This assay was based on an earlier method reported by Ellman et al. [37] (link) based on the ability of the acetylcholinesterase enzyme to hydrolyse acetylthiocholine producing a thiocholine that reacts with DTNB which gives off a sharp yellow colour. Brie y, 2.85ml of the buffer was placed on the test tube followed by 50 µL of DTNB, 50 µL sample and 20 µL of 78 mM acetylthiocholine iodide and the rate of absorbance increase was monitored over 5 minutes at 412 nm using a spectrophotometer and was expressed as µmoles of substrate hydrolysed/min/mg protein.