Recombinant E. coli Methionine Aminopeptidase/METAP, CF

• Activation Buffer: 50 mM HEPES, 0.1 mM CoCl₂, 0.1 M NaCl, pH 7.5
• Assay Buffer: 25 mM Tris, pH 8.0
• Recombinant E. coli Methionine Aminopeptidase (reMAP) (Catalog # 1628-ZN)
• Recombinant Human DPPIV/CD26 (rhDPPIV) (Catalog # 9168-SE)
• Substrate: H-Met-Gly-Pro-AMC (Catalog # ES017)
• F16 Black Maxisorp Plate (Nunc Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute reMAP to 40 µg/mL in Activation Buffer.
2. Dilute Substrate to 200 µM in Activation Buffer.
3. Mix 100 µL of 40 µg/mL reMAP with 100 µL 200 µM Substrate. Include a Substrate Blank containing 100 µL of Activation Buffer and 100 µL 200 µM Substrate.
4. Incubate at 37 °C for 15 minutes.
5. Stop the reaction by heating for 5 minutes at 95 to 100 °C.
6. Place reactions on ice for 3 minutes, then spin briefly.
7. Dilute rhDPPIV/CD26 to 2 µg/mL in Assay Buffer.
8. Load 50 µL of reaction mixture into a plate and add 50 µL of 2 µg/mL rhDPPIV/CD26.
9. Incubate plate with shaking for 10 minutes at room temperature.
10. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively in endpoint mode.
11. Calculate specific activity:

    Specific Activity (pmol/min/µg) =	Adjusted Fluorescence* (RFU) x Conversion Factor** (pmol/RFU)
    	Incubation time (min) x amount of enzyme (µg)

         *Adjusted for Substrate Blank

         **Derived using calibration standard 7-Amino, 4-Methyl Coumarin (AMC) (Sigma, Catalog # A9891).

Per Well:

• reMAP: 1 µg
• rhDPPIV/CD26: 0.100 µg
• Substrate: 50 µM