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Determination of acetylcholinesterase activity by chronopotentiometry

2019-07-17 来源:亚科官网


Acetylcholinesterase (AChE) is an enzyme related to neurological function. In medicine, blood cholinesterase activity is an important reference for the diagnosis and treatment of acute organophosphorus pesticide poisoning. At present, the common method for determining the activity of cholinesterase is the colorimetric method (Ellman method). The method generally takes 60 to 90 minutes from sample processing to completion, and the analysis speed is slow. Therefore, the method for rapidly determining the activity of acetylcholinesterase is very necessary.
Instruments and reagents
LK98A-microcomputer electrochemical analysis system; XW-80A vortex mixer; SYC-15 super constant temperature water bath; constant temperature electrolytic cell (homemade); electrode: platinum counter electrode, saturated calomel electrode.
Acetylcholinesterase (EC, type VI-S, from electroporation, 500 U/mg, Sigma); substrate: acetylthiocholine iodide (alias: thioacetylcholine iodide, cas: 1866-15-5, sub-item number: D0007), used in 0.1 mol/L, pH 7.4 Tris-HCl buffer solution to prepare a 0.002 mol/L solution, stored at 4 °C. The water is double distilled water.
Experimental method
1. Measurement conditions
Gear selection: constant current; sensitivity selection: 10mA; filtering parameters: 10~Z; step current: 0.020mA; magnification: 1; sampling points: 8000; sampling interval: 320ms; water bath temperature: 26°C
2. Experimental steps
Pipette 8.0 mL of the substrate solution into a constant temperature electrolytic cell and stir for 5 min. A certain amount of AChE was added, and an Et curve was recorded by electrolysis 9 under the above conditions, and the slope ΔE/Δt of the jump portion on the Et curve was measured.
Experimental principle
Electrolysis was carried out by applying a constant microcurrent to the two platinum electrodes of the electrolytic cell using iodinated acetylthiocholine as a substrate. A stable potential (about 0.45 V) was obtained because I- was oxidized to I2 in the substrate. When acetylcholinesterase is added, the thiol produced by enzymatic hydrolysis has greater electrical activity than the substrate, and can be oxidized at a lower potential (about 0.25 V), thus forming a drop in potential. The change of potential E with time t during electrolysis was recorded to obtain a typical S-shaped curve. The slope dE/dt of the depolarization curve reflects the rate of hydrolysis, ie the rate of oxidation of the enzyme. Under certain conditions, the stronger the enzyme activity, the faster the enzymatic reaction, and the larger the slope dE/d of the depolarization curve. Therefore, the activity of the enzyme to be tested can be calculated using a calibration curve.
Vitality calibration curve
Under the optimal conditions determined above, different quantities (0.02U, 0.03U, 0.04U, 0.05U, 0.06U) of commercial AChE were added, and the ΔE/Δt values were determined to linearly regress the data to obtain the linear equation. It is Y=-0.3236+35.59X, and its correlation coefficient is r=0.9920. There is a good linear relationship between the slope value ΔE/Δt of the jump portion on the E~t curve and the enzyme amount. The sample can be measured using a calibration curve.
Determination of activity of immobilized enzyme tablets
The AChE was immobilized on a commercial carrier membrane by glutaraldehyde cross-linking method to prepare an immobilized enzyme tablet, and the enzyme tablet activity was determined under the optimal conditions determined above, and the results are shown in Table 1.
It can be seen from the data in Table 1 that the immobilized acetylcholinesterase activity was determined by electrochemical chronopotentiometry, and the method was stable and reliable.
The activity of acetylcholinesterase was determined by electrochemical chronopotentiometry. The linear relationship between the amount of enzyme and its activity value △E/Δt was obtained. The results of satisfactory determination of immobilized acetylcholinesterase tablets were obtained. Compared with other methods, the method is simple, fast and reproducible.
Related links: acetylthiocholine iodide
Edited by Suzhou Yacoo Science Co., Ltd.