A versatile polyacrylamide gel electrophoresis based sulfotransferase assay.
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-PAGE) is described. This assay has been successfully applied to substrates as small as alpha-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.


CONCLUSIONS
The 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.
Wu
Zhengliang L
ZL
R&D Systems Inc, 614 McKinley Place NE, Minneapolis, MN 55413, USA. leon.wu@rndsystems.com
Ethen
Cheryl M
CM
Larson
Sara
S
Prather
Brittany
B
Jiang
Weiping
W
D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006801:Humans; D009284:Naphthols / Q000302:isolation & purification; D011506:Proteins / Q000302:isolation & purification*; D011509:Proteoglycans / Q000302:isolation & purification; D015238:Sulfotransferases / Q000737:chemistry*
D009284:Naphthols; D011506:Proteins; D011509:Proteoglycans; C029350:1-naphthol; D015238:Sulfotransferases