R&D Systems Inc, 614 McKinley Place NE, Minneapolis, MN 55413, USA.
BMC Biotechnol. 2010 Feb 10;10:11. doi: 10.1186/1472-6750-10-11.
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.
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.
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.
磺基转移酶是一大类酶，通过与 3'-磷酸腺苷-5'-磷酸硫酸（PAPS）供体磺化，调节广泛底物的生物活性或可及性。这些酶的测定一直很困难。为了更好地了解这些酶，需要一种方便的测定方法。
描述了一种基于十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）的通用磺基转移酶测定方法。该测定法已成功应用于小至α-萘酚和大至蛋白聚糖的底物。例如，我们展示了重组人 CHST4、TPST1、CHST3 和 HS6ST1 的测定法。为了评估小分子是否适用于这种类型的测定法，还提出了一种估计分子与 PAPS 相对迁移率的方法。由 SULT1A1、SULT1E1、SULT2A1 和 CHST4 产生的各种磺化小分子的估计相对迁移率在实际相对迁移率的+/-0.2 范围内。
当前方法的多功能性来自 SDS-PAGE 能够根据不同参数分离蛋白质和小分子的能力。虽然 SDS-PAGE 中蛋白质的迁移率与其大小成反比，但小分子的迁移率与其电荷/质量比成正比。产物与 PAPS 的预测相对迁移率是磺基转移酶是否可以用 SDS-PAGE 测定的良好指标。由于磷酸化在化学上与磺化最相似，因此该方法可能也适用于激酶。
