武漢云克隆科技股份有限公司

IDO (Indoleamine 2,3-dioxygenase 1) is a heme enzyme that catalyzes the first and rate-limiting step in tryptophan catabolism to N-formyl-kynurenine. This enzyme acts on multiple tryptophan substrates including D-tryptophan, L-tryptophan, 5-hydroxy-tryptophan, tryptamine, and serotonin. Thus, bioactivity of recombinant human IDO was measured through its ability to oxidize L-tryptophan to N-formyl-kynurenine, using Methylene Blue as indicator. The reaction was performed in 50 mM MES, pH 6.5 (Assay Buffer), initiated by addition 50 μL of various concentrations of IDO (diluted by Assay Buffer) to 50 μL substrate mixture of 800 μM L-tryptophan, 9000 units/mL catalase （RPC418Hu05）, and 40 μM Methyene Blue in assay buffer with equal volume of 80 mM ascorbic acid in 0.405 M Tris, pH 8.0. The final well serves as a negative control with no IDO, replaced with 50μL assay buffer. The absorbance was read in 321 nm in kinetic mode for 5 minutes. The result indicated that recombinant human IDO can oxidize L-tryptophan, the specific activity is 10581 pmol/min/μg.