Direct Fluorescence Evaluation of D-Amino Acid Oxidase Activity Using a Synthetic D-Kynurenine Derivative

D-Amino acid oxidase (DAO) has been suggested to be associated with the central nervous system diseases, such as schizophrenia. We newly synthesized a nonfluorescent 5-methylthio-D-kynurenine (MeS-D- KYN), which was converted to blue-fluorescent 6-MeS-kynurenic acid (MeS-KYNA, lambda ex = 364 nm, lambda em = 450 nm) through a one-step reaction by incubation with DAO. It was revealed that fluorescence intensity increased accompanied by commercial porcine kidney DAO activity (unit) with a good correlation (R2 = 0.9972), suggesting that the fluorometric evaluation of DAO activity using MeS-D-KYN is feasible. MeS-D-KYN was applied to fluorescent DAO imaging in cultured LLC-PK1 cells, and the blue fluorescence of MeS-KYNA overlapped considerably with the location of peroxisomes, which was suggested to be the location of DAO in the cells. Because fluorescence was diminished in the presence of 6-chloro-1,2benzisoxazol-3(2H)-one (CBIO), a DAO inhibitor, it was considered that DAO activity in cells could be directly evaluated using MeS-D-KYN as the substrate.

Automated summary

D-Amino acid oxidase (DAO) has been suggested to be associated with central nervous system diseases. A nonfluorescent substrate, MeS-D-KYN, was synthesized and showed potential for evaluating DAO activity by converting to blue-fluorescent MeS-KYNA. Fluorescence imaging confirmed the overlap of MeS-KYNA fluorescence with the distribution of DAO in cells, indicating the direct evaluation of cellular DAO activity using MeS-D-KYN as a substrate.