Recombinant Human PNMT Protein, CF

• Assay buffer: 25 mM MES, pH 6.0
• Hydrolysis buffer: 100 mM HEPES, pH 7.0
• Recombinant Human PNMT (rhPNMT) (Catalog # 7854-MT)
• Substrate: DL-Norepinephrine hydrochloride (Sigma, Catalog # A7256), 0.5 M stock in deionized water
• Recombinant Human Adenosylhomocysteinase/AHCY (rhAHCY) (Catalog # 6466-AH)
• Recombinant Human Adenosine Deaminase/ADA (rhADA) (Catalog # 7048-AD)
• S-adenosylmethionine (AdoMet) (Sigma, Catalog # A7007), 20 mM stock in deionized water
• Glutathione, reduced (Amresco, Catalog # 399), 250 mM stock in deionized water.
• ThioGlo® 3 Fluorescent Thiol Reagent (Covalent Associates, Inc., Catalog # T-003), 10 mM stock in DMSO
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rhPNMT to 80 µg/mL in Assay Buffer.
2. Create a Substrate Mixture containing 400 µM AdoMet and 1 mM DL-Norepinephrine hydrochloride in Assay Buffer.
3. Combine equal volumes of rhPNMT and Substrate Mixture. As a Control, replace rhPNMT with Assay Buffer.
4. Incubate reactions and control at room temperature for 30 minutes.
5. Boil reactions and control at 95‑100 °C for 5 minutes. Cool samples on ice for 3 minutes.
6. Create a Hydrolysis Mixture containing 25 µg/mL rhAHCY and 2.5 µg/mL rhADA in Hydrolysis Buffer.
7. Add an equal volume of Hydrolysis Mixture to reactions and control.
8. Incubate reactions and control at 37 °C for 1 hour.
9. Prepare standard curve during incubation (step 7).
10. Dilute Glutathione to 20 µM in Assay Buffer.
11. Continue standard curve by performing six additional ½ dilutions in Assay Buffer.
12. Load 25 µL of each curve point into the plate in triplicate. Include a standard blank of Assay Buffer only in triplicate.
13. Add 25 µL of Hydrolysis Buffer to the curve wells.
14. Load 50 µL of reaction(s) and control into empty wells in triplicate.
15. Dilute ThioGlo to 100 µM in DMSO.
16. Add 50 µL of 100 µM ThioGlo to all wells, including standard curve.
17. Incubate sealed plate at room temperature for 5 minutes in the dark.
18. Read at excitation and emission wavelengths of 380 nm and 445 nm (top read), respectively, in endpoint mode.
19. Calculate specific activity: 

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

Per Well:

• rhPNMT: 1 µg
• DL-Norepinephrine hydrochloride: 125 µM
• AdoMet: 50 µM
• rhAHCY: 6.25 µg/mL
• rhADA: 0.625 µg/mL
• ThioGlo: 50 µM
• Glutathione Curve: 500, 250, 125, 62.4, 31.2, 15.6, 7.8 and 0 pmol