The determination of the cholinesterase activity using 3,3′,5,5′-tetramethylbenzidine as an indicator




acetylcholinesterase, acetylcholine, photometric detection, 3,3′,5,5′-tetramethylbenzidine


Aim. To develop a new method, which has a good reproducibility of the experimental results, is fast, cheap and provides safe working conditions during the analysis, in order to determine the activity of cholinesterase.
Experimental part. The light absorption of the test and control samples was measured using a CPhC-3-01 photoelectric photometer (420 nm, l = 3 cm). The reaction rate was characterized by the value of the optical density of the solution in 10 min (the fixed time method). Measurements were performed at +37 °C, the temperature of the reaction mixture was maintained by thermostatіng in water, the pH of the solutions was monitored potentiometrically using a glass electrode. The determination was repeated five times with each solution of a certain concentration of the enzyme. According to the average values obtained, the calibration graph of the specific activity of the enzyme (in international units – activity unit (AU/mg) – kmol/min to 1 mg of the substance) on the optical density of the solution was constructed. Using the mean value of five measurements of the optical density of the test solution the specific activity of the enzyme (U) was found by the calibration graph.
Results and discussion. The essence of the method is the photometrical measurement of the rate of the enzymatic hydrolysis of acetylcholine in a buffer medium using 3,3′,5,5′-tetramethylbenzidine (TMB). The enzymatic hydrolysis reaction of the substrate was performed at pH 8.3, and in 10 min after the start the rate of enzymatic hydrolysis of acetylcholine was measured. The linear dependence of the optical density on the specific activity of the enzyme (U) was observed in the range of 3.5 – 28 AU/mg (activity unit/mg). The activity of the enzyme, according to the average results of 5 measurements, was 27.9 AU/mg. The declared activity the enzyme in accordance with the quality certificate was 28 AU/mg. The limit of quantification was 0.2 AU/mg. Metrological characteristics of the method were as follows: RSD = 1.8 % (n = 5; P = 0.95), accuracy – 0.45 %. These values indicate that the method proposed for determining the activity of cholinesterase is characterized by high sensitivity, reliability and reproducibility of the results. At the same time, it was proven that there was no systematic error in determining the activity of cholinesterase by the method developed.
Conclusions. As a result of the research conducted a new method for determining the activity of the cholinesterase enzyme has been developed; it is characterized by high sensitivity, reliability and reproducibility of the results, and also provides safe working conditions during the analysis.


Ramsay, R. R.; Tipton, K. F. Assessment of Enzyme Inhibition: A Review with Examples from the Development of Monoamine Oxidase and Cholinesterase Inhibitory Drugs. Molecules 2017, 22 (7), 1192.

Hopkins, A. L.; Groom, C. R. The druggable genome. Nature Reviews Drug Discovery 2002, 1 (9), 727 – 730.

Nikolic, K.; Mavridis, L.; Bautista-Aguilera, O. M.; Marco-Contelles, J.; Stark, H.; do Carmo Carreiras, M.; Rossi, I.; Massarelli, P.; Agbaba, D.; Ramsay, R. R.; Mitchell, J. B. Predicting targets of compounds against neurological diseases using cheminformatic methodology. J Comput Aided Mol Des 2015, 29 (2), 183-198.

Consalvi, S.; Biava, M.; Poce, G. COX inhibitors: a patent review (2011 - 2014). Expert Opin Ther Pat 2015, 25 (12), 1357-1371.

Ng, H.-L.; Chen, S.; Chew, E.-H.; Chui, W.-K. Applying the designed multiple ligands approach to inhibit dihydrofolate reductase and thioredoxin reductase for anti-proliferative activity. Eur. J. Med. Chem. 2016, 115, 63 – 74.

Men’shikov, V. V., Ed. Laboratornye metody issledovaniya v klinike [Laboratory research methods in the clinic, in Russian]; Medicine: Moscow, 1987.

Kamyshnikov, V. S.; Volotovskaya, O.A.; Hodyukova, A. B.; Dal’nova, T. S.; Vasi liu-Svetlickaya, S. G.; Zubovskaya, E. T.; Alekhnovich, L. I. Metody klinicheskih laboratornyh issledovanij [Methods of clinical laboratory research, in Russian], 8th ed.; MEDpress-inform: Moscow, 2016.

Hestrin, S. The reaction of acetylcholine and other carboxylic acid derivatives with hydroxylamine, and its analytical application. J. Biol. Chem. 1949, 180 (1), 249 – 261.

Yakovlev, V. A. Kinetika fermentativnogo kataliza [Kinetics of Enzymatic Catalysis]; Nauka: Moscow, 1965; p. 144 (in Russian).

Hanker, J. S.; Gelberg, A.; Witten, B. Colorimetric Estimation of Some Cholinergic Esters: Application to the Demonstration of Acetylcholinesterase Activity. Journal of the American Pharmaceutical Association (Scientific ed.) 1958, 47 (10), 728 – 730.

Derzhavna farmakopeia Ukrainy: v 3 tomakh, 2 vydannia [The State Pharmacopoeia of Ukraine: in 3 volumes, 2nd ed., in Ukrainian]; State Enterprise “Ukrainian Scientific Pharmacopoeial Center for Quality of Medicines”: Kharkiv, 2015; Vol. 1.

Sigma-Aldrich. Safety data sheet according to Regulation (EC) No. 1907/2006. (accessed Jun 11, 2021).

Kapustnyk, V. A.; Kostiuk, I. F.; Bondarenko, H. O.; Ihnatiev, O. M.; Parpaliei, I. O.; Rodionova, V. V.; Khukhlina, O. S.; Kalmykov, O. O. Profesiini khvoroby [Occupational diseases, in Ukrainian], 5th ed.; Medicina: Kyiv, 2017.




How to Cite

Koval’ska, O. V.; Blazheyevskіy M. Y.; Diadchenko, V. V. The Determination of the Cholinesterase Activity Using 3,3′,5,5′-Tetramethylbenzidine As an Indicator. J. Org. Pharm. Chem. 2021, 19, 34-39.



Original researches