12 research outputs found
Catalytic mechanism of Golgi-resident human tyrosylprotein sulfotransferase-2: A mass spectrometry approach
Toward an Artificial Golgi: Redesigning the Biological Activities of Heparan Sulfate on a Digital Microfluidic Chip
Inhibition of influenza virus replication via small molecules that induce the formation of higher-order nucleoprotein oligomers
A Mass Spectrometric Approach to the Study of DNA-Binding Proteins: Interaction of Human TRF2 with Telomeric DNA
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 phar-macologically important heparan sulfate and heparin. Re-cently, 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 as-says 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 pro-vides 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