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