THE KINETICS OF CHOLINESTERASES MEASURED FLUOROMETRICALLY

Abstract

A new, simple, sensitive method is described for the fluorometric assay of cholinesterase activity based upon the hydrolysis of 1-naphthyl esters and the measurement of the fluorescence of 1-naphthol. This allows the study of the kinetics of cholinesterases and inhibitors with histochemical substrates and permits assessment of the parameters of the enzyme reaction under conditions approximating those in the histochemical system. 1-Naphthylacetate is a substrate for AChE3 and ChE, while 1-naphthyl butyrate is selective for ChE. The application of the procedure to the study of inhibition by hydrolyzable as well as nonhydrolyzable, nonfluorogenic, inhibitors is demonstrated. ACh was found to be a mixed inhibitor of eel AChE in this system. Edrophonium was found to be a more potent competitive inhibitor of AChE than either physostigmine or pyridostigmine, but a much weaker inihibitor of ChE than the latter two. Ambenonium behaves as a noncompetitive inhibitor of ChE; it is at least 10,000 times more effective on AChE and is 300 times more potent an inhibitor of AChE than is physostigmine. The use of edrophonium and ambenonium as selective inhibitors of AChE is suggested.