CHEMBL3706281

Colorimetric Assay: The inhibition efficacy of PAD4 inhibitors were determined by colorimetric measurement of citrulline generated by PAD4 catalyzed citrullination of BAEE. 0.2 μg PAD4 was pre-incubated with inhibitors in 100 μl buffer containing 50 mM Tris-HCl pH7.6, 5 mM CaCl2, 2 mM DTT for 0.5 hr at 37°C. The reaction was started by the addition of BAEE to 5 mM and halted 1.5 hr later with the addition of 25 μl 5 M HClO4. Then the samples were briefly centrifuged at 12000 rpm for 2 min at 4°C. and the supernatant (120 μl) were assayed for citrulline by mixing with 120 μl reagent A (0.5% w/v diacetyl monoxime and 15% w/v NaCl in water) and 240 μl reagent B (1% w/v antipyrine, 0.15% w/v ferric chloride, 25% v/v H2SO4 and 25% v/v H3PO4). The mixtures were boiled for 15 min and cooled to room temperature in ice bath. The absorbance of the reagent mixtures at 464 nm was measured by Thermo BioMate 3 spectrophotometer.