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

Area of Science:

• Analytical Chemistry
• Biochemistry
• Enzyme Kinetics

Background:

• Traditional spectrophotometric methods limit enzymatic assays to specific substrates and conditions.
• Cholinesterase enzymes are crucial in various physiological processes and drug development.
• There is a need for methods that allow enzymatic analysis under physiological conditions using natural substrates.

Purpose of the Study:

• To demonstrate the first-time application of ion-selective electrodes for comparative enzymatic analysis.
• To develop and validate a kinetic-potentiometric method for studying cholinesterases.
• To compare the binding affinities and kinetic parameters of cholinesterases with natural and synthetic substrates.

Main Methods:

• Kinetic-potentiometric analysis using ion-selective electrodes.
• Enzymatic assays of acetylcholinesterase and butyrylcholinesterase.
• Monitoring ionic substrate concentration during enzymatic reactions.
• Inhibition studies using neostigmine.

Main Results:

• Ion-selective electrodes successfully enabled comparative enzymatic analysis of cholinesterases.
• Natural substrates (acetylcholine, butyrylcholine) showed higher binding affinity than thiol derivatives.
• Enzyme inhibition studies revealed higher inhibition of acetylcholinesterase by neostigmine.
• Maximum hydrolysis rates were substrate-independent for each enzyme.

Conclusions:

• The developed kinetic-potentiometric method offers a viable alternative to traditional spectrophotometric assays for cholinesterases.
• This approach allows enzymatic analysis using natural substrates under physiological conditions.
• The method's applicability can be extended to a broader range of enzymatic assays.