Recombinant Human Enteropeptidase/Enterokinase Protein, CF

•  Assay Buffer: 50 mM Tris, 150 mM NaCl, 10 mM CaCl₂, 0.05% Brij-35, pH 7.5 (TCNB)
• Recombinant Human Enteropeptidase/Enterokinase, active (rhEnterokinase) (Catalog # 10438-SE)
• Substrate: thiobenzyl benzyloxycarbonyl-L-lysinate (Z-Lys-SBzl) (Bachem, Catalog # M-1300), 10 mM stock in DMSO
• 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) (Sigma, Catalog # D8130), 10 mM stock in DMSO
• 96-well clear plate (Catalog # DY990)
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute rhEnterokinase to 0.04 µg/mL in Assay Buffer.
2. Dilute Substrate to 400 µM in Assay Buffer containing 400 µM of DTNB.
3. In a plate, load 50 µL of the diluted rhEnterokinase, and start the reaction by adding 50 µL of Substrate/DTNB mixture.  Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of Substrate/DTNB mixture.
4. Read at an absorbance of 405 nm in kinetic mode of 5 minutes.
5. 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

• rhEnterokinase: 0.002 µg
• DTNB: 200 µM
• Substrate: 200 µM