Assays for determining heparan sulfate and heparin O-sulfotransferase activity and specificity
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Authors
Sterner, Eric
Li, Lingyun
Paul, Priscilla
Beaudet, Julie M.
Liu, Jian
Linhardt, Robert J.
Dordick, Jonathan S.
Issue Date
2014-01-01
Type
Article
Language
ENG
Keywords
Biology , Chemistry and chemical biology , Chemical and biological engineering , Biomedical engineering
Alternative Title
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
Note : if this item contains full text it may be a preprint, author manuscript, or a Gold OA copy that permits redistribution with a license such as CC BY. The final version is available through the publisher’s platform.
Note : if this item contains full text it may be a preprint, author manuscript, or a Gold OA copy that permits redistribution with a license such as CC BY. The final version is available through the publisher’s platform.
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
Publisher
Springer
Terms of Use
Journal
Volume
Issue
PubMed ID
DOI
ISSN
16182650
16182642
16182642