Recombinant Human Enteropeptidase/Enterokinase Protein, CF

• Assay Buffer: 50 mM Tris, 0.15 M NaCl, 10 mM CaCl₂, 0.05% Brij-35, pH 7.5 (TCNB)
• Recombinant Human Enteropeptidase/Enterokinase (rhEnterokinase) (Catalog # 1585-SE)
• Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
• 1,10 Phenanthroline (Sigma, Catalog # 320056), 0.6 M stock in DMSO
• Substrate: thiobenzyl benzyloxycarbonyl-L-lysinate (Z-Lys-SBzl) (Bachem, Catalog # M-1300), 10 mM stock in DMSO
• 96 well Clear Plate (Costar, Catalog #  92592)
• 5,5’-dithio-bis(2-nitrobenzoic acid) (DTNB) (Sigma, Catalog # D-8130)
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Activate rhEnterokinase with Thermolysin.
1. Dilute rhEnterokinase to 100 μg/mL in Assay Buffer.
2. Dilute Thermolysin to 3.16 μg/mL in Assay buffer.
3. Mix equal volumes of diluted rhEnterokinase and Thermolysin.
4. Incubate at 37 °C for 30 minutes.
5. Stop the reaction by adding an equal volume of 20 mM 1,10 Phenanthroline to the reaction tube.
2. Dilute rhEnterokinase to 0.1 μg/mL in Assay Buffer.
3. Dilute Substrate to 200 μM in Assay Buffer with 200 μM of DTNB.
4. Load in a 96 well clear plate 50 μL of the diluted rhEnterokinase. For a Substrate Blank load 50 μL of the Assay Buffer.
5. Start the reaction by adding 50 μL of the Substrate/DTNB mixture to wells.
6. Read in kinetic mode for 5 minutes at an absorbance of 405 nm.
7. Calculate specific activity:

     *Adjusted for Substrate Blank

     **Using the extinction coefficient 13260 M^-1cm^-1

     ***Using the path correction 0.32 cm

     Note: the output of many spectrophotometers is in mOD

Per Well:

• rhEnterokinase: 0.005 μg
• DTNB: 100 μM
• Substrate: 100 μM