False positiveness in a choline acetyltransferase assay caused by nonionic reducing reagents

Area of Science:

• Biochemistry
• Enzyme kinetics
• Neuroscience

Background:

• Choline acetyltransferase (ChAT) is crucial for acetylcholine synthesis.
• Dithiothreitol (DTT) and 2-mercaptoethanol are common protective agents during ChAT purification.
• Potential interference of protective agents with enzyme assays is a concern.

Purpose of the Study:

• To investigate the interaction between DTT/2-mercaptoethanol and acetyl-CoA during ChAT purification.
• To identify alternative protective agents for ChAT purification that avoid assay interference.

Main Methods:

• Enzyme purification of choline acetyltransferase.
• Acetyl-CoA reaction assays.
• Acetylcholine quantification using organic scintillation liquid.

Main Results:

• Dithiothreitol and 2-mercaptoethanol react with acetyl-CoA.
• Reaction products are co-extracted with acetylcholine, leading to false high enzyme activity readings.
• Sodium thioglycolate, an anionic reducing agent, does not interfere with the assay.

Conclusions:

• Standard protective agents like DTT and 2-mercaptoethanol can artifactually inflate choline acetyltransferase activity measurements.
• Sodium thioglycolate is a suitable alternative protective agent for ChAT purification, ensuring assay accuracy.