A new method for determining the cholinesterase activity
Keywords:acetylcholinestherase; acetylcholine; photometric detection
Aim. To develop a principally new method, which would allow achieving the necessary accuracy and reproducibility of the analysis results, for determining the activity of the blood cholinesterase; to create safe working conditions when performing the analysis.
Results and discussion. The kinetic method proposed for determining the activity of cholinesterase consists in photometric measurement of the rate of the enzymatic hydrolysis of the acetylcholine substrate (by its residue) in the phosphate buffer using p-phenetidine as an indicator. The rate of the enzymatic hydrolysis of acetylcholine was determined by the tangent of the inclination angle of the linear part of the kinetic curve in the А–t coordinates at a wavelength of 358 nm. The linear dependence of the conditional reaction rate (tgα) on the enzyme concentration was observed in the concentration range of 0.12 – 0.36 mg/mL. Metrological characteristics of the method developed were: RSD = 2.0 % (n = 5; P = 0.95), correctness 0.4 %. These values indicate that the method for determining the activity of blood cholinesterase is sensitive, reliable and reproducible.
Experimental part. The experiments on determining the rate of the enzymatic hydrolysis were repeated three times with a specific concentration of the enzyme. Using the data obtained the kinetic curves were constructed in the А–t coordinates; on their basis the tangents of the angles of inclination in min-1 were calculated. The calibration graph was constructed using the average values of the tangents of the angles of inclination, which corresponded to a certain concentration of the solution of the working standard sample of the enzyme. The equation of the calibration dependence of tgα-enzyme concentration was calculated by the method of least squares and found to be tgα (min-1) = –0.17с + 9.13 (r = 0.999).
Conclusions. As a result of the studies conducted, a new method for determining the activity of the cholinesterase enzyme has been developed. The method is characterized by a high sensitivity, reliability and reproducibility and provides safe working conditions when performing the analysis.
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Блажеєвський, М. Є.; Ковальська, О. В.; Дядченко, В. В. (Національний фармацевтичний університет). Спосіб визначення активності холінестерази крові. Патент України 117474, 26.06.2017.
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