Fluorescence assay for protein post-translational tyrosine sulfation.

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

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. [Figure not available: see fulltext.]

FLUORESCENCE; SULFATION; PROTEIN-protein interactions; TYROSYLPROTEIN sulfotransferase; SULFOTRANSFERASES; DROSOPHILA melanogaster; PEPTIDES; GLUTATHIONE transferase

Analytical & Bioanalytical Chemistry, 2013, Vol 405, Issue 4, p1425

1618-2642

Academic Journal

10.1007/s00216-012-6540-3