Assay-of-Acetylcholinesterase-Activity-in-the-Brain -...

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142 Ever since the discovery of acetylcholine (ACh) as a neurotransmitter by Sir Henry Dale and Otto Loewi (for which they were awarded the Nobel Prize in 1936), its function in health and dysfunction in disease has been increasingly recognized. In the recent past, the role of ACh in learning and memory has been demonstrated indubitably. Further, pharmacological manipulation of cholinergic function has been found useful in the treatment of CNS disorders like Alzheimer’s and Parkinson’s disease. Thus, assessing cholinergic function is considered as an important tool in neuroscience research. Acetylcholine per se has a very short half-life and direct estimation of ACh is a little difficult in brain homogenates. There are several approaches to evaluate cholinergic function indirectly. Estimating the expression of choline acetyl transferase (ChAT) and acetylcholinesterase (AChE) by immunochemical and histochemical techniques provide information on the cholinergic function, but are tedious and time taking. Estimation of AChE activity provides a relatively easy and valuable assessment of cholinergic function. The method of AChE activity estimation is popularly known as Ellman’s method named after George Ellman who developed this method in 1961 (Ellman et al., 1961). The esterase activity is measured by providing an artificial substrate, acetylthiocholine (ATC). Thiocholine released because of the cleavage of ATC by AChE is allowed to react with the -SH reagent 5,5’- dithiobis-(2-nitrobenzoic acid) (DTNB), which is reduced to thionitrobenzoic acid, a yellow coloured anion with an absorption maxima at 412nm (Figure 1). The extinction coefficient of the thionitro benzoic acid is 1.36 × 104/molar/ centimeter. The concentration of thionitro benzoic acid detected using a UV spectrophotometer is then taken as a direct estimate of the AChE activity. Methodology Reagents 1. 0.1M Phosphate buffer Solution A: 5.22g of K 2 HPO 4 and 4.68g of NaH 2 PO 4 are dissolved in 150 ml of distilled water. Solution B: 6.2g NaOH is dissolved in 150ml of distilled water. Solution B is added to solution A to get the desired pH (pH 8.0 or 7.0) and then finally the volume is made up to 300ml with distilled water. 2. DTNB Reagent 39.6 mg of DTNB with 15 mg NaHCO 3 is dissolved in 10 ml of 0.1M phosphate buffer (pH 7.0). 3. Acetylthiocholine (ATC) 21.67 mg of acetylthiocholine is dissolved in 1 ml of distilled water. Figure 1: The steps involved in estimation of AChE activity using Ellman’s reaction. Acetylthiocholine is broken down in the presence of AChE to release thiocholine, which reacts with the -SH reagent 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB) to form thionitro benzoic acid. ASSAY OF ACETYLCHOLINESTERASE ACTIVITY IN THE BRAIN
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This note was uploaded on 10/18/2010 for the course BIO 204 taught by Professor O'neal during the Fall '07 term at SUNY Stony Brook.

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Assay-of-Acetylcholinesterase-Activity-in-the-Brain -...

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