A method for the determination of the concentration of catalytic centres in low activity and unstable preparations of acetylcholinesterase.

A simple and rapid method has been developed for the titration of catalytic centres of acetylcholinesterase of low activity and stability in homogenates of larvae of the cattle tick Boophilus microplus. It is based on the difference in uptake of the labeled organophosphate inhibitor ¹⁴C]coroxon between substrateprotected and unprotected enzyme. The excess coroxon is removed rapidly by solvent extraction of the acidified enzyme medium with acetone and toluene. The method was validated by the use of bovine erythrocyte acetylcholinesterase, with only 6 × 10^?12 catalytic centre mole equivalents of this enzyme being required for a single accurate assay. The turnover number at pH 7.6 and 37°C was 1.22 × 10⁶ molecules of acetylcholine hydrolysed per min per active centre. The catalytic efficiency of enzyme of larvae of the cattle tick was markedly different, being onetenth of that of bovine erythrocyte enzyme. Advantages of the method are discussed.