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Optimal detection of cholinesterase activity in biological samples: modifications to the standard Ellman's assay

D. Dingova, J. Leroy, A. Check, V. Garaj, E. Krejci, A. Hrabovska,

. 2014 ; 462 (-) : 67-75.

Jazyk angličtina Země Spojené státy americké

Typ dokumentu časopisecké články, práce podpořená grantem

Perzistentní odkaz   https://www.medvik.cz/link/bmc15023330

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.

Citace poskytuje Crossref.org

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$a Ellman's assay is the most commonly used method to measure cholinesterase activity. It is cheap, fast, and reliable, but it has limitations when used for biological samples. The problems arise from 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), which is unstable, interacts with free sulfhydryl groups in the sample, and may affect cholinesterase activity. We report that DTNB is more stable in 0.09 M Hepes with 0.05 M sodium phosphate buffer than in 0.1M sodium phosphate buffer, thereby notably reducing background. Using enzyme-linked immunosorbent assay (ELISA) to enrich tissue homogenates for cholinesterase while depleting the sample of sulfhydryl groups eliminates unwanted interactions with DTNB, making it possible to measure low cholinesterase activity in biological samples. To eliminate possible interference of DTNB with enzyme hydrolysis, we introduce a modification of the standard Ellman's assay. First, thioesters are hydrolyzed by cholinesterase to produce thiocholine in the absence of DTNB. Then, the reaction is stopped by a cholinesterase inhibitor and the produced thiocholine is revealed by DTNB and quantified at 412 nm. Indeed, this modification of Ellman's method increases butyrylcholinesterase activity by 20 to 25%. Moreover, high stability of thiocholine enables separation of the two reactions of the Ellman's method into two successive steps that may be convenient for some applications.
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$a Leroy, Jacqueline $u CNRS UMR 8257 MD 4, COGNAC G, Université Paris Descartes, 75014 Paris, France.
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$a Check, Abel $u CNRS UMR 8257 MD 4, COGNAC G, Université Paris Descartes, 75014 Paris, France.
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$a Garaj, Vladimir $u Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Comenius University, Bratislava, Slovakia.
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$a Krejci, Eric $u CNRS UMR 8257 MD 4, COGNAC G, Université Paris Descartes, 75014 Paris, France.
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$a Hrabovska, Anna $u Department of Pharmacology and Toxicology, Faculty of Pharmacy, Comenius University in Bratislava, Slovakia; Biomedical Research Centre, University Hospital Hradec Kralove, Sokolska 581, 500 05 Hradec Kralove, Czech Republic. Electronic address: anna.hrabovska@gmail.com.
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