Recombinant Human Ketohexokinase Protein, CF Summary
Product Specifications
Met1-Val298, with C-terminal 6-His tag
Analysis
Product Datasheets
Carrier Free
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.
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.
8177-HK
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl, Brij-35 and DTT. |
| Shipping | The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below. |
| Stability & Storage: | Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
|
Assay Procedure
- Universal Kinase Activity Kit (Catalog # EA004)
- Assay Buffer: 25 mM HEPES, 300 mM KCl, 10 mM MgCl2, 10 mM CaCl2, pH 7.0 (Note: Assay Buffer contains 300 mM KCl, which differs from buffer supplied in kit
- Recombinant Human Ketohexokinase (rhKHK) (Catalog # 8177-HK)
- D-(-)-Fructose (Sigma, Catalog # F0127), 500 mM stock in deionized water
- 96-well Clear Plate (Catalog # DY990)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute 1 mM Phosphate Standard provided by the Universal Kinase Activity Kit by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of Assay Buffer for a 100 µM stock. The is the first point of the standard curve.
- Prepare standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock in Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
- Load 50 µL of each dilution of the standard curve into a plate in triplicate. Include a curve blank containing 50 μL of Assay Buffer.
- Prepare Substrate Mixture composed of 0.33 mM ATP and 166.6 mM D-(-)-Fructose in Assay Buffer.
Dilute rhKHK to 25 ng/µL in Assay Buffer. - Load 10 µL of the 25 ng/µL rhKHK into the plate in triplicate. Include a Control containing 10 µL of Assay Buffer.
- Dilute Coupling Phosphatase 4 (supplied in kit) to 10 µg/mL in Assay Buffer.
- Add 10 µL of 10 µg/mL Coupling Phosphatase 4 to wells containing enzyme and Control, excluding the standard curve.
- Add 30 µL of Substrate Mixture to the wells, excluding the standard curve.
- Incubate sealed plate at room temperature for 10 minutes.
- Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
- Add 100 µL of deionized water to all wells. Mix briefly.
- Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
- Read plate at 620 nm (absorbance) in endpoint mode.
- Calculate specific activity:
|
Specific Activity (pmol/min/µg) = |
Adjusted phosphate released* (nmol) x (1000 pmol/nmol) |
| Incubation time (min) x amount of enzyme (µg) x coupling rate** |
*Derived from the phosphate standard curve using linear fitting and adjusted for Control.
**The assumed coupling rate is 0.475 under these conditions.
- rhKHK: 0.25 µg
- Coupling Phosphatase 4: 0.1 µg
- ATP: 0.2 mM
- D-(-)-Fructose: 100 mM
Reconstitution Calculator
Background: Ketohexokinase
KHK1 catalyzes conversion of fructose to fructose-1-phosphate (1). It is the first enzyme that catabolizes dietary fructose. Mutation of this protein is the molecular basis for essential fructosuria, a clinically benign condition characterized by the incomplete metabolism of fructose in the liver, leading to its excretion in urine (2, 3). Essential fructosuria does not have any clinical manifestations and no treatment is required. However, deficiency of aldolase B, the second enzyme involved in the metabolism of fructose results in the accumulation of fructose-1-phosphate in the blood, which causes fructosemia or hereditary fructose intolerance (4). High level of fructose-1-phosphate inhibits the production of glucose and results in diminished regeneration of adenosine triphosphate. Patients with fuctosemia have symptoms of elevated uric acid, growth abnormalities, and coma if untreated. Therefore, inhibition of KHK1 may lead to a cure for fructosemia. High level of expression of KHK1 is found in liver, kidney, gut, spleen and pancreas. Low levels of expression of KHK1 is found in heart, muscle, brain, and eye (3). The enzymatic activity of recombinant human KHK1 is measured using a phosphatase-coupled method (5).
- Trinh, C.H. et al. (2009) Acta. Crystallogr. D Biol Crystallogr. 65:201.
- Zhang, X. et al. (2011) Bioorg. Med. Chem. Lett. 21:4762.
- Bonthron, D.T. et al. (1994) Hum. Mol. Genet. 3:1627.
- Kaiser, U.B. and Hegele, R. A. (1991). Am. J. Med. Sci. 302: 364.
- Wu, Z.L. (2011) PLoS One 6:e23172.
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