Recombinant Human Plasma Kallikrein/KLKB1 Catalog Number: 2497-SE DESCRIPTION Source Mouse myeloma cell line, NS0-derived human Plasma Kallikrein/KLKB1 protein Gly20-Ala638, with a C-terminal 6-His tag Accession # P03952 N-terminal Sequence Gly20 Analysis Structure / Form Pro and processed forms Predicted Molecular 70 kDa Mass SPECIFICATIONS SDS-PAGE 80 kDa, reducing conditions Activity Measured by its ability to cleave a flourogenic peptide substrate Pro-Phe-Arg-7-amido-4-methylcoumarin (PFR-AMC). The specific activity is >1,000 pmol/min/µg, as measured under the described conditions. Endotoxin Level <1.0 EU per 1 μg of the protein by the LAL method. Purity >90%, by SDS-PAGE under reducing conditions and visualized by silver stain. Formulation Supplied as a 0.2 μm filtered solution in Sodium Acetate and NaCl. See Certificate of Analysis for details. Activity Assay Protocol Materials Activation Buffer: 100 mM Tris, 10 mM CaCl2, 150 mM NaCl, pH 7.5 Assay Buffer: 50 mM Tris, 250 mM NaCl, pH 7.5 Recombinant Human Plasma Kallikrein/KLKB1 (rhKLKB1) (Catalog # 2497-SE) Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN) EDTA (Sigma, Catalog # E-4884) Substrate Pro-Phe-Arg-AMC (Bachem, Catalog # I-1295) F16 Black Maxisorp Plate (Nunc, Catalog # 475515) Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent Assay 1. Dilute rhKLKB1 to 200 µg/mL in Activation Buffer. 2. Dilute Thermolysin to 20 µg/mL in Activation Buffer. 3. Combine equal volumes of 200 µg/mL rhKLKB1 and 20 µg/mL Thermolysin for final concentrations of 100 µg/mL and 10 µg/mL respectively. 4. Incubate at 37 °C for 30 minutes. 5. Stop the reaction with 50 µM EDTA. 6. Dilute incubated rhKLKB1 to 0.5 ng/µL in Assay Buffer. 7. Dilute Substrate to 200 µM in Assay Buffer. 8. Load 50 μL of the 0.5 ng/µL rhKLKB1 in a plate, and start the reaction by adding 50 µL of 200 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 200 µM Substrate. 9. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively in kinetic mode for 5 minutes. 10. Calculate specific activity: Adjusted Vmax * (RFU/min) x Conversion Factor** (pmol/RFU) Specific Activity (pmol/min/µg) = amount of enzyme (µg) *Adjusted for Substrate Blank **Derived using calibration standard, 7-amino-4-methylcoumarin (Sigma, Catalog # A9891). Final Assay Per Well: Conditions rhKLKB1: 0.025 µg Substrate: 100 µM PREPARATION AND STORAGE Shipping The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below. Stability & Storage Use a manual defrost freezer and avoid repeated freeze-thaw cycles. 6 months from date of receipt, -20 to -70 °C as supplied. 3 months, -20 to -70 °C under sterile conditions after opening. Rev. 5/20/2019 Page 1 of 2 Recombinant Human Plasma Kallikrein/KLKB1 Catalog Number: 2497-SE BACKGROUND Human Plasma Kallikrein, a serine protease, is synthesized in the liver and circulates in the plasma by binding to high molecular weight (HMW) kininogen or as a free zymogen. Once activated by its physiological activator, factor XII, it displays endopeptidase activity towards peptide bonds after arginine (preferred) and lysine. It cleaves HMW kininogen, its major physiological substrate, to release the potent vasodilator peptide bradykinin. It is also able to cleave a number of inactive precursor proteins to generate active products, such as plasminogen and prourokinase. Thus, it plays an important role in blood pressure regulation, fibrinolysis, and neutrophil activation (1). Human Plasma Kallikrein precursor contains a signal peptide (residues 1 to 19) and a pro form sequence (residues 20 to 638). Upon activation, the pro form is converted to a heavy chain and a light chain, which is linked by disulfide bonds and the latter contains the catalytic domain (2). The human Plasma Kallikrein pro form was expressed in the NS0 cells with a foreign signal peptide. The purified enzyme is mainly the pro form. When activated by thermolysin, it displays activity against a fluorogenic peptide substrate as described in Activity Assay Protocol. This activity can be inhibited by Human Serpin G1/C1 Inhibitor (Catalog # 2488-PI). References: 1. Coleman, R. (2004) in Handbook of Proteolytic Enzymes, Barrett, A.J. et al. (eds.) p. 1644, Academic Press, San Diego. 2. Chung, N.W. et al. (1986) Biochemistry 25:2410. Rev. 5/20/2019 Page 2 of 2 .