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

Authors
Sterner, Eric
Li, Lingyun
Paul, Priscilla
Beaudet, Julie M.
Liu, Jian
Linhardt, Robert J.
Dordick, Jonathan S.
ORCID
https://orcid.org/0000-0003-2219-5833
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Issue Date
2014-01-01
Keywords
Biology , Chemistry and chemical biology , Chemical and biological engineering , Biomedical engineering
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Full Citation
Assays for determining heparan sulfate and heparin O-sulfotransferase activity and specificity, E. Sterner, L. Li, P. Paul, J. M. Beaudet, J. Liu, R. J. Linhardt, J. S. Dordick, Analytical and Bioanalytical Chemistry, 406, 525–536, 2014
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 [(35)S] 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.
Description
Analytical and Bioanalytical Chemistry, 406, 525–536
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Department
The Linhardt Research Labs.
The Shirley Ann Jackson, Ph.D. Center for Biotechnology and Interdisciplinary Studies (CBIS)
Publisher
Springer
Relationships
The Linhardt Research Labs Online Collection
Rensselaer Polytechnic Institute, Troy, NY
Analytical and Bioanalytical Chemistry
https://harc.rpi.edu/
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