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Assessment of Acetylcholinesterase Activity Using Indoxylacetate and Comparison with the Standard Ellman’s Method
Faculty of Military Health Sciences, University of Defense, Trebesska 1575, 50001 Hradec Kralove, Czech Republic
CEA-Grenoble, LITEN/DTNM/LCRE, 17 rue des Martyrs, 38000 Grenoble, France
* Author to whom correspondence should be addressed.
Received: 20 February 2011; in revised form: 1 March 2011 / Accepted: 29 March 2011 / Published: 18 April 2011
Abstract: Assay of acetylcholinesterase (AChE) activity plays an important role in diagnostic, detection of pesticides and nerve agents, in vitro characterization of toxins and drugs including potential treatments for Alzheimer’s disease. These experiments were done in order to determine whether indoxylacetate could be an adequate chromogenic reactant for AChE assay evaluation. Moreover, the results were compared to the standard Ellman’s method. We calculated Michaelis constant Km (2.06 × 10−4 mol/L for acetylthiocholine and 3.21 × 10−3 mol/L for indoxylacetate) maximum reaction velocity Vmax (4.97 × 10−7 kat for acetylcholine and 7.71 × 10−8 kat for indoxylacetate) for electric eel AChE. In a second part, inhibition values were plotted for paraoxon, and reactivation efficacy was measured for some standard oxime reactivators: obidoxime, pralidoxime (2-PAM) and HI-6. Though indoxylacetate is split with lower turnover rate, this compound appears as a very attractive reactant since it does not show any chemical reactivity with oxime antidots and thiol used for the Ellman’s method. Thus it can be advantageously used for accurate measurement of AChE activity. Suitability of assay for butyrylcholinesterase activity assessment is also discussed.
Keywords: acetylcholinesterase; indoxylacetate; 5,5’-dithio-bis-2-nitrobenzoic acid; Alzheimer’s disease; oxime reactivator; enzyme activity; nerve agents
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Pohanka, M.; Hrabinova, M.; Kuca, K.; Simonato, J.-P. Assessment of Acetylcholinesterase Activity Using Indoxylacetate and Comparison with the Standard Ellman’s Method. Int. J. Mol. Sci. 2011, 12, 2631-2640.
Pohanka M, Hrabinova M, Kuca K, Simonato J-P. Assessment of Acetylcholinesterase Activity Using Indoxylacetate and Comparison with the Standard Ellman’s Method. International Journal of Molecular Sciences. 2011; 12(4):2631-2640.
Pohanka, Miroslav; Hrabinova, Martina; Kuca, Kamil; Simonato, Jean-Pierre. 2011. "Assessment of Acetylcholinesterase Activity Using Indoxylacetate and Comparison with the Standard Ellman’s Method." Int. J. Mol. Sci. 12, no. 4: 2631-2640.