • Iduronic Acid
  • Glucuronic Acid
  • Heparitin Sulfate
  • Carbohydrate Epimerases