Methylene Blue (Sigma, Catalog # 28514), 10 mM stock in deionized water
96-well Clear Plate (Costar, Catalog # 92592)
Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
Prepare the Substrate Mixture.
Dilute Ascorbic Acid to 80 mM in 0.405 M Tris, pH 8.0.
Prepare a mixture of 800 μM L-Tryptophan, 9000 units/mL catalase, and 40 μM Methyene Blue in Assay Buffer.
Mix equal volumes of 1a and 1b for final concentrations of 40 mM Ascorbic Acid, 400 μM L-Tryptophan, 4500 units/mL Catalase, and 20 μM Methylene Blue.
Dilute rhIDO to 16 ng/μL in Assay Buffer.
Load into a plate 50 μL of 16 ng/μL rhIDO, and start the reaction by adding 50 μL of Substrate Mixture.
Include a Substrate Blank containing 50 μL of Assay Buffer and 50 μL of Substrate Mixture.
Read at 321 nm in kinetic mode for 5 minutes.
Specific Activity (pmol/min/µg) =
Adjusted Vmax* (OD/min) x well volume (L) x 1012 pmol/mol
ext. coeff** (M-1cm-1) x path corr.*** (cm) x amount of enzyme (µg)
*Adjusted for Substrate Blank **Using the extinction coefficient 3750 M-1cm-1 ***Using the path correction 0.32 cm Note: the output of many spectrophotometers is in mOD. Per Well:
rhIDO: 0.800 µg
Ascorbic Acid: 20 mM
L-Tryptophan: 200 μM
Catalase: 225 units
Methylene Blue: 10 µM
Background: Indoleamine 2,3-dioxygenase/IDO
Indoleamine 2,3-dioxygenase (IDO) is a heme-containing intracellular dioxygenase catalyzing the degradation of the essential amino acid L-tryptophan to N‑formyl‑kynurenine (1). This degradation is the first and rate-limiting step of the L-kynurenine pathway (2). IDO is widely expressed in dendritic cells, macrophages, microglia, eosinophils, fibroblasts, endothelial cells, and most tumor cells. In immune cells, its expression is mainly induced by cytokines such as IFN‑ gamma, IFN‑ alpha, IFN‑ beta, and IL‑10. IDO has an antimicrobial function due to its decreasing the availability of the essential amino acid tryptophan in inflammatory environments (3). Recent studies have demonstrated that IDO induces immunosuppression during infection, pregnancy, transplantation, autoimmunity, and neoplasia (3‑5).
Lewis-Ballester, A. et al. (2009) Proc. Natl. Acad. Sci. USA. 106:17371.
Costantino, G. (2009) Expert Opin. Ther. Targets 13:247.
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