• Purity

  >95%, by SDS-PAGE under reducing conditions and visualized by silver stain

• Endotoxin Level

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

• Activity

  Measured by its ability to convert the substrate 3, 4-dihydroxy L-phenylalanine (L-Dopa) to 3, 4-dihydroxyphenylethylamine (dopamine). The dopamine product is measured by its absorbance at 340 nm after derivatization with trinitrobenzene sulfonic acid. Sherald, F.     et al. (1973) Anal. Biochem.     56:300. The specific activity is >1500 pmol/min/µg, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com

• Source

  Spodoptera frugiperda,     Sf 21 (baculovirus)-derived Met1-Glu480, with a C-terminal 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  No result obtained

• Predicted Molecular Mass

  55 kDa

• SDS-PAGE

  55 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 50 mM HEPES, 100 mM NaCl, pH 7.2

• Recombinant Human Dopa Decarboxylase/DDC (rhDopa) (Catalog # 3564-DC)

• Pyridoxal 5’-Phosphate hydrate (Sigma, Catalog # P9255)

• 3,4-dihydroxy L-phenylalanine (L-DOPA) (Sigma, Catalog # D9628)

• 2,4,6-Trinitrobenzene sulfonic acid (TNBS), 5% picryl solution (Sigma, Catalog # P2297)

• Benzene (Sigma, Catalog # 319953)

• 96 well clear UV-transparent microplate (Corning, Catalog # 3635)

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

1. Prepare 5 mM stock solutions of L-DOPA and pyridoxal phosphate in deionized water. Use vigorous vortexing and mixing to get both reagents into solution.

2. Dilute rhDopa to 5 ng/µL in Assay Buffer.

3. Combine 160 µL of 5 ng/μL rhDopa with 20 µL 5 mM L-DOPA, 4 µL 5 mM pyridoxal phosphate, and 16 µL Assay Buffer. Include a control containing 20 µL 5 mM L-DOPA, 4 µL 5 mM pyridoxal phosphate, and 176 µL Assay Buffer without any rhDopa.

4. Incubate for 30 minutes at 37 °C.

5. Stop the reaction by heating for 2 minutes at 95 - 100 °C. After heating, cool on ice.

6. Add 10 µL of 5% TNBS to each tube and vortex.

7. Incubate for 20 minutes at 37 °C. Ensure that all samples undergo derivitization for the same length of time, stagger the addition of TNBS and the benzene extraction (next step) if necessary.

8. In a fume hood, add 300 µL of benzene to each reaction in the same order the 5% TNBS was added. Vortex for 30 seconds.

9. Centrifuge all reactions at 13,000 rpm in a microcentrifuge for 5 minutes to separate the aqueous and organic solvents.

10. Carefully pipet out 200 µL of the benzene phase (the upper layer) of each reaction and load into a UV-transparent microplate. It is recommended that the microplate be kept covered as much as possible to minimize evaporation of the benzene.

11. Read at an absorbance of 340 nm in endpoint mode.

12. Calculate specific activity:

     *Adjusted for Substrate Blank

     **Derived using calibration standard dopamine (3,4-dihydroxyphenylethylamine) (Sigma, Catalog # H8502).

Per Reaction:

• rhDopa: 0.800 µg

• L-DOPA: 500 µM

• Pyridoxal Phosphate: 100 µM

Background: Dopa Decarboxylase/DDC

Dopa Decarboxylase (DDC), also known as aromatic amino acid decarboxylase, is a group II decarboxylase (1). The enzyme catalyzes the decarboxylation of aromatic L-amino acids to produce the corresponding amines. DDC produces the neurotransmitters dopamine and serotonin from L-Dopa and L-5-hydroxytryptophan, respectively. The inhibition of DDC could be used for the treatment of schizophrenia and Parkinson's disease (2).

• References:

1. Sandmeier, E. et al. (1994) Eur. J. Biochem. 221:997.

2. Bertoldi, M. et al. (1996) J. Biol. Chem. 271:23954.

• Entrez Gene IDs:

  1644 (Human)

• Alternate Names:

  AADC; AADCaromatic-L-amino-acid decarboxylase; DDC; dopa decarboxylase (aromatic L-amino acid decarboxylase); Dopa Decarboxylase; EC 4.1.1; EC 4.1.1.28