• Purity

  >80%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane

• Endotoxin Level

  <0.1 EU per 1 μg of the protein by the LAL method.  

• Activity

  Measured by its ability to phosphorylate thymidine. The specific activity is >550 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com

• Source

  E. coli-derived Ser2-Asn234, with C-terminal 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Ser2 & Cys3

• Predicted Molecular Mass

  26 kDa

• SDS-PAGE

  25-30 kDa, reducing conditions

Assay Procedure

Materials

• Universal Kinase Activity Kit (Catalog # )

• 10X Assay Buffer  (supplied in kit): 250 mM HEPES, 1500 mM NaCl, 100 mM MgCl₂, 100 mM CaCl₂ pH 7.0

• Recombinant Human Thymidine Kinase 1 (rhTK-1) (Catalog # 8180-TK)

• Acceptor substrate: Thymidine (Sigma, Catalog # T9250), 100 mM stock in deionized water

• 96-well Clear Plate (Catalog # )

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

1. Prepare 1X Assay Buffer containing 0.5 mM DTT.

2. 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. This is the first point of the standard curve.

3. 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.

4. Prepare a substrate mixture containing 0.5 mM ATP and 12.5 mM Thymidine in Assay Buffer.

5. Dilute Coupling Phosphatase 4 (supplied in kit) to 10 µg/mL in Assay Buffer.

6. Dilute rhTK-1 to 25 µg/mL in Assay Buffer.

7. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.

8. Load 20 µL of the 25 µg/mL rhTK-1 into the plate. Include a Control containing 20 µL of Assay Buffer.

9. Add 10 µL of 10 µg/mL Coupling Phosphatase 4 to the wells, excluding the standard curve.

10. Add 20 µL of substrate mixture to the wells, excluding the standard curve.

11. Cover the plate with a plate sealer and incubate at room temperature for 10 minutes.

12. Add 30 µL of Malachite Green Reagent A to all wells. Mix briefly.

13. Add 100 µL of deionized water to all wells. Mix briefly.

14. Add 30 µL of Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.

15. Read plate at 620 nm (absorbance) in endpoint mode.

16. Calculate specific activity:

     *Derived from the phosphate standard curve using linear fitting and adjusted for Control.

      ** The coupling rate is 0.475 under these conditions.

Per Reaction:

• rhTK-1: 0.5 µg

• Coupling Phosphatase 4: 0.1 μg

• ATP: 10 nmol (0.2 mM)

• Thymidine: 250 nmol (5 mM)
  

Background: Thymidine Kinase 1

Thymidine kinases are 2'-deoxythymidine kinases that phosphorylate deoxythymidine and generate deoxythymidine 5’-phosphate (1). Thymidine kinases have a key function in the synthesis of DNA and thereby in cell division, as they are part of the unique reaction chain to introduce deoxythymidine into the DNA. Two forms of thymidine kinase are present in mammalian cells, TK1 and TK2. TK1 is cell cycle-dependent and is present in the cytoplasm only in anticipation of cell division (2, 3); whereas TK2 is located in mitochondria and is cell cycle-independent (4). TK1 is synthesized by the cell during the S phase of cell division and is degraded after cell division is completed (5). TK1 normally occurs in tissue as a dimer and can be converted to more active tetrameric form by ATP binding (6). TK1 is feedback inhibited by thymidine triphosphate, the product of the further phosphorylation of thymidine (7). Because tumor cells replicate much more frequently than normal cells and requires faster DNA synthesis and higher TK1 activity, TK1 is a cancer maker especially for hematologic malignancies (8, 9). In clinical chemistry TK1 is used as a proliferation marker in the diagnosis, control of treatment and follow-up of malignant disease (10). In addition, thymidine kinase is required for the action of many antiviral drugs (11). The enzymatic activity of recombinant human TK1 is measured using a phosphatase-coupled method (12).

• References:

1. Wintersberger, E. (1997) Biochem. Soc. Trans. 25:303.

2. Littelfield, J.W. (1966) Biochim. Biophys. Acta 114:398.

3. Flemington, E, et al. (1987) Gene 52:267.

4. Berk, A.J. et al. (1973) Arch. Biochem. Biophys. 154:563.

5. Zhu, C. et al. (2006) Nucleosides Nucleotides Nucleic Acids 25:1185.

6. Munch-Petersen, B. et al. (1993) J. Biol. Chem. 268:15621.

7. Mikkelsen, N. E. et al. (2003). Biochemistry 42:5706.

8. Doi S, et al. (1990) Nagoya J. Med. Sci. 52:19.

9. Ellims PH, et al. (1981). Cancer Res. 41: 691.

10. Lin TS, et al. (1976) J. Med. Chem.19:495.

11. Baba M, et al. (1987). Biochem. Biophys. Res. Commun. 142:128.

12. Wu, Z.L. (2011) PLoS One 6:e23172.

• Entrez Gene IDs:

  7083 (Human); 21877 (Mouse)

• Alternate Names:

  EC 2.7.1.21; thymidine kinase 1 soluble isoform; Thymidine Kinase 1; thymidine kinase 1, soluble; thymidine kinase, cytosolic; thymidine kinase-1; TK1; TK2