Comparative enzymatic studies using ion-selective electrodes. The case of cholinesterases

Abstract

The application of traditional ion-selective electrodes for comparative enzymatic analysis was demonstrated for the first time in this study. A kinetic-potentiometric method based on the monitoring of the concentration of the ionic substrate involved in the enzymatic reaction catalyzed by different cholinesterases is used for this purpose. A comparative study was performed comprising both enzymatic assays using different ionic substrates and the corresponding inhibited reactions in presence of neostigmine (a synthetic anticholinesterase). The developed approach is used to obtain valuable comparative results through calculation of kinetic parameters, such as Michaelis and inhibition constants. Interesting results were obtained for acetylcholinesterase and butyrylcholinesterase enzymes, which were selected as proof-of-concept: (i) the binding affinity that these enzymes have for their natural substrates showed to be higher (acetylcholine and butyrylcholine respectively) than for their corresponding thiol derivatives (acetylthiocholine and butyrylthiocholine), which are traditionally used in spectrophotometric enzymatic assays; (ii) as expected, the maximum hydrolysis rate found in the assays of each enzyme was independent of the substrate used; (iii) acetylcholinesterase enzyme inhibition due to neostigmine was found to be higher (higher inhibition constant). Advantageously, the use of ion-selective electrodes permits to perform cholinesterases’ enzymatic assays using their natural substrates and under physiological conditions, unlike the traditional spectrophotometric methods used in routine enzymatic assays. Importantly, while well-known enzymes are use throughout this work, this approach can be extended to other types of enzymatic assays as a tangible alternative to traditional spectrophotometric methods.