A versatile polyacrylamide gel electrophoresis based sulfotransferase assay

Abstract

BackgroundSulfotransferases are a large group of enzymes that regulate the biological activity or availability of a wide spectrum of substrates through sulfation with the sulfur donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS). These enzymes are known to be difficult to assay. A convenient assay is needed in order to better understand these enzymes.ResultsA universal sulfotransferase assay method based on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is described. This assay has been successfully applied to substrates as small as α-naphthol and as big as proteoglycans. As examples, we present the assays for recombinant human CHST4, TPST1, CHST3 and HS6ST1. In order to assess whether a small molecule can be applicable to this type of assay, a method to estimate the relative mobility of a molecule to PAPS is also presented. The estimated relative mobilities of various sulfated small molecules generated by SULT1A1, SULT1E1, SULT2A1 and CHST4 are in the range of ± 0.2 of the actual relative mobilities.ConclusionThe versatility of the current method comes from the ability that SDS-PAGE can separate proteins and small molecules according to different parameters. While mobilities of proteins during SDS-PAGE are inversely related to their sizes, mobilities of small molecules are positively related to their charge/mass ratios. The predicted relative mobility of a product to PAPS is a good indicator of whether a sulfotransferase can be assayed with SDS-PAGE. Because phosphorylation is most similar to sulfation in chemistry, the method is likely to be applicable to kinases as well.

New Targets and Inhibitors of Mycobacterial Sulfur Metabolism

“…Historically, sulfotransferase assays have often been conducted using 35 S labeled substrate in combination with chromatography, electrophoresis, or filter binding [162–165]. Non-radioactive assays using spectrophotometry and mass spectrometry have also been reported [166–168].…”

Recent advances in sulfotransferase enzyme activity assays

“…These methods have found application with substrates ranging from small molecules being acted on by cytosolic STs, for example α-naphthol, to large molecules being acted on by Golgi STs, for example proteoglycans, in which sulfated products and donor substrate PAP 35 S are separated by electrophoresis using SDS-PAGE. Electrophoresis-based separations have been demonstrated with both carbohydrate STs and TPSTs [24]. Dot-blotting radiometric activity assays have been used in high-throughput screening, demonstrating the activity of two carbohydrate STs, heparan sulfate N- deacetylase/ N- ST and high-endothelial cell (HEC) GlcNAc-6 -O- ST on PAP 35 S and de- N- sulfonated heparin and N- acetylglucosamine, respectively [17] (Scheme 3).…”

Engineering microbial factories for synthesis of value-added products

“…In a recent study, Brown and coworkers investigated the hydrolysate inhibitor tolerance of both Z. mobilis and S. cerevisiae in regards to the expression level of a conserved bacterial member of the Sm-like family of RNA-binding proteins Hfq and its homologue Lsm. Their results indicate that these regulator proteins are very important for pretreatment inhibitor tolerance [146]. …”