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

FEBS Open Bio

卷:16

Method

Kahoko Miyake¹  Yuki Enoki¹  Yuka Nakazawa¹  Kazuaki Taguchi¹  Kazuaki Matsumoto¹ 

[1] Division of Pharmacodynamics, Faculty of Pharmacy, Keio University, Tokyo, Japan

关键词: ‐amino acid;    ‐amino acid oxidase;    redox dye;    uremic toxins;    WST‐8;   

DOI  :  10.1002/2211-5463.70217

 received in 2025-05-13, accepted in 2026-02-10,  发布年份 2026

来源: Wiley

【 摘 要 】

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.

【 授权许可】

CC BY   
© 2026 The Author(s). FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

【 图 表 】

Figure 3. Detection of positive cells in vivo at different ages.

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