原创力文档a versatile polyacrylamide gel electrophoresis based sulfotransferase assay基于通用的聚丙烯酰胺凝胶电泳sulfotransferase化验.pdfVIP
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a versatile polyacrylamide gel electrophoresis based sulfotransferase assay基于通用的聚丙烯酰胺凝胶电泳sulfotransferase化验
Wu et al. BMC Biotechnology 2010, 10:11
/1472-6750/10/11
METHODOLOGY ARTICLE Open Access
A versatile polyacrylamide gel electrophoresis
based sulfotransferase assay
*
Zhengliang L Wu , Cheryl M Ethen, Sara Larson, Brittany Prather, Weiping Jiang
Abstract
Background: Sulfotransferases 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.
Results: A universal sulfotransferase assay method based on sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS) is described. This assay has been successfully applied to substrates as small as a-
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.
Conclusion: The versatility of the current method comes from the ability that SDScan separate proteins and
small molecules according to different parameters. While mobilities of proteins during SDSare 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 metho
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