Assay Procedure
Materials
Assay Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
Human alpha 2-Macroglobulin (h alpha 2-Macroglobulin) (Catalog # 1938-PI)
Recombinant Human Serpin F2 (rhSerpin F2) (Catalog # 1470-PI)
Recombinant Human Active Trypsin 3/PRSS3 (rhTrypsin 3) (Catalog # 3714-SE)
Substrate: MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(DNP)-NH2 (Catalog # ES002)
F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
Dilute rhTrypsin 3 to 1.28 ug/mL with Assay Buffer.
Prepare a curve of h alpha 2-Macroglobulin with Assay Buffer. Dilute h alpha 2-Macroglobulin (MW: 720000 Da) to the following concentrations: 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, and 0.3125 nM.
Combine 30 uL of h alpha 2-Macroglobulin curve with 30 uL of 1.28 ug/mL rhTrypsin 3. Include a control containing 30 uL of Assay Buffer with 30 uL of rhTrypsin 3 in duplicate. Also include a h alpha 2-Macroglobulin control in duplicate for each sample tested containing 30 uL of 200 nM h alpha 2-Macroglobulin and 30 uL Assay Buffer.
Incubate at 37 degrees C for 1 hour.
Dilute rhSerpin F2 to 40 ug/mL with Assay Buffer.
Add 60 uL of 40 ug/mL rhSerpin F2 to each reaction.
Incubate at 37 degrees C for 15 minutes.
Dilute Substrate to 20 uM in Assay Buffer.
Load into plate 50 uL of h alpha 2-Macroglobulin curve containing rhTrypsin 3 and rhSerpin F2, and start the reaction by adding 50 uL of 20 uM Substrate to each well.
Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
Derive the 50% inhibiting concentration (IC50) of h alpha 2-Macroglobulin, by its trapping of rhTrypsin 3, toward rhSerpin F2 activity by plotting RFU/min (or specific activity) vs. concentration (h alpha 2-Macroglobulin) with 4-PL fitting.
The specific activity for rhTrypsin 3 at each point may be determined using the following formula (if needed):
Specific Activity (pmol/min/ug) =
Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU) / amount of enzyme (ug)
*Adjusted for h alpha 2-Macroglobulin control
**Derived using calibration MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).
Per Well:
h alpha 2-Macroglobulin: 25, 12.5, 6.25, 3.125, 1.563, 0.781, 0.391, and 0.0391 nM
rhTrypsin 3: 0.016 ug
rhSerpin F2: 1 ug
Substrate: 10 uM
