Recombinant Human Adenosylhomocysteinease/AHCY Protein, CF

• Assay Buffer: 50 mM HEPES, 2 mM MgCl₂, 1 mM EDTA, pH 7.0
• Recombinant Human Adenosylhomocysteinase/AHCY (rhAHCY) (Catalog # 6466-AH)
• Glutathione, reduced (Amresco, Catalog # 399)
• Adenosylhomocysteine (Sigma, Catalog # A9384), 10 mM stock in 10 mM HCl
• Recombinant Human Adenosine Deaminase/ADA (rhADA) (Catalog # 7048-AD)
• ThioGlo®3 Fluorescent Thiol Reagent (Covalent Associates, Inc., Catalog # T-003), 10 mM stock in DMSO
• DMSO (Sigma, Catalog # 154938)
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Prepare a 10 mM stock of reduced glutathione in deionized water.
2. Prepare the standard curve by diluting the 10 mM reduced glutathione (10,000 pmol/µL) to 10 pmol/µL in Assay Buffer and  performing six additional ½ serial dilutions.
3. Dilute rhAHCY to 0.667 μg/mL in Assay Buffer.
4. Dilute adenosylhomocysteine to 1 mM in Assay Buffer.
5. Dilute rhADA to 0.24 mg/mL in Assay Buffer.
6. Load the microplate as follows:
   a. Reactions: 15 µL of 0.667 μg/mL rhAHCY, 15 µL of 0.24 mg/mL rhADA, and 20 µL of 1 mM adenosylhomocysteine
   b. Substrate Blanks: 15 µL of Assay Buffer, 15 µL of 0.24 mg/mL rhADA, and 20 µL of 1 mM adenosylhomocysteine
   c. Standard Curve: 50 µL per well
7. Cover microplate and incubate at 37 °C for 30 minutes.
8. Dilute ThioGlo® to 100 µM in DMSO.
9. After incubation, add 50 µL of 100 µM ThioGlo® to each well.
10. Incubate at room temperature for 5 minutes in the dark.
11. Read the plate in endpoint mode at excitation and emission wavelengths of 380 nm and 445 nm, respectively.
12. Calculate specific activity:

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

Per Well:

• rhAHCY: 0.010 µg
• Adenosylhomocysteine: 200 µM
• rhADA: 3.6 μg
• ThioGlo®: 50 µM