Colorimetric determination of the activity of acetylcholinesterase and its inhibitors by exploiting the iodide-catalyzed oxidation of 3,3',5,5'-tetramethylbenzidine by hydrogen peroxide

We describe a sensitive and selective colorimetric method for the determination of the activity of the enzyme acetylcholinesterase (AChE) and its inhibitors. Detection is based on the fact that acetylthiocholine iodide (ATCI) catalyzes the oxidation of the substrate 3,3',5,5'-tetramethylbenzidine (TMB) by H2O2 to form a blue product (ox-TMB) with an absorption peak at 652 nm, but that oxidation is suppressed if ACTI previously is hydrolyzed by AChE to form thiocholine which decolorizes ox-TMB. In the presence of inhibitor, the activity of AChE is inhibited, thereby inducing the recovery of the blue coloration. Based on these findings, a highly sensitive method is developed for the determination of AChE and its inhibitors. The assay only requires mixing of buffer, solutions of ATCI, TMB, H2O2 and a sample containing AChE and photometric measurement. It works in the 0.05 to 5 mUaEuro cent mL(-1) enzyme activity range and has a detection limit as low as 30 mu UaEuro cent mL(-1). The inhibitor neostigmine causes 50 % enzyme inhibition in 14.5 nM concentration. This analytical system has a wide scope in that it may be applied to the determination of the activity of various other hydrolases with proper substrates.