Sulfatase activity assay using an activity-based probe by generation of N-methyl isoindole under reducing conditions.
Sulfatases catalyze the hydrolysis of sulfate esters that are present in a range of biomolecules. This is an important step in several biological processes such as cellular degradation, hormone regulation, and cell signaling. We have developed a new activity-based sulfatase probe (probe 1) that generates a fluorescent N-methylisoindole upon hydrolysis by sulfatase. Because of the autoxidation of N-methylisoindole, the sulfatase activity was also tested under reducing conditions, containing either glutathione (GSH) or tris(2-carboxyethyl)phosphine (TCEP), exhibiting little change in kinetic parameters compared to non-reducing conditions. Probe 1 displayed reasonable kinetic parameters under both non-reducing and reducing conditions, among which the use of Tris buffer and Tris buffer containing GSH appeared to be appropriate conditions for inhibitor screening. Probe 1 showed stronger intensity upon treatment with sulfatase under neutral conditions than under acidic conditions, but it still has limitations in the selectivity for a specific sulfatase. Nevertheless, the fluorescent signal generated as a result of the release of N-methylisoindole after treatment of probe 1 with sulfatase provides a new assay for measuring sulfatase activity that could be adapted for high throughput screening.
Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-747, Republic of Korea.
Hey Young
HY
Yoon
Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-747, Republic of Korea. Electronic address: jihong@snu.ac.kr.
Jong-In
JI
Hong
D005453:Fluorescence; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D054833:Isoindoles / Q000378:metabolism*; D007700:Kinetics; D010720:Phosphines / Q000378:metabolism*; D013379:Substrate Specificity; D013429:Sulfatases / Q000378:metabolism*
D005978:Glutathione; D054833:Isoindoles; D010720:Phosphines; D013429:Sulfatases; C080938:tris(2-carboxyethyl)phosphine
Activity-based probe; N-methyl isoindole; Reducing agents; Sulfatase