title: Assays for determining heparan sulfate and heparin O-sulfotransferase activity and specificity.
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.
authors.affiliation: Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA.
authors.forename: Eric
authors.initials: E
authors.lastname: Sterner
authors.forename: Lingyun
authors.initials: L
authors.lastname: Li
authors.forename: Priscilla
authors.initials: P
authors.lastname: Paul
authors.forename: Julie M
authors.initials: JM
authors.lastname: Beaudet
authors.forename: Jian
authors.initials: J
authors.lastname: Liu
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Linhardt
authors.forename: Jonathan S
authors.initials: JS
authors.lastname: Dordick
mesh_terms: D001681:Biological Assay; D002240:Carbohydrate Sequence; D002853:Chromatography, Liquid; D003124:Colorimetry; D006493:Heparin / Q000737:chemistry*; D006497:Heparan Sulfate / Q000737:chemistry*; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009578:Nitrobenzenes / Q000737:chemistry; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000032:analysis* / Q000737:chemistry
chemical_list: D006493:Heparin; D006497:Heparan Sulfate; D009578:Nitrobenzenes; D015238:Sulfotransferases; C023379:4-nitrophenyl sulfate; C077557:tyrosine-ester sulfotransferase