CHEMBL3887709

Inhibition Assay: To test for enzyme inhibition, the hemoglobin capture assay was used to measure nitric oxide production. The assay was performed at 37° C. in HEPES buffer (100 mM, with 10% glycerol, pH 7.4) in the presence of 10 μM L-arginine. Also included were 100 μM NADPH, 0.83 mM CaCl2, approximately 320 units/mL of calmodulin, 10 μM tetrahydrobiopterin, and human oxyhemoglobin (3 μM). For iNOS, CaCl2 and calmodulin were omitted and replaced with HEPES buffer (as neither are required for activation of iNOS). This assay was performed in 96-well plates using a Synergy 4 BioTek hybrid reader, and the dispensing of NOS enzyme and hemoglobin were automated; after 30 sec (maximum delay), NO production was read by monitoring the absorbance at 401 nm (resulting from the conversion of oxyhemoglobin to methemoglobin).