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

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 meas...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Olena V. Koval’ska, Mykola Ye. Blazheyevskіy, Vladislav V. Diadchenko
Formato:	article
Lenguaje:	EN RU UK
Publicado:	National University of Pharmacy (Kharkiv, Ukraine) 2021
Materias:	acetylcholinesterase acetylcholine photometric detection tetramethylbenzidine Chemistry QD1-999
Acceso en línea:	https://doi.org/10.24959/ophcj.21.240776 https://doaj.org/article/72a105ab835e4c3ebdd6125ba2051f15
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:72a105ab835e4c3ebdd6125ba2051f15
record_format	dspace
spelling	oai:doaj.org-article:72a105ab835e4c3ebdd6125ba2051f152021-11-14T22:30:06ZThe determination of the cholinesterase activity using 3,3′,5,5′-tetramethylbenzidine as an indicatorhttps://doi.org/10.24959/ophcj.21.2407762308-83032518-1548https://doaj.org/article/72a105ab835e4c3ebdd6125ba2051f152021-09-01T00:00:00Zhttp://ophcj.nuph.edu.ua/article/view/240776https://doaj.org/toc/2308-8303https://doaj.org/toc/2518-1548Aim. 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.Olena V. Koval’skaMykola Ye. BlazheyevskіyVladislav V. DiadchenkoNational University of Pharmacy (Kharkiv, Ukraine)articleacetylcholinesteraseacetylcholinephotometric detectiontetramethylbenzidineChemistryQD1-999ENRUUKЖурнал органічної та фармацевтичної хімії, Vol 19, Iss 3, Pp 34-39 (2021)
institution	DOAJ
collection	DOAJ
language	EN RU UK
topic	acetylcholinesterase acetylcholine photometric detection tetramethylbenzidine Chemistry QD1-999
spellingShingle	acetylcholinesterase acetylcholine photometric detection tetramethylbenzidine Chemistry QD1-999 Olena V. Koval’ska Mykola Ye. Blazheyevskіy Vladislav V. Diadchenko The determination of the cholinesterase activity using 3,3′,5,5′-tetramethylbenzidine as an indicator
description	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.
format	article
author	Olena V. Koval’ska Mykola Ye. Blazheyevskіy Vladislav V. Diadchenko
author_facet	Olena V. Koval’ska Mykola Ye. Blazheyevskіy Vladislav V. Diadchenko
author_sort	Olena V. Koval’ska
title	The determination of the cholinesterase activity using 3,3′,5,5′-tetramethylbenzidine as an indicator
title_short	The determination of the cholinesterase activity using 3,3′,5,5′-tetramethylbenzidine as an indicator
title_full	The determination of the cholinesterase activity using 3,3′,5,5′-tetramethylbenzidine as an indicator
title_fullStr	The determination of the cholinesterase activity using 3,3′,5,5′-tetramethylbenzidine as an indicator
title_full_unstemmed	The determination of the cholinesterase activity using 3,3′,5,5′-tetramethylbenzidine as an indicator
title_sort	determination of the cholinesterase activity using 3,3′,5,5′-tetramethylbenzidine as an indicator
publisher	National University of Pharmacy (Kharkiv, Ukraine)
publishDate	2021
url	https://doi.org/10.24959/ophcj.21.240776 https://doaj.org/article/72a105ab835e4c3ebdd6125ba2051f15
work_keys_str_mv	AT olenavkovalska thedeterminationofthecholinesteraseactivityusing3355tetramethylbenzidineasanindicator AT mykolayeblazheyevskíy thedeterminationofthecholinesteraseactivityusing3355tetramethylbenzidineasanindicator AT vladislavvdiadchenko thedeterminationofthecholinesteraseactivityusing3355tetramethylbenzidineasanindicator AT olenavkovalska determinationofthecholinesteraseactivityusing3355tetramethylbenzidineasanindicator AT mykolayeblazheyevskíy determinationofthecholinesteraseactivityusing3355tetramethylbenzidineasanindicator AT vladislavvdiadchenko determinationofthecholinesteraseactivityusing3355tetramethylbenzidineasanindicator
_version_	1718428992188448768

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

Ejemplares similares