A microfluorometric assay for cholinesterases, suitable for multiple kinetic determinations of picomoles of released thiocholine

A highly sensitive microfluorometric assay for cholinesterases has been developed. Enzymatic activity is measured by monitoring the thiocholine produced by specific hydrolysis of acetylthiocholine. This is carried out by reacting the thiocholine formed with the fluorogenic compound N-(4-(7 diethylamino-4-methylcoumarin-3-yl)phenyl)maleimide to yield an intensely fluorescent product. The assay is linear over a range extending from a few picomoles to nanomoles of thiocholine. The specificity and accuracy of this microfluorometric assay were examined using microgram quantities of rat brain tissue as a source for cholinesterases. The specific activities and the K_m values determined by this new method for both cholinesterase activities present in the brain (acetylcholine hydrolase, EC 3.1.1.7, and "nonspecific" cholinesterase-acylcholine acylhydrolase, EC 3.1.1.8) were identical to those reported earlier using the less sensitive spectrophotometric and radiometric methods. The background emission caused by nonenzymatic hydrolysis of the substrate is relatively low, and does not exceed background values encountered in other methods. The assay may be used for monitoring the kinetics of enzymatic activities in microscale reaction mixtures, providing a linear determination of the thiocholine produced over a period of at least 30 h at room temperature. The method can also be adapted for use in other enzymatic assays where reagents containing thiol groups can be produced or consumed.