A versatile polyacrylamide gel electrophoresis based sulfotransferase assay

Zhengliang L Wu,Brittany Prather,Weiping Jiang,Cheryl Ethen,Sara Larson

doi:10.1186/1472-6750-10-11

Zhengliang L Wu, Brittany Prather + Show 3 more

Open Access

https://doi.org/10.1186/1472-6750-10-11

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Journal: BMC Biotechnology	Publication Date: Jan 1, 2010
Citations: 44	License type: cc-by

Affiliation: R&D Systems (United States)

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.

Highlights

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)
Protein sulfation is known to be important in proteinprotein interactions, such as leukocyte adhesion molecule P-selectin glycoprotein ligand-1 (PSGL-1) binding to P-selectin on activated endothelium [10,11]
We have developed a versatile electrophoresis-based sulfotransferase assay that can be applied to substrates ranging from small molecules, such as a-naphthol, to large molecules, such as proteoglycans, by taking advantage of the fact that both small and large molecules can be separated from PAPS during SDS-PAGE (Figure 1)

Summary

Introduction

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. Sulfation is a ubiquitous post-translational modification that affects the biological activity of a wide variety of substrates, ranging in molecular mass from less than 103 to greater than 106 Da. The reaction is catalyzed by sulfotransferases using 3’-phosphoadenosine-5’-phosphosulfate (PAPS) as the sulfate donor [1]. Golgi resident sulfotransferases are involved in modifying numerous glycans and proteins on the cell membranes and within extracellular matrix. Protein sulfation is known to be important in proteinprotein interactions, such as leukocyte adhesion molecule PSGL-1 binding to P-selectin on activated endothelium [10,11]

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Conclusion