A Rapid, Nonradioactive Assay for Measuring Heparan Sulfate C-5 Epimerase Activity Using Hydrogen/Deuterium Exchange-Mass Spectrometry

  • Ponnusamy BabuEmail author
  • Xylophone V. Victor
  • Karthik Raman
  • Balagurunathan Kuberan
Part of the Methods in Molecular Biology book series (MIMB, volume 1229)


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.

Key words

Glycosaminoglycans Heparan sulfate C5-epimerase H/D exchange Mass spectrometry 



This work was supported in part by NIH grants (P01HL107152 and R01GM075168) to B.K. and by the NIH fellowship F31CA168198 to K.R. P.B. thanks Centre for Cellular and Molecular Platforms for support.


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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Ponnusamy Babu
    • 1
    Email author
  • Xylophone V. Victor
    • 2
    • 3
  • Karthik Raman
    • 2
    • 3
  • Balagurunathan Kuberan
    • 2
    • 3
  1. 1.Glycomics and GlycoproteomicsCentre for Cellular and Molecular Platforms, NCBS-TIFRBangaloreIndia
  2. 2.Department of Medicinal ChemistryUniversity of UtahSalt Lake CityUSA
  3. 3.Department of BioengineeringUniversity of UtahSalt Lake CityUSA

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