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Recombinant Human Kynurenine 3-Monooxygenase/KMO Protein, CF

Catalog #: 8050-KM
2 Citations Datasheet / COA / SDS
Catalog # Availability Size / Price Qty
8050-KM-025
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Citations (2)
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Recombinant Human Kynurenine 3-Monooxygenase/KMO Protein, CF Summary

Product Specifications

Purity
>80%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
Activity
Measured by the consumption of NADPH by hydroxylation of L-kynurenine to 3-hydroxy-kynurenine. The specific activity is >290 pmol/min/μg, as measured under the described conditions.
Source
Spodoptera frugiperda, Sf 21 (baculovirus)-derived human Kynurenine 3-Monooxygenase/KMO protein
Asp2-Leu441, with an N-terminal Met and 6-His tag
Accession #
O15229
N-terminal Sequence
Analysis
Inconclusive results, intact N-terminus verified by anti-poly-His Western
Predicted Molecular Mass
51 kDa
SDS-PAGE
40-45 kDa, reducing conditions

Product Datasheets

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8050-KM

Product Datasheet
COA
SDS

Carrier Free

What does CF mean?

CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.

What formulation is right for me?

In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.

8050-KM

Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl and Brij-35.
Shipping The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.

Assay Procedure

Materials
  • Assay Buffer: 50 mM Sodium Phosphate, 0.1% (w/v) Brij-35, pH 7.5
  • Recombinant Human Kynurenine 3-Monooxygenase/KMO (rhKMO) (Catalog # 8050-KM)
  • beta -Nicotinamide adenine dinucleotide phosphate reduced, tetrasodium salt ( beta -NADPH) (Sigma, Catalog # N7505), 10 mM stock in deionized water
  • L-Kynurenine (Tocris, (Catalog # 4393), 40 mM stock in 80 mM HEPES, pH 8.0
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rhKMO to 20 ng/μL in Assay Buffer.
  2. Prepare a Reaction Mixture containing 400 μM beta -NADPH and 600 μM L-Kynurenine in Assay Buffer.
  3. In a plate, load 50 μL of 20 ng/μL rhKMO, and start the reaction by adding 50 μL of Reaction Mixture.
  4. Include a Substrate Blank containing 50 μL of Assay Buffer and 50 μL of Reaction Mixture.
  5. Read at an absorbance of 340 nm in kinetic mode for 10 minutes.
  6. Calculate specific activity:

     Specific Activity (pmol/min/µg) =

 Adjusted Vmax* (OD/min) x -1 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 6270 M-1cm-1 
     ***Using the path correction 0.32 cm
     Note: the output of many spectrophotometers is in mOD Per Well:
  • rhKMO: 1 μg
  • beta -NADPH: 200 μM
  • L-Kynurenine: 300 μM
Reconstitution Calculator

Reconstitution Calculator

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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Background: Kynurenine 3-Monooxygenase/KMO

Kynurenine 3-monooxygenase (KMO), also known as kynurenine 3-hydroxylase, is a part of the kynurenine pathway of tryptophan degradation (1). KMO catalyzes the NADPH- and flavin adenine dinucleotide (FAD)-dependent 3-hydroxylation of kynurenine to 3-hydroxykynurenine (3-HK). 3-HK is neurotoxic via the generation of hydrogen peroxide (2) and through the excitotoxic effects of its downstream metabolite quinolinic acid (3). The levels of 3-HK and quinolinic acid are increased in the brain with Alzheimer's disease and Huntington's disease (1). Inhibition of KMO was shown to reverse cognitive and motor deficits in mouse models of those diseases via an increase in neuroprotective kynurenic acid (4). KMO is found in the mitochondrial outer membrane of microglial cells in the brain and dendritic cells and macrophages in the periphery (1). This recombinant human KMO was expressed as a C-terminally truncated protein.

References
  1. Schwarcz, R. et al. (2012) Nat. Rev. Neurosci. 13:465.
  2. Okuda, S. et al. (1996) Proc. Natl. Acad. Sci. 93:12553.
  3. Stone, T.W. and M.N. Perkins. (1981) Eur. J. Pharmacol. 72:411.
  4. Zwilling, D. et al. (2011) Cell 145:863.
Entrez Gene IDs
8564 (Human); 98256 (Mouse); 59113 (Rat)
Alternate Names
KMO; kynurenine 3-monooxygenase (kynurenine 3-hydroxylase); Kynurenine 3Monooxygenase; Kynurenine 3-Monooxygenase

Citations for Recombinant Human Kynurenine 3-Monooxygenase/KMO Protein, CF

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

2 Citations: Showing 1 - 2
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  1. Kynurenic Acid Protects Against Ischemia/Reperfusion-Induced Retinal Ganglion Cell Death in Mice
    Authors: RB Nahomi, MH Nam, J Rankenberg, S Rakete, JA Houck, GC Johnson, DL Stankowska, MB Pantcheva, PS MacLean, RH Nagaraj
    Int J Mol Sci, 2020-03-05;21(5):.
    Species: Mouse
    Sample Types: Tissue Homogenates
    Applications: Bioassay
  2. Development of a Rapid Fluorescence-Based High-Throughput Screening Assay to Identify Novel Kynurenine 3-Monooxygenase Inhibitor Scaffolds
    Authors: KR Jacobs, GJ Guillemin, DB Lovejoy
    SLAS Discov, 2018-02-08;0(0):2472555218757.
    Species: Human
    Sample Types: Small Molecule
    Applications: Bioassay

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