Fluorescence assay for protein post-translational tyrosine sulfation

Bo Han Chen, Chen Chu Wang, Lu Yi Lu, Kuo Sheng Hung, Yuh Shyong Yang

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12 引文 斯高帕斯(Scopus)


We developed a fluorescent assay to conveniently determine the kinetics of protein sulfation, which is essential for understanding interface between protein sulfation and protein-protein interactions. Tyrosylprotein sulfotransferase (TPST) catalyzes protein sulfation using 3′-phosphate 5′-phosphosulfate (PAPS) as sulfuryl group donor. In this report, PAPS was regenerated following sulfuryl group transfer between adenosine 3′,5′-diphosphate and 4-methylumbelliferyl sulfate catalyzed by phenol sulfotransferase (PST). The TPST and PST coupled enzyme platform continuously generated fluorescent 4-methylumbelliferone (MU) that was used to real-time monitor protein sulfation. Using a recombinant N utilization substance protein A fused Drosophila melanogaster tyrosylprotein sulfotransferase, we demonstrated that the activity of TPST determined through MU fluorescence directly correlated with protein sulfation. Kinetic constants obtained with small P-selectin glycoprotein ligand-1 peptide (PSGL-1 peptide, MW 1541) or its large glutathione S-transferase fusion protein (GST-PSGL-1, MW 27833) exhibited significant variation. This assay can be further developed to a high-throughput method for the characterization of TPSTs and for the identification and screening of their protein substrates.

頁(從 - 到)1425-1429
期刊Analytical and Bioanalytical Chemistry
出版狀態已發佈 - 2月 2013

ASJC Scopus subject areas

  • 分析化學
  • 生物化學


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