New Findings about Ellman’s Method to Determine Cholinesterase Activity

Abstract

The original Ellman's spectrophotometrical method for cholinesterase activity determination uses 5,5Ј-dithiobis-2-nitrobenzoic acid (DTNB, Ellman's reagent) as a chromogen and records the level of cholinesterase activity as an increase of absorbance at 412 nm. Although this procedure usually poses no problem, exceptions arise when the concentration of DTNB is far higher than the concentration of acetylthiocholine (ATCH). It was found that the ratio of concentrations of DTNB/ATCH is an important parameter for the ATCH hydrolysis course: high excess of DTNB decreases the hydrolysis rate resulting in a lower measured enzyme activity. Our experiments indicate that this influence of DTNB concentration can be explained by the inhibition of ATCH hydrolysis by DTNB.

Neurite Outgrowth and Neuroprotective Effects of Quercetin fromCaesalpinia mimosoidesLamk. on Cultured P19-Derived Neurons

“…Ellman's colorimetric method [18, 19] and modified method using 96-well microplates [20] were used. Briefly, acetylthiol, the product of hydrolysis reaction of ACh by cholinesterase, will react with DTNB to give 2-nitro-5-thiobenzoate (NTB − ), which ionizes to the NTB 2- dianion (in water at pH 8).…”

“…The inhibition activity of samples on this cell line was investigated by the modified Ellman's colorimetric method [17, 18]. The condition was slightly modified to enable the cellular enzyme to work properly [19]. Briefly, 25  μ L of sample dissolved in buffer containing 50% methanol, 100  μ L of 3 mM DTNB in buffer containing 0.1 M NaCl and 0.02 M MgCl 2 ·2H 2 O, and 25  μ L of 15 mM ATCI in deionized water were added to the well containing 100  μ L of the cells.…”

Inhibition effect of graphene oxide on the catalytic activity of acetylcholinesterase enzyme

“…A modified Ellman colorimetric method was used to collect the measured data. The modification involved the concentration ratio, DTNB/ATChI, which should not be too high because otherwise the enzyme activity could be inhibited.…”

Cholinesterase assay by an efficient fixed time endpoint method

“…This difference of activities can be explained because DTNB interacts with the protein [5] in the usual kinetic Ellman's method because DTNB is in the medium during the enzymatic reaction.…”

“…In these methods, the chromogen reagent 5,5′dithiobis nitro benzoic acid (DTNB) is added with the substrate and colour is monitored. Some authors pointed that the chromogen can alter cholinesterase activity [5].A modification of Bellino's method is proposed for acetylcholine-hydrolyzing activity determinations that is based on stopping the reaction after a fixed substrate reaction time using a mixture of detergent SDS and DTNB.The method may be adapted to the user needs by modifying the enzyme concentration and applied for simultaneously testing many samples in parallel; i.e. for complex experiments of kinetics assays with organophosphate inhibitors in different tissues.…”