HPLC JN-01 KAT-II IC50

KAT-II (0.5 μg) was incubated at 37 °C for 10 min in a 50 μL reaction mixture containing 50 μM PLP, 5 mM α-ketoglutarate, 5 mM l-kynurenine in PBS, pH 7.4, with the inhibitor being studied (1–2000 μM). Equal volume of formic acid (0.8M) was added to terminate the reaction, and 50 uL of this mixture was diluted to 1 mL. Kynurenine and KYNA produced during the reaction was analyzed by HPLC using a C18 reverse-phase column, with 50% (<i>v</i>/<i>v</i>) methanol and 50% (<i>v</i>/<i>v</i>) water used as the mobile phase. Kynurenine and KYNA were detection by UV detection at a wavelength of 330 nm.将0.5 μg的犬尿氨酸氨基转移酶II（KAT-II）置于50 μL的反应体系中，于37 ℃条件下孵育10 min。该反应体系以pH 7.4的磷酸盐缓冲液（Phosphate Buffered Saline, PBS）为缓冲介质，包含50 μM 磷酸吡哆醛（Pyridoxal 5'-phosphate, PLP）、5 mM α-酮戊二酸、5 mM L-犬尿氨酸，以及浓度范围为1~2000 μM的待研究抑制剂。向体系中加入等体积的0.8 M甲酸以终止反应，随后取50 μL该混合液稀释至1 mL。采用高效液相色谱（High Performance Liquid Chromatography, HPLC）结合C18反相色谱柱对反应生成的犬尿氨酸与犬尿喹啉酸（KYNA）进行分析，流动相为50%（v/v）甲醇与50%（v/v）水的混合溶液。通过波长330 nm的紫外检测法完成犬尿氨酸与KYNA的检测。