Recombinant Human Glycine N-methyltransferase/GNMT, CF

• Methylation Buffer: 20 mM Tris, 2 mM MgCl₂, 1 mM EDTA, pH 9.0
• Hydrolysis Buffer: 100 mM HEPES, 2 mM MgCl₂, 1 mM EDTA, pH 7.0
• Recombinant Human Glycine N‑Methyltransferase/GNMT (rhGNMT) (Catalog # 6526-MT)
• Recombinant Human Adenosylhomocysteinase/AHCY (rhAHCY) (Catalog # 6466-AH)
• Glutathione, reduced (Amresco, Catalog # 399), 250 mM stock in deionized water
• S-Adenosylmethionine (AdoMet) (Sigma, Catalog # A7007), 10 mM stock in 50% DMSO, 50% deionized water (v/v)
• Glycine (Sigma, Catalog # G7126), 1 M in deionized water
• Recombinant Human Adenosine Deaminase/ADA (rhADA) (Catalog # 7048-AD)
• ThioGlo® 3 Fluorescent Thiol Reagent (Covalent Associates, Inc., Catalog # T-003)
• DMSO (Sigma, Catalog # 154938)
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute AdoMet to 800 µM in Methylation Buffer.
2. Dilute Glycine to 20 mM in Methylation Buffer.
3. Form Substrate Mixture by combining equal volumes of 800 µM AdoMet and 20 mM Glycine.
4. Dilute rhGNMT to 0.8 µg/mL in Methylation Buffer.
5. Prepare methylation reaction by combining 50 µL of 0.8 µg/mL rhGNMT with 50 µL of Substrate Mixture. Also prepare a Substrate Blank containing Methylation Buffer in place of rhGNMT.
6. Incubate methylation reaction and substrate blank for 30 minutes at room temperature.
7. Stop methylation reaction by heating at 95-100 °C for 5 minutes. After stopping the reaction, cool on ice for 1 minute.
8. Prepare a standard curve by diluting the 250 mM reduced glutathione (250,000 pmol/µL) to 10 pmol/µL in Hydrolysis Buffer and performing six additional ½ serial dilutions.  Include a standard curve blank consisting of Hydrolysis Buffer only.
9. Dilute rhAHCY to 10 ng/µL in Hydrolysis Buffer.
10. Dilute rhADA to 71.1 μg/mL in Hydrolysis Buffer.
11. Form Enzyme Mixture by combining equal volumes of 10 ng/µL rhAHCY and 71.1 μg/mL rhADA.
12. Prepare hydrolysis reaction by adding 100 µL of Enzyme Mixture to the stopped methylation reaction and blank.
13. Incubate the hydrolysis reaction, Substrate Blank, standard curve and standard curve blank for 1 hour at 37 °C.
14. Load 50 µL of hydrolysis reaction, Substrate Blank, and standard curve into wells of a black microplate.
15. Dilute ThioGlo® to 100 µM in DMSO.
16. Add 50 µL of 100 µM ThioGlo® to each well.
17. Incubate microplate at room temperature for 5 minutes in the dark.
18. Read the plate in endpoint mode at excitation and emission wavelengths of 380 nm and 445 nm, respectively.
19. Calculate specific activity:
    

    Specific Activity (pmol/min/µg) =	Thiol produced (pmol)
    	Incubation time (min) x amount of enzyme (µg)

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

Per Well:

• rhGNMT: 0.010 µg
• rhAHCY: 0.125 µg
• AdoMet: 100 µM
• Reduced glutathione curve: 500, 250, 125, 62.5, 31.25, 15.625, and 7.813 pmol
• rhADA: 0.889 μg
• ThioGlo®: 50 µM