Assay protocol

Principle: Measured by its ability to inhibit active Cathepsin L cleavage of a fluorogenic peptide substrate Z-LR-AMC.

Materials

1.Assay Buffer: 50 mM MES, 5 mM DTT, pH 6.0

2.SerpinB3/SCCA Protein, Human

3.Recombinant Human Cathepsin L

4.Substrate: Z-Leu-Arg-AMC (Catalog # ES008)

5.96 ELISA Removable Plate, Black, High binding (GENEVER, Catalog # GMO2-96H)

6.Plate Reader (PerkinElmer, excitation 380 nm and emission 460 nm)

Produce

1.Dilute rhCathepsin L to 40 μg/mL in Assay Buffer.

2.Incubate 40 μg/mL rhCathepsin L for 15 minutes on ice to activate.

3.Dilute activated rhCathepsin L to 0.2 μg/mL in Assay Buffer.

4.Prepare a curve of rhSerpin B3 (MW = 45.9 KDa) in Assay Buffer. Make the following serial dilutions: Top Conc.=1000, 333, 111, 37, 12, 4, 1.4, 0.5, 0.15, 0.05 nM.

5.Combine 25 μL of each dilution with 25 μL of the 0.2 μg/mL rhCathepsin L. Include an enzyme control containing Assay Buffer in place of rhSerpin B3.

6.Incubate reaction mixtures at 37 °C for 15 minutes.

7.Dilute reactions by adding 200 μL Assay Buffer to each.

8.Dilute Substrate to 20 μM in Assay Buffer.

9.Load into a black well plate 50 μL of the diluted incubated mixtures and start the reaction by adding 50 μL substrate.

10.Read at excitation and emission wavelengths of 380 nm and 460 nM (top read), respectively, in kinetic mode for 10 minutes.

11.Derive the 50% inhibition concentration (IC50) value for rhSerpin B3 by plotting RFU/min vs. concentration with 4-PL fitting.

12.Calculate the specific activity for rhCathepsin L 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.