Recombinant Human Nicotinamide N-Methyltransferase/NNMT, CF

• Assay Buffer: 20 mM Tris, pH 8.5
• Hydrolysis Buffer: 100 mM HEPES pH 7.0
• Recombinant Human Nicotinamide N‑Methyltransferase/NNMT (rhNNMT) (Catalog # 7736-MT)
• Glutathione, reduced (Amresco, Catalog # 399), 250 mM stock in deionized water
• Recombinant Human Adenosylhomocysteinase/AHCY (rhAHCY) (Catalog # 6466-AH)
• Recombinant Human Adenosine Deaminase/ADA, (rhADA) (Catalog # 7048-AD)
• S-adenosylmethionine (Sigma, Catalog # A7007), 10 mM stock in 50% DMSO in deionized water
• Nicotinamide (Sigma, Catalog # 72340), 100 mM stock in 50 mM Tris, 100 mM NaCl, 30% Isopropanol, pH 8.0
• 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 the stock of reduced glutathione to 40 µM (40 pmol/µL) in Assay Buffer. This is the first point of the standard curve.
2. Continue standard curve by performing six ½ serial dilutions of the 40 µM glutathione in Assay Buffer. The standard curve has a range of 31.25 to 2000 pmol per well.
3. Dilute rhNNMT to 2 µg/mL in Assay Buffer.
4. Create Substrate Mixture containing 200 µM S-adenosylmethionine and 4 mM Nicotinamide in Assay Buffer.
5. Combine equal volumes of 2 µg/mL rhNNMT and Substrate Mixture. As a Control, combine equal volumes of Substrate Mixture with Assay Buffer.
6. Incubate reactions at 37 °C for 30 minutes.
7. To stop the reaction, boil samples at 100 °C for 5 minutes. Then cool reactions on ice for 1 minute.
8. Create a Hydrolysis Mixture containing 25 µg/mL rhAHCY and 2.5 µg/mL rhADA in Hydrolysis Buffer.
9. Combine equal volumes of cooled reactions from step 7 and Hydrolysis Mixture.
10. Incubate mixtures at 37 °C for one hour.
11. Load 50 µL of each point of the standard curve into a plate. Include a curve blank containing 50 µL of Assay Buffer.
12. Load 50 µL of each reaction mixture into a plate.
13. Dilute ThioGlo to 100 µM in DMSO.
14. Add 50 µL of 100 µM ThioGlo to each well.
15. Incubate at room temperature for 5 minutes in the dark.
16. Read the plate in endpoint mode at excitation and emission wavelengths of 380 nm and 445 nm, respectively.
17. Calculate specific activity:

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

• rhNNMT: 0.025 µg
• rhAHCY: 0.625 µg
• rhADA: 0.0625 µg
• S-adenosylmethionine: 0.025 mM
• Nicotinamide: 0.5 mM
• ThioGlo: 50 µM