- Dilute recombinant human Meprin alpha Subunit (R&D Systems, Catalog # 3220-ZN) to 0.1 mg/mL in TCNB buffer [50 mM Tris, 150 mM NaCl, 10 mM CaCl2 (Sigma, Catalog # C-5080), 0.05% Brij® 35, pH 7.5] containing 0.1 µg/mL Trypsin. Mix well by gently vortexing.
- Incubate the mixture at 37° C for 3 hours in a heat block covered with foil. Stop trypsin activity by adding 1 mM AEBSF (R&D Systems, Catalog # EI001).
- Dilute activated MEP1A to 0.4 µg/mL in Meprin alpha Subunit assay buffer (50 mM Tris, pH 7.5). Note: Include a control that has not been activated with trypsin if the activity of the non-activated enzyme needs to be determined.
- Dilute the substrate, Mca-Y-V-A-D-A-P-K(Dnp)-OH (R&D Systems, Catalog # ES007) to 20 µM with Meprin alpha Subunit assay buffer.
- Load the plate in 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).
||4.2 Relative Fluorescence Units (RFU)/sec
|Conversion Factor =
|Enzyme Amount =
|Specific Activity =
||Rate x Conversion factor x 60 sec/min
||4.2 RFU/sec x 0.18 pmole/RFU x 60 sec/min
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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