Kynurenine 3-hydroxylase Binding Affinity

The Surface Plasmon Resonance (SPR) method was used in conjunction with Biacore T200 (GE Healthcare, Uppsala, Sweden) to measure the binding affinities at 25 ºC. Recombinant human Kynurenine 3-hydroxylase (KMO) protein (OriGene Technologies Inc., Rockville, MD, USA) was immobilized on the CM5 sensor chip (GE Healthcare, Uppsala, Sweden) by amine-coupling method to achieve the target density of 10000 resonance units (RU). The PBS-P (10 mM sodium phosphate, 150 mM NaCl, 0.005% Tween-20, pH 7.4) was used as a running buffer. Then different concentrations of the compounds (0.1953125 µM, 0.390625 µM, 0.78125 µM, 1.5625 µM, 3.125 µM) were injected at a flow rate of 30 μL/min. Chip platforms were washed with a running buffer and 50% DMSO. The binding affinities were analyzed with the Biacore evaluation software (T200 Version 2.0) by curve fitting using a 1:1 binding model 33 (link).