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

2021 ◽  
Vol 19 (3(75)) ◽  
pp. 34-39
Author(s):  
Olena V. Koval’ska ◽  
Mykola Ye. Blazheyevskіy ◽  
Vladislav V. Diadchenko
Keyword(s):  
Enzymatic Hydrolysis ◽  
Optical Density ◽  
Working Conditions ◽  
Specific Activity ◽  
High Sensitivity ◽  
New Method ◽  
Limit Of Quantification ◽  
Activity Unit ◽  
Hydrolysis Of ◽  
Safe Working

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.

scholarly journals The Optimised Statistical Model for Enzymatic Hydrolysis of Tapioca by Glucoamylase Immobilised on Mesostructured Cellular Foam Silica

2019 ◽  
Vol 14 (2) ◽  
pp. 380
Author(s):  
Joni Agustian ◽  
Lilis Hermida
Keyword(s):  
Enzymatic Hydrolysis ◽  
Large Scale ◽  
Specific Activity ◽  
Agitation Speed ◽  
Quadratic Model ◽  
Lack Of Fit ◽  
Km Value ◽  
Hydrolysis Of ◽  
Central Composite ◽  
Better Than

Enzymatic hydrolysis of starches using free glucoamylase to reducing sugars have difficulties in recovering and recycling of the enzyme, hence immobilisation on inert supports were widely studied. However, effectiveness of the immobilised glucoamylase were merely observed only on soluble starches. It was considered a valuable thing to know performance of glucoamylase on Mesostructured Cellular Foam (MCF) silica in hydrolysing of tapioca. An optimised study on enzymatic hydrolysis of tapioca using glucoamylase on MCF silica (9.2T-3D) and its kinetics were described including justification of the predicted model as it was required to develop in large scale operations. Central Composite Design was used to model the process by studying effects of three factors on DE values after enzyme immobilisation.  Immobilisation of glucoamylase on this support gave up to 82% efficiency with the specific activity of 1,856.78 U.g-1. Its used to hydrolysis of tapioca resulted DE values of 1.740-76.303% (w/w) where the highest DE was obtained at pH of 4.1, temperature of 70 ℃ and agitation speed of 140 rpm. The optimisation produced a polynomial quadratic model having insignificant lack-of-fit and low standard deviation, so that it was applicable and reliable in simulating the DE with only 0.80% of data were not described. Temperature affected the process highly, but the buffer pH, agitation speed and factorial interactions were considered not important. KM value for immobilised enzyme was better than the free glucoamylase, however, its reaction rate was slower than the free glucoamylase catalysis. Copyright © 2019 BCREC Group. All rights reserved 

scholarly journals Sensing activity of cholinesterases through a luminescence response of the hexarhenium cluster complex [{Re6S8}(OH)6]4−

The Analyst ◽  
2016 ◽  
Vol 141 (13) ◽  
pp. 4204-4210 ◽  
Author(s):  
Julia G. Elistratova ◽  
Asiya R. Mustafina ◽  
Konstantin A. Brylev ◽  
Konstantin A. Petrov ◽  
Michael A. Shestopalov ◽  
...  
Keyword(s):  
Enzymatic Hydrolysis ◽  
New Method ◽  
Cluster Complex ◽  
Hydrolysis Of

A new method to sense enzymatic hydrolysis of acetylcholine through a cluster luminescence.

A micromethod for quantitative determination of iodoamino acids in thyroglobulin

1997 ◽  
Vol 153 (1) ◽  
pp. 99-104 ◽  
Author(s):  
N Baudry ◽  
B Mallet ◽  
P J Lejeune ◽  
L Vinet ◽  
J L Franc
Keyword(s):  
Acetic Acid ◽  
Enzymatic Hydrolysis ◽  
Mobile Phase ◽  
High Sensitivity ◽  
The Other ◽  
Reverse Phase Hplc ◽  
Reverse Phase ◽  
Spectrophotometric Detection ◽  
Hydrolysis Of

Abstract We describe a new method for quantification of iodoamino acids after enzymatic hydrolysis of thyroglobulin. The procedure involves separation of monoiodotyrosine (MIT), diiodotyrosine, tri-iodothyronine and thyroxine by reverse phase HPLC with a Vydac C18 stationary phase and a mobile phase of water–acetonitrile–acetic acid. The separation is monitored by sensitive spectrophotometric detection through a 96-well microplate system based on the catalytic Sandell–Kolthoff reaction of iodide on the oxidation of arsenic(III) by cerium(IV). This new microassay is particularly convenient because of its high sensitivity and its rapidity (less than 2 h). It can detect 1 pmol MIT and 0·5 pmol of the other three iodoamino acids with a recovery higher than 96%. Moreover, the 96-well microplate system allows many samples to be tested simultaneously and avoids the use of radiolabeled iodine. Journal of Endocrinology (1997) 153, 99–104

scholarly journals A New Method of Investigating the Enzymatic Hydrolysis of Sucrose. II.

1963 ◽  
Vol 17 ◽  
pp. 992-1000 ◽  
Author(s):  
Kate Bak ◽  
Bjørn Andersen ◽  
Bengt Lindberg ◽  
James McKay ◽  
E. Meisingseth
Keyword(s):  
Enzymatic Hydrolysis ◽  
New Method ◽  
Hydrolysis Of

On the Preparation of Bovine α-Thrombin

1978 ◽  
Vol 39 (01) ◽  
pp. 193-200 ◽  
Author(s):  
Erwin F Workman ◽  
Roger L Lundblad
Keyword(s):  
Immobilized Enzyme ◽  
Catalytic Properties ◽  
Specific Activity ◽  
Guanidine Hydrochloride ◽  
Low Molecular Weight ◽  
Reversible Process ◽  
Gel Beads ◽  
Tissue Thromboplastin ◽  
C 50 ◽  
Hydrolysis Of

SummaryAn improved method for the preparation of bovine α-thrombin is described. The procedure involves the activation of partially purified prothrombin with tissue thromboplastin followed by chromatography on Sulfopropyl-Sephadex C-50. The purified enzyme is homogeneous on polyacrylamide discontinuous gel electrophoresis and has a specific activity toward fibrinogen of 2,200–2,700 N.I.H. U/mg. Its stability on storage in liquid media is dependent on both ionic strenght and temperature. Increasing ionic strength and decreasing temperature result in optimal stability. The denaturation of α-thrombin by guanidine hydrochloride was found to be a partially reversible process with the renatured species possessing properties similar to “aged” thrombin. In addition, the catalytic properties of a-thrombin covalently attached to agarose gel beads were also examined. The activity of the immobilized enzyme toward fibrinogen was affected to a much greater extent than was the hydrolysis of low molecular weight, synthetic substrates.

Enhancement of Platelet Sensitivity to ADP-Aggregation by Isometric Exercise in Arteriosclerotic Patients and its Prevention

1977 ◽  
Vol 37 (02) ◽  
pp. 329-338 ◽  
Author(s):  
Tadahiro Sano ◽  
Takeshi Motomiya ◽  
Hiroh Yamazaki ◽  
Takio Shimamoto
Keyword(s):  
Cyclic Amp ◽  
Mechanical Stress ◽  
Optical Density ◽  
Platelet Function ◽  
Isometric Exercise ◽  
Phosphodiesterase Inhibitor ◽  
New Method ◽  
Platelet Aggregability ◽  
Control Subjects ◽  
Healthy Control

SummaryA new method for assessment of platelet sensitivity to ADP-aggregation was devised. Its reproducibility and the correlations between the values obtained by this method, the optical density (O. D.) method, and the screen filtration pressure (SFP) method were assessed. In summary, this method may be said to have three main points:1. It can be performed without centrifugation, avoiding mechanical stress to platelets, using only 0.8 ml. of blood and inexpensive equipment.2. It may reflect different aspects of platelet function from the O. D. method and the SFP method, despite the positive significant correlations between the values obtained by these three methods.3. It was proved to be highly reproducible and is thought to be useful clinically.By using this method, the effect of sustained isometric exercise by handgripping on platelet aggregability was assessed in coronary sclerotic and cerebral arteriosclerotic patients on placebo and EG-626, a newly synthesized cyclic AMP phosphodiesterase inhibitor. On placebo, an enhancement of platelet sensitivity was observed after isometric exercise in coronary and cerebral arteriosclerotic patients but not in healthy control subjects. The enhancement was prevented by pretreatment of EG-626, administered orally 1.5 hours prior to exercise.

Microwave-assisted enzymatic hydrolysis of starch

2009 ◽  
Author(s):  
Marcin Lukasiewicz ◽  
Anna Osowiec ◽  
Magdalena Marciniak
Keyword(s):  
Enzymatic Hydrolysis ◽  
Microwave Assisted ◽  
Hydrolysis Of

Actual issues of hygienic regulation and creation of safe working conditions at pharmaceutical manufacturing enterprises

2020 ◽  
Vol 60 (10) ◽  
pp. 640-644
Author(s):  
Vadim M. Vasilkevich ◽  
Ruslan V. Bogdanov ◽  
Elena V. Drozdova
Keyword(s):  
Pharmaceutical Industry ◽  
Working Conditions ◽  
Experimental Studies ◽  
Pharmaceutical Products ◽  
Laboratory Animals ◽  
Production Environment ◽  
Basic Principles ◽  
Toxicological Studies ◽  
Health Disorders ◽  
Safe Working

Introduction. The working conditions of pharmaceutical industry workers are characterized by the combined effect of unfavorable factors of the production environment, among which the leading one is chemical. The aim of study is to substantiate the basic principles and criteria for hygienic regulation of pharmaceutical products in their production to ensure safe working conditions for employees based on the results of their own research and existing requirements of technical regulations. Materials and methods. Analysis of working conditions and the prevalence of health disorders in pharmaceutical workers (according to literature data), toxicological studies of pharmaceutical substances on laboratory animals, scientific justification of hygiene standards in the air of the working area. Results. Among employees of the pharmaceutical industry, the predominant forms of production-related health disorders are diseases of the respiratory system, as well as skin dermatitis of allergic origin, liver and biliary tract diseases. Based on the results of experimental studies of domestic pharmaceutical products for the treatment of cardiovascular, oncological and mental diseases that have priority socio-economic significance, the basic principles and features of the practice of justifying the hygienic standards of medicines in the air of the working area are developed and systematized. Conclusions. During hygienic rationing of medicines, it is necessary to use a differentiated approach that allows, based on the analysis of information about the chemical structure, physical and chemical characteristics, production conditions, pharmacotherapeutic activity, and the results of studying the toxic effect in an experiment on laboratory animals, to determine the maximum permissible content in the air of the working area of medicines or to justify the prohibition of isolation with reasoned recommendations for their safe production.

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