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

Abstract

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, λex = 364 nm, λem = 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-1,2-benzisoxazol-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.