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Application of thio‐detecting fluorescent probes in high throughput functional screen of organophosphonate hydrolase
Author(s) -
Zhang Jun,
Wang Beilin,
Cashman John,
Xia Gang
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.2_supplement.235
Subject(s) - thio , nerve agent , esterase , chemistry , fluorescence , reagent , high throughput screening , combinatorial chemistry , catalysis , acetylcholinesterase , biochemistry , enzyme , stereochemistry , organic chemistry , physics , quantum mechanics
Development of prophylactic reagent to prevent organophosphonate (OP) nerve agent toxicity is an essential approach to protect military personnel and civilian emergency crews to unexpected nerve agent exposure. The long term goal of our research is to use molecular evolution approach to evolve candidate human cholinesterase (ChE) into highly efficient catalytic OP bioscavengers. To achieve this goal, we established a high throughput functional screen platform to identify OP catalytic esterase variants from highly diversified mutation libraries using a series of thio‐methyl‐OP compounds as model OP substrates. Due to low catalytic activity of ChE against OP compounds, traditional Ellman reaction is not sensitive enough for direct measurement of OP hydrolysis activity in a high throughput format. To identify a more sensitive method, we evaluated a number of compounds that become fluorescent after reacting with thiol group in the aspects of sensitivity, signal stability, and compatibility with high throughput assay platform. We report here our application of these free thio‐detecting fluorescent probes in a novel assay system that enable direct measurement of OP hydrolysis catalyzed by ChE mutants indentified from our functional screen. Supported by NIH grant 5U54NS058183 (JZ).