An Efficient In Vitro Assay for Acetylcholinesterase Reactivators Using Immobilized Enzyme

Abstract

A new and efficient in vitro assay for evaluating reactivators of organophosphate-inhibited acetylcholinesterase has been developed. Low density polyethylene beads (4 mm) were func-tionalized to terminal aldehydes and used to immobilize acetylcholinesterase (AChE, Electrophorus electricus, E.G. 3.1.1.7) via a stable Schiff base link. AChE activity in columns containing immobilized enzyme could be continuously monitored spectrophotometrically in a closed loop flow system using acetylthiocholine and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB). Immobilized enzyme exhibited good esterase activity (0.5 units/bead), which could be retained on storage at −16°C for four months. The kinetics for substrate hydrolysis were flow-rate dependent below substrate saturation levels. This system allowed for independent inhibition and reactivation of the enzyme. Immobilized enzyme could be inhibited with diisopropylfluorophosphate (DFP) and 20–90% of original activity restored with several oximes in less than 20 minutes. The extent of reactivation was dependent on the concentration of the reactivators. This system has advantages over previously reported procedures, because hydrolysis of substrate due to reactivator is minimized and inhibitor-reactivator interactions are eliminated, subsequently minimizing the need to correct experimental results.