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A Robust and Convenient Fluorimetric Assay for Measuring Acetylcholinesterase Activity and Screening Acetylcholinesterase Inhibitors
Author(s) -
Liao Jinfang,
Zhao Qin,
Diwu Zhenjun
Publication year - 2010
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.24.1_supplement.681.11
Subject(s) - acetylcholinesterase , acetylthiocholine , chemistry , plate reader , aché , chromatography , enzyme , biochemistry , fluorescence , physics , quantum mechanics
Acetylcholinesterase (AchE) is one of the most crucial enzymes for nerve responses and neurological functions. AChE inhibitors are the key drugs for management of Alzheimer's disease and myasthenia gravis. Although AchE is a proven drug development target, the existing technologies for screening AChE inhibitors are a compromise between throughput, sensitivity and physiological relevance. We have developed a robust fluorimetric Thiolite Green–based assay for measuring AchE activities and screening AchE inhibitors. This new fluorimetric method is much more sensitive for detecting AChE activity than the existing colorimetric methods that is generally based on DNTB. The assay is based on the fluorogenic reaction of Thiolite Green with thiocholine. This specific fluorogenic reaction can be conveniently used to quantify thiocholine produced from the AChE–induced hydrolysis of acetylthiocholine. The fluorescence intensity of Thiolite Green‐thiocholine adduct is proportional to the formation of thiocholine, therefore the AChE activity. The Assay provides a continuous and ultrasensitive fluorimetric assay to detect as little as 0.1 mU/mL AChE, which is 100 times more sensitive than the DNTB colorimetric method. Its signal can be easily read by a fluorescence microplate reader with Ex/Em = 490/520 nm.