Recombinant Human Serpin B4 Pr

Materials

• Maturation Buffer: 50 mM MES, pH 5.5
• Cathepsin Buffer: 50 mM MES, 50 mM NaCl, 5 mM DTT, pH 5.5
• Assay Buffer: 20 mM Tris, 2 M KCl, 0.02% (v/v) Triton® X-100, pH 9.0
• Recombinant Human Serpin B4 (rhSerpin B4) (Catalog # 9437-PI)
• Recombinant Human Chymase/CMA1 (rhChymase) (Catalog # 4099-SE)
• Recombinant Mouse Active Cathepsin C/DPPI (rmCathepsin C) (Catalog # 2336-CY)
• Heparin (Tocris, Catalog # 2812), 20 mg/mL stock in deionized water
• Substrate:  Suc-Ala-Ala-Pro-Phe-AMC (Bachem, Catalog # I-1465), 10 mM stock in DMSO
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: Gemini EM by Molecular Devices) or equivalent

1. Dilute rhChymase-1 to 20 µg/mL in Maturation Buffer.
2. Dilute rmCathepsin C to 20 µg/mL in Cathepsin Buffer.
3. Dilute Heparin to 2.5 mg/mL in deionized water.
4. Combine equal volumes of 20 µg/mL rhChymase and 20 µg/mL rmCathepsin.
5. Add 2.5 mg/mL Heparin to the rhChymase mixture for a final concentration of 49 µg/mL.
6. Incubate rhChymase mixture at room temperature for 1 hour.
7. Dilute the rhChymase mixture to 4 µg/mL of rhChymase in Assay Buffer and incubate at room temperature for 5 minutes.
8. Prepare a curve of rhSerpin B4 (MW: 45,677 Da) in Assay Buffer. Make the following serial dilutions: 2000, 500, 250, 125, 62.5, 31.25, 15.624, 7.812, 3.908 and 0.3908 nM.
9. Combine equal volumes of each point of the rhSerpin B4 curve with 4 µg/mL rhChymase. Include an enzyme control containing equal volumes of Assay Buffer and 4 µg/mL CMA1.
10. Incubate reaction mixtures at room temperature for 30 minutes.
11. Dilute Substrate to 200 µM in Assay Buffer.
12. Load 50 µL of the incubated mixtures into empty wells of a plate, and start the reaction by adding 50 µL of 200 µM Substrate.  Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of 200 µM Substrate.
13. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
14. Derive the 50% inhibition concentration (IC50) value for rhSerpin B4 by plotting RFU/min (or specific activity) versus concentration with 4-PL fitting.
15. Calculate specific activity for rhChymase at each point using the following formula (if needed):

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

  *Adjusted for Substrate Blank.
**Derived using calibration standard 7-amino, 4-Methyl Coumarin (Sigma, Catalog # A9891).

Per Well:
rhSerpin B4: 500, 125, 62.5, 31.25, 15.625, 7.812, 3.906, 1.953, 0.977, and 0.0977 nM
rhChymase-1: 0.1 µg
Substrate: 100 µM