title: Fluorometric method for inorganic pyrophosphatase activity detection and inhibitor screening based on click chemistry.
abstract: A fluorometric method for pyrophosphatase (PPase) activity detection was developed based on click chemistry. Cu(II) can coordinate with pyrophosphate (PPi), the addition of pyrophosphatase (PPase) into the above system can destroy the coordinate compound because PPase catalyzes the hydrolysis of PPi into inorganic phosphate and produces free Cu(II), and free Cu(II) can be reduced by sodium ascorbate (SA) to form Cu(I), which in turn initiates the ligating reaction between nonfluorescent 3-azidocoumarins and terminal alkynes to produce a highly fluorescent triazole complex, based on which, a simple and sensitive turn on fluorometric method for PPase can be developed. The fluorescence intensity of the system has a linear relationship with the logarithm of the PPase concentration in the range of 0.5 and 10 mU with a detection limit down to 0.2 mU (S/N = 3). This method is cost-effective and convenient without any labels or complicated operations. The proposed system was applied to screen the potential PPase inhibitor with high efficiency. The proposed method can be applied to diagnosis of PPase-related diseases.
authors.affiliation: Ministry of Education Key Laboratory of Analysis and Detection for Food Safety, Fujian Provincial Key Laboratory of Analysis and Detection for Food Safety, Fuzhou University , Fuzhou, Fujian 350116, China.
authors.forename: Kefeng
authors.initials: K
authors.lastname: Xu
authors.forename: Zhonghui
authors.initials: Z
authors.lastname: Chen
authors.forename: Ling
authors.initials: L
authors.lastname: Zhou
authors.forename: Ou
authors.initials: O
authors.lastname: Zheng
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Wu
authors.forename: Longhua
authors.initials: L
authors.lastname: Guo
authors.forename: Bin
authors.initials: B
authors.lastname: Qiu
authors.forename: Zhenyu
authors.initials: Z
authors.lastname: Lin
authors.forename: Guonan
authors.initials: G
authors.lastname: Chen
mesh_terms: D057930:Click Chemistry; D003300:Copper / Q000737:chemistry*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005470:Fluorometry / Q000379:methods*; D057166:High-Throughput Screening Assays; D006868:Hydrolysis; D043564:Inorganic Pyrophosphatase / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D057230:Limit of Detection
chemical_list: D004791:Enzyme Inhibitors; D003300:Copper; D043564:Inorganic Pyrophosphatase