Analytical and Bioanalytical Chemistry

, Volume 406, Issue 2, pp 525–536 | Cite as

Assays for determining heparan sulfate and heparin O-sulfotransferase activity and specificity

  • Eric Sterner
  • Lingyun Li
  • Priscilla Paul
  • Julie M. Beaudet
  • Jian Liu
  • Robert J. Linhardt
  • Jonathan S. Dordick
Research Paper


O-sulfotransferases (OSTs) are critical enzymes in the cellular biosynthesis of the biologically and pharmacologically important heparan sulfate and heparin. Recently, these enzymes have been cloned and expressed in bacteria for application in the chemoenzymatic synthesis of glycosaminoglycan-based drugs. OST activity assays have largely relied on the use of radioisotopic methods using [35S] 3′-phosphoadenosine-5′-phosphosulfate and scintillation counting. Herein, we examine alternative assays that are more compatible with a biomanufacturing environment. A high throughput microtiter-based approach is reported that relies on a coupled bienzymic colorimetric assay for heparan sulfate and heparin OSTs acting on polysaccharide substrates using arylsulfotransferase-IV and p-nitrophenylsulfate as a sacrificial sulfogroup donor. A second liquid chromatography-mass spectrometric assay, for heparan sulfate and heparin OSTs acting on structurally defined oligosaccharide substrates, is also reported that provides additional information on the number and positions of the transferred sulfo groups within the product. Together, these assays allow quantitative and mechanistic information to be obtained on OSTs that act on heparan sulfate and heparin precursors.


Herapan sulfate O-sulfotranferase coupled enzyme colorimetric assay


Enzymes Mass spectrometry Bioassays Sulfotransferases Coupled assay Heparin Heparan sulfate 



2-O sulfotransferase


3-O sulfotransferase isoform 1


6-O sulfotransferase isoform 1


6-O sulfotransferase isoform 3






Antithrombin III


C5 epimerase


Completely de-O-sulfonated heparin


Current good manufacturing process


Fourier transform mass spectrometry




Glucuronic acid




Hydrophilic interaction liquid chromatography


Iduronic acid


Liquid chromatography-mass spectrometry


N-deacetylase N-sulfotransferase


3′-adenosine 5′-phosphate


3′-adenosine 5′-phosphosulfate







Supplementary material

216_2013_7470_MOESM1_ESM.pdf (856 kb)
ESM 1 (PDF 855 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Eric Sterner
    • 1
  • Lingyun Li
    • 2
  • Priscilla Paul
    • 1
  • Julie M. Beaudet
    • 2
  • Jian Liu
    • 6
  • Robert J. Linhardt
    • 1
    • 2
    • 3
    • 4
  • Jonathan S. Dordick
    • 1
    • 3
    • 4
    • 5
  1. 1.Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary StudiesRensselaer Polytechnic InstituteTroyUSA
  2. 2.Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary StudiesRensselaer Polytechnic InstituteTroyUSA
  3. 3.Department of Biomedical Engineering, Center for Biotechnology and Interdisciplinary StudiesRensselaer Polytechnic InstituteTroyUSA
  4. 4.Department of Biology, Center for Biotechnology and Interdisciplinary StudiesRensselaer Polytechnic InstituteTroyUSA
  5. 5.Department of Material Sciences, Center for Biotechnology and Interdisciplinary StudiesRensselaer Polytechnic InstituteTroyUSA
  6. 6.Department of Chemical Biology and Medicinal Chemistry, Eshelman School of PharmacyUniversity of North CarolinaChapel HillUSA

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