• Purity

  >90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie? Blue Staining.

• Endotoxin Level

  <1.0 EU per 1 μg of the protein by the LAL method.  

• Activity

  Measured by its ability to oxidize L-tryptophan to N-formyl-kynurenine. The specific activity is >1,000 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on .

• Source

  E. coli-derived Ala2-Pro407, with an N-terminal Met and 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Met

• Predicted Molecular Mass

  46 kDa

• SDS-PAGE

  41 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 50 mM MES, pH 6.5

• 0.405 M Tris, pH 8.0

• Recombinant Mouse Indoleamine 2,3-dioxygenase/IDO (rmIDO) (Catalog # 9157-AO)

• Ascorbic Acid (Sigma, Catalog # 255564), 500 mM stock in deionized water

• L-Tryptophan (Sigma, Catalog # T0254), 10 mM stock in deionized water

• Catalase (Sigma, Catalog # C30), 100,000 units/mL stock in Assay Buffer

• Methylene Blue (Sigma, Catalog # 28514), 10 mM stock in deionized water

• 96-well Clear Plate (Catalog # )

• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Prepare the Substrate Mixture.
   a. Dilute Ascorbic Acid to 80 mM in 0.405 M Tris, pH 8.0.
   b. Prepare a mixture of 800 μM L-Tryptophan, 9000 units/mL Catalase, and 40 μM Methylene Blue in Assay Buffer.
   c. 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.

2. Dilute rmIDO to 8 ng/μL in Assay Buffer.

3. Load 50 μL of 8 ng/μL rmIDO to clear plate, and start the reaction by adding 50 μL of Substrate Mixture. Include a Substrate Blank containing 50 μL Assay Buffer and 50 μL Substrate Mixture.

4. Read plate in kinetic mode for 5 minutes at an absorbance of 321 nm.

     *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:

• rmIDO: 0.40 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 that catalyzes the degradation of the essential amino acid L-tryptophan to N formyl kynurenine. This degradation is the first and rate-limiting step of the L-kynurenine pathway (1). IDO expression is induced in macrophages, plasmacytoid dendritic cells (pDC), and microglia by multiple inflammatory stimuli including IFN-g, TNF-a, CpG DNA, HIV-1 Tat protein, microbial infection, or GITR Ligand activation (2-9). It can be expressed by tumor cells, pDC in tumor draining lymph nodes, and MDSC in breast cancer (10-12). IDO expression is critical for the ability of these cells to induce the activation of resting Treg and the support of immune tolerance to apoptotic cell debris (7, 11, 13). These cells also suppress anti-tumor immunity (10, 12), the development of autoimmunity (13), and the replication of Chlamydia, Herpes simplex virus, and measles virus (3, 4, 14). In the absence of IDO function, activation of pDC does not induce immunosuppression but instead triggers IL-6 production and the redirecting of Treg to produce IL-17 (7). Mouse IDO shares 61% and 87% amino acid sequence identity with human and rat IDO, respectively (15).

• References:

1. Munn, D.H. and A.L. Mellor (2016) Trends Immunol. 37:193.

2. Thomas, S.R. et al. (1994) J. Biol. Chem. 269:14457.

3. Carlin, J.M. et al. (1989) J. Interferon Res. 9:329.

4. Adams, O. et al. (2004) Microbes Infect. 6:806.

5. Planes, R. and E. Bahraoui (2013) PLoS One 8:e74551.

6. O'Connor, J.C. et al. (2009) J. Neurosci. 29:4200.

7. Baban, B. et al. (2009) J. Immunol. 183:2475.

8. Grohmann, U. et al. (2007) Nat. Med. 13:579.

9. Loughman, J.A. and D.A. Hunstad (2012) J. Infect. Dis. 205:1830.

10. Holmgaard, R.B. et al. (2015) Cell Rep. 13:412.

11. Sharma, M.D. et al. (2007) J. Clin. Invest. 117:2570.

12. Yu, J. et al. (2013) J. Immunol. 190:3783.

13. Ravishankar, B. et al. (2012) Proc. Natl. Acad. Sci. USA 109:3909.

14. Obojes, K. et al. (2005) J. Virol. 79:7768.

15. Habara-Ohkubo, A. et al. (1991) Gene 105:221.

• Entrez Gene IDs:

  3620 (Human); 15930 (Mouse)

• Alternate Names:

  3dioxygenase; EC 1.13.11.52; IDO; IDO1; IDOIDO-1; INDO; INDOindole 2,3-dioxygenase; Indoleamine 2; indoleamine 2,3-dioxygenase 1; Indoleamine 2,3-dioxygenase; indoleamine-pyrrole 2,3 dioxygenase; Indoleamine-pyrrole 2,3-dioxygenase