A Polarographic Method for following the Rates of Cholinesterase-catalyzed Hydrolyses of Acetylthiocholine.

In the past few years there has been considerable interest in the kinetic effects of certain species, such as Mylaxim and THA (g-amino1,2,3,4-tetrahydroaminoacridine) (1)) which act as inhibitors in t,he acetylcholinesterase-catalyzed hydrolysis of acetylcholine. Both compounds are essentially associated (completely inhibit enzyme activity) with t.he enzyme in less than a few minutes (2 to 4 min). As available assay methods are incapable of following the rates of such rapid reactions, it was of considerable interest to develop a simple rapid technique for these studies. The kinetic-based method for measurement of acetylcholinesterase activity for inhibition studies is essentially a problem in products analyses, which, under normal conditions, arc acetic acid and choline. As a sensitive analytical method for the alcoholic OH group of the choline molecule is not available, previous methods for following the hydrolysis rates have monitored the rate of production of t,he protons generated in the reaction. All of these methods are unsuit.able for following fast reactions, however. The Warburg manometrie method (2)) while being the standard method of determining the enzyme activity, is inherently slow in response and limits the choice of medium to a bicarbonate buffer. Titrimetric methods, employing a pH-Stat, such as the methods of Wilson (3) and Main and Dauterman (41, require t.he use of unbuffered media and are extremely slow because of the very long response time of the glass electrode and the mechanical servo system controlling the addition of NaOH (specially designed pH stats, however, are available which have response times of less than 10 set). Volume