engleski

Limitations of the Ellman method in the cholinesterase reactivation assay

The Ellman method is widely used for spectrophotometric cholinesterase (ChE) assay due to its simplicity and reliability. The method utilizes acetylthiocholine ester (ATCh) as ChE substrate and DTNB (5, 5’ -dithiobis(2-nitrobenzoic acid)) as the thiol reagent. It is a two-step process in which enzyme hydrolyses ATCh in the first step, and the released thiocholine reacts with DTNB in the second step. Enzyme activity is measured indirectly through increase in the final product (TNB anion) absorption. However, another nucleophile like hydroxide or oxime can react with ATCh, producing thiocholine and finally TNB anion. Oximes are used as reactivators of ChE inhibited by organophosphorus compounds. To get accurate enzyme activity, separately measured reactions of the oxime with ATCh (oximolysis) must be subtracted from the enzyme ATCh hydrolysis when oxime is present. ATCh showed the same rate of oximolysis (k=11 M-1min-1, pH=7.4, 25 °C) with three different oximes (HI-6, TMB-4 and K027) when the Ellman method was employed. This seemed odd and to evaluate this we directly measured the reaction between the oxime and ATCh, without DTNB at different medium pH (7.4-9.2) by measuring the decrease in the oxime anion absorption over time. Oximes are proton donors, and the increase in the medium pH increases the concentration fraction of deprotonated oximes. Therefore, the rate constants of directly measured oximolysis were 22, 230 and 306 M-1 min-1 for HI-6, TMB-4 and K027, respectively. To explain the discrepancy in rate constants between the Ellman method and directly measured oximolysis, we repeated the Ellman method measurements with K027 (in our case the fastest reacting oxime), ATCh and DTNB, but this time using different medium pHs because thiocholine is a proton donor, just like the oximes. The obtained rate constant of the Ellman reaction was 82 M-1 min-1, after taking thiocholine anion concentration into account. This result showed that oximolysis, reaction between oxime and ATCh, could be faster than the Ellman reaction between thiocholine and DTNB. Therefore, one who measures ChE activity in the presence of an oxime must be aware of that.

ellman method; cholinesterase; DTNB; oximes

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