engleski

Acetylcholinesterase and butyrylcholinesterase activity measurements by the Ellman method: I. Evaluation of procedure protocol

We suggest the following procedure for measuring erythrocyte acetylcholinesterase (AChE ; EC 3.1.1.7) and plasma butyrylcholinesterase (BChE, EC 3.1.1.8) activities in human blood. After centrifugation of whole blood and separation of plasma, the erythrocytes are suspended in water (in a volume corresponding to the initial volume of blood), diluted 60-fold with phosphate buffer and the suspension frozen in order to haemolyse the erythrocytes. After thawing, the suspension is further diluted with phosphate buffer (0.1 M, pH 7.4, 37 oC) and the thiol reagent DTNB added (0.33 mM final conc.). After 10 min, acetylthiocholine (ATCh) is added (1.0 mM final conc.) and the increase in absorbance read at 412 nm. The BChE activity in plasma is also measured with 1.0 mM ATCh. The final dilution of the erythrocytes during enzyme assay is 600-fold and of the plasma 150-fold. The optical density of the erythrocyte suspension is not stable. The absorbance decreases about 0.006 over 10 min. at 412 nm ; the extent of that decrease is too small to interfere with the enzyme assay. DTNB reacts with thiol groups present in erythrocytes ; under the above conditions, this reaction is completed over about 10 min. and the reaction reduces the DTNB concentration by about 0.5%. For that reason the substrate is added 10 min. after DTNB.

acetylcholinesterase; butyrylcholinesterase; activity mesurement; human blood

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