- Dilute recombinant mouse ACE-2 (R&D Systems, Catalog # 3437-ZN) to 0.5 µg/mL in ACE-2 assay buffer [75 mM Tris (Amresco, Catalog # 0826), 1 M NaCl (J.T. Baker, Catalog # 3624-07), pH 7.5].
- Dilute the substrate, Mca-Y-V-A-D-A-P-K(Dnp)-OH (R&D Systems, Catalog # ES007) to 20 µM with ACE-2 assay buffer.
- Load a plate into the plate reader.
- Add 50 µL of enzyme to each well.
- Add 50 µL of substrate to each well.
- Read in kinetic mode on a fluorescence plate reader (excitation 320 nm, emission 405 nm) for 5 minutes.
- Calculate the specific activity (pmole/min/µg).
Example:
Rate = 4.2 Relative Fluorescence Units (RFU)/sec Conversion Factor = 0.18 pmole/RFU Enzyme Amount = 0.1 µg Specific Activity = Rate x Conversion factor x 60 sec/min Enzyme amount = 4.2 RFU/sec x 0.18 pmole/RFU x 60 sec/min 0.1 µg = 453.6 pmole/min/µg
Calculating the Conversion Factor
The conversion factor will depend on the instrument and settings. To determine the conversion factor, a standard curve should be constructed using a peptide fragment linked with only the fluorescent group. Add the peptide fragments in wells at different amounts (for example, for MCA peptide vary between 0 to 200 pmoles). Read RFUs at the correct excitation and emission wavelengths (320 nm/405 nm for MCA peptide). Plot RFUs (y-axis) versus the amount of peptide (x-axis) and fit by linear regression. In the resulting equation, y = mx + b, the slope of the line (m) corresponds to RFUs at 1 pmole. In the example above, 1 pmole = 5.56 RFU. Then 1 RFU = 0.18 pmole. Ideally, the conditions for constructing the standard curve should be as close to the assay conditions as possible, which include pH, salt concentration, buffer, etc. The peptide fragment should be similar to the cleavage product containing the fluorescent group. We use Mca-Pro-Leu (Bachem, Catalog # M1975) for all our Mca-containing protease substrates (ES001, ES002, ES003, ES004, ES005, ES007, and ES010).
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