| Assay Method Information | |
| | In-vitro IDO2 Enzyme (Indoleamine 2,3-dioxygenase2) Assay |
| Description: | Human indoleamine 2,3-dioxygenase-2 (hIDO2) catalyzes the oxidative cleavage of the pyrrole ring of the indole nucleus of tryptophan to yield N-formylkynurenine which can be converted to kynurenine (KYN) by deformylation. hIDO2 with an C-terminal His tag (Sino Biological Inc (China)) was expressed in E. coli cells and the protein was purified using standard methods well known in the art. Unless otherwise stated, all materials were procured from Sigma Aldrich, Mo., USA.The assay monitoring the conversion of L-tryptophan to KYN was carried out as follows. hIDO2 (160 nM) was incubated with tryptophan (5000 04) in the presence of ascorbic acid (20 mM), methylene blue (10 04) and catalase (100 μg/mL) in potassium phosphate buffer (100 mM; pH 7.5) at 37 degrees C. for 30 min. The reaction was terminated with 30% trichloroacetic acid (TCA) and further incubated at 65 degrees C. for 15 min to fully convert N-formylkynurenine to KYN. The reaction mixture was then centrifuged to remove sediments, and the KYN in the supernatant was measured by UV-visible absorption spectroscopy at 360 nm using a Waters HPLC system fitted with a C-18 column or by LC/MS/MS (C. J. D Austin et. Al, Amino Acid 2009, 565-578).Percent inhibition at each concentration of test compounds was determined by determining the decrease in KYN with reference to the reaction control with 1% DMSO vehicle. Data were analyzed using nonlinear regression to generate IC50 values using Graph Pad Prism 5. Compounds 2 and 184 were tested as described above. |
| Affinity data for this assay | |
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