Recombinant Human Dopa Decarboxylase/DDC Protein, CF

• 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).

• rhDopa: 0.800 µg
• L-DOPA: 500 µM
• Pyridoxal Phosphate: 100 µM