Optimal detection of cholinesterase activity in biological samples: modifications to the standard Ellman's assay
Jazyk angličtina Země Spojené státy americké Médium print-electronic
Typ dokumentu časopisecké články, práce podpořená grantem
PubMed
24929086
DOI
10.1016/j.ab.2014.05.031
PII: S0003-2697(14)00250-4
Knihovny.cz E-zdroje
- Klíčová slova
- Acetylcholinesterase, Biological samples, Butyrylcholinesterase, DTNB, Ellman’s assay,
- MeSH
- butyrylcholinesterasa metabolismus MeSH
- ELISA metody MeSH
- enzymatické testy metody MeSH
- kyselina dithionitrobenzoová metabolismus MeSH
- lidé MeSH
- thiocholin metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- butyrylcholinesterasa MeSH
- kyselina dithionitrobenzoová MeSH
- thiocholin MeSH
Ellman's assay is the most commonly used method to measure cholinesterase activity. It is cheap, fast, and reliable, but it has limitations when used for biological samples. The problems arise from 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), which is unstable, interacts with free sulfhydryl groups in the sample, and may affect cholinesterase activity. We report that DTNB is more stable in 0.09 M Hepes with 0.05 M sodium phosphate buffer than in 0.1M sodium phosphate buffer, thereby notably reducing background. Using enzyme-linked immunosorbent assay (ELISA) to enrich tissue homogenates for cholinesterase while depleting the sample of sulfhydryl groups eliminates unwanted interactions with DTNB, making it possible to measure low cholinesterase activity in biological samples. To eliminate possible interference of DTNB with enzyme hydrolysis, we introduce a modification of the standard Ellman's assay. First, thioesters are hydrolyzed by cholinesterase to produce thiocholine in the absence of DTNB. Then, the reaction is stopped by a cholinesterase inhibitor and the produced thiocholine is revealed by DTNB and quantified at 412 nm. Indeed, this modification of Ellman's method increases butyrylcholinesterase activity by 20 to 25%. Moreover, high stability of thiocholine enables separation of the two reactions of the Ellman's method into two successive steps that may be convenient for some applications.
CNRS UMR 8257 MD 4 COGNAC G Université Paris Descartes 75014 Paris France
Department of Pharmaceutical Chemistry Faculty of Pharmacy Comenius University Bratislava Slovakia
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