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Fluorescence assay for protein post-translational tyrosine sulfation

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Abstract

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.

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Acknowledgments

This work was supported by National Science Council (NSC99-2311-B-009-004-MY3) and MOE-ATU Program, Taiwan.

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Authors and Affiliations

  1. Department of Biological Science and Technology, National Chiao Tung University, 75 Po-Ai Street, Hsinchu, 30050, Taiwan

    Bo-Han Chen, Chen-Chu Wang, Lu-Yi Lu & Yuh-Shyong Yang

  2. Department of Neurosurgery, Center of Excellence for Clinical Trial and Research, Taipei Medical University-Wan Fang Medical Center, Taipei, 116, Taiwan

    Kuo-Sheng Hung

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  1. Bo-Han Chen
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  2. Chen-Chu Wang
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  3. Lu-Yi Lu
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  4. Kuo-Sheng Hung
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  5. Yuh-Shyong Yang
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Corresponding author

Correspondence to Yuh-Shyong Yang.

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Chen, BH., Wang, CC., Lu, LY. et al. Fluorescence assay for protein post-translational tyrosine sulfation. Anal Bioanal Chem 405, 1425–1429 (2013). https://doi.org/10.1007/s00216-012-6540-3

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  • DOI: https://doi.org/10.1007/s00216-012-6540-3

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