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% (v/v) methanol and 50% (v/v) water used as the mobile phase. Kynurenine and KYNA were detection by UV detection at a wavelength of 330 nm.