engleski

Limitation of the Ellman method: Cholinesterase activity measurement in the presence of oximes

The Ellman method for assaying thiols is widely used for cholinesterase activity measurement. The cholinesterase activity is measured indirectly quantifying the concentration of 5-thio-2-nitrobenzoic acid (TNB) ion formed in the reaction between thiol reagent DTNB (5, 5'-dithiobis(2-nitrobenzoic acid)) and thiocholine, a product of substrate i.e. acetylthiocholine (ATCh) hydrolysis by the cholinesterase. Oximes, reactivators of inhibited cholinesterase, are nucleophiles that also react with ATCh (oximolysis), producing thiocholine and indirectly TNB ion. The aim of this study was to characterize ATCh oximolysis. Therefore we measured the oximolysis between oximes (K027 or HI-6) and ATCh in the presence of DTNB at different pH, taking into account the final concentration of a product which is thiocholine. To confirm oximate ion involvement in the nucleophilic attack we also determined the reaction rate between the oximes and ATCh, without DTNB, at different pH by measuring the decrease in oximate ion absorption over time. Oximate ion of K027 reacted 14 times faster with ATCh (306 M-1 min-1) than oximate ion of HI-6 (22 M-1 min-1). However the rate constants obtained with the Ellman method were 84 M-1 min-1 for K027 and 22 M-1 min-1 for HI-6. Our results confirmed that the rate obtained with K027 using the Ellman method is actually rate of the Ellman reaction itself. This suggests that the Ellman method can not be used uncritically to evaluate oxime reaction with choline esters, in particular when oximolysis is faster than the Ellman reaction itself at a given pH.

acetylcholinesterase; acetylthiocholine; reactivation; oximolysis; DTNB

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