I use modified Ellman's method for determine cholinesterase inhibitory activities of some compounds. But i suspect that some thiourea derivetives form thiol group and give false negative yellow color. Is there any alternative method or modification for standart Ellman's procedure.
Dear Burcu,
We have done AChE estimation by this method in our lab. Below is the protocol, hope you find some assistance.
AChE Assay
AChE activity was determined sepectropotometrically at 412 nm by measuring the rate of hydrolysis of substrate, according to the method of Ellman et al., (1961). Which is base on the principle of the measurement of rate of production of thiocholine as acetylcholine is hydrolyses.
Solutions:
Solution - A. Di-Sodium hydrogen phosphate (Na2 H.PO4 2H2O) 8.89 gm was dissolved in 500 ml double distilled water.
Solution - B. Sodium di-hydrogen phosphate (Na H2.PO4 2H2O) 7.80 gm was dissolved in 500 ml double distilled water.
Solutions A and B were mixed and pH was maintained at 7.4 just before the use.
Substrate solution to use fresh.
This is based on the principal of measurement of the rate of hydrolysis of thiocholine as acetylthiocholine to hydrolyze. The chemical reaction is complete the thiocholine with 5: 5-dithio-bis-2-nitrobenzoic acid ion to produce the yellow anion of 5-thio.2-nitrobenzoic acid. The rate of the colors production measured at 412 nm in a spectrophotometer.
Method: Aliquots of homogenates were diluted with 2.6 ml 0.1 M phosphate buffer (pH 7.4) to a total volume 2.8 ml which contained 100 mM DTNB and 75 mM substrate (ATCI). The enzyme activity was calculated by extinction coefficient (ΔE= 1.36 X 104M-1Cm-1) for the yellow anion of 5-thi-2 nitrobenzoic acid. The AChE activity was expressed as µM of ATCI hydrolyzed /min/milligram of protein. All sepectropotometrically measurement was done in duplicate. In each experiment blank without sample was used to estimation of enzyme activity. The inhibition % was determined and compared for different regions of the brain and serum.
Further you can access one of our papers on link below:
Article Anticholinesterase Efficacy of Bacopa monnieri against the B...
6 minutes ago
Ellman's method is the best to determine cholinesterase inhibitory activity. If you suspect false negative results, tray to change some of your protocol experimental parameters including temperature and essay tubes and of course repeat each test at least twice.
Ellman's method is the most assured method for ChEs inhibition. But if ur getting problem with your compounds try using another blank with just compound and DTNB along with the bioassay. And the absorbance you get from this blank you can minus that from your bioassay absorbance results and then try for calculations.
Either increase the volume of DTNB so that your test compounds interference is negligilbe.
Or make a dummy/ blank of DTNB and test compounds. Later subtract the value from your enzyme inhibition assay
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