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

Abstract

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.

Figure

Herapan sulfate O-sulfotranferase coupled enzyme colorimetric assay

Keywords

Enzymes Mass spectrometry Bioassays Sulfotransferases Coupled assay Heparin Heparan sulfate 

Abbreviations

2-OST

2-O sulfotransferase

3-OST-1

3-O sulfotransferase isoform 1

6-OST-1

6-O sulfotransferase isoform 1

6-OST-3

6-O sulfotransferase isoform 3

Ac

Acetyl

AST-IV

Aryl-sulfotransferase

AT

Antithrombin III

C5-epi

C5 epimerase

CDSNS HP

Completely de-O-sulfonated heparin

cGMP

Current good manufacturing process

FTMS

Fourier transform mass spectrometry

GAGs

Glycosaminoglycans

GlcA

Glucuronic acid

GlcN

Glucosamine

HILIC

Hydrophilic interaction liquid chromatography

IdoA

Iduronic acid

LC-MS

Liquid chromatography-mass spectrometry

NDST

N-deacetylase N-sulfotransferase

PAP

3′-adenosine 5′-phosphate

PAPS

3′-adenosine 5′-phosphosulfate

PNP

para-nitrophenol

PNPS

para-nitrophenylsulfate

S

Sulfo

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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