Fluorescence assay for protein post-translational tyrosine sulfation

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

研究成果: 雜誌貢獻文章

8 引文 (Scopus)

摘要

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.

原文英語
頁(從 - 到)1425-1429
頁數5
期刊Analytical and Bioanalytical Chemistry
405
發行號4
DOIs
出版狀態已發佈 - 2013

指紋

Tyrosine
Assays
Fluorescence
Proteins
Arylsulfotransferase
Hymecromone
Phosphates
Peptides
Kinetics
Diphosphates
Staphylococcal Protein A
Glutathione Transferase
Drosophila melanogaster
Adenosine
Screening
Fusion reactions
Throughput
protein-tyrosine sulfotransferase
Substrates
Enzymes

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry

引用此文

Fluorescence assay for protein post-translational tyrosine sulfation. / Chen, Bo Han; Wang, Chen Chu; Lu, Lu Yi; Hung, Kuo Sheng; Yang, Yuh Shyong.

於: Analytical and Bioanalytical Chemistry, 卷 405, 編號 4, 2013, p. 1425-1429.

研究成果: 雜誌貢獻文章

Chen, Bo Han ; Wang, Chen Chu ; Lu, Lu Yi ; Hung, Kuo Sheng ; Yang, Yuh Shyong. / Fluorescence assay for protein post-translational tyrosine sulfation. 於: Analytical and Bioanalytical Chemistry. 2013 ; 卷 405, 編號 4. 頁 1425-1429.
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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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AU - Lu, Lu Yi

AU - Hung, Kuo Sheng

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N2 - 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.

AB - 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.

KW - Fluorescence enzyme assay

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KW - Protein sulfation

KW - Tyrosylprotein sulfotransferase (TPST)

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