Establishing an assay to evaluate d-amino acid oxidase enzyme kinetics and inhibition using WST-8 redox dye

d-Amino acid oxidase (DAO) inhibitors are novel candidate therapeutic agents for neurodegenerative diseases. In this study, a novel inhibition assay using WST-8 as a detection reagent was developed and its effectiveness for evaluating redox-based enzyme kinetics was tested. Furthermore, using this assay, we also investigated the inhibitory activities of uremic toxins and endogenous amino acid metabolites. Briefly, a mixture of DAO, WST-8, and d-amino acids was allowed to react in phosphate buffer and DAO activity was detected as an increase in absorbance when both d-serine and DAO were present. However, as reducing compounds could potentially cause false-positive results, we confirmed that the detection of DAO activity in this system was mediated by the redox reaction of WST-8. Critically, we verified that this assay could be used to evaluate the inhibitory activity of known DAO inhibitors, including uremic toxins which showed weak DAO inhibition. This study establishes an alternative assay system that can be utilized for assessing DAO-inhibitory activity and Other enzymatic reactions mediated by redox reactions.

d‐amino acid; d‐amino acid oxidase; WST‐8; redox dye; uremic toxins.