Recombinant Human Kininogen HMW (HKa) Protein, CF

• Reactivity: Hu
• Applications: Inhibition Activity
• Format: Carrier-Free

Recombinant Human Kininogen HMW (HKa) Protein, CF Summary

• Details of Functionality: Measured by its ability to inhibit papain cleavage of a fluorogenic peptide substrate Z-FR-AMC (Catalog # ES009). The IC₅₀ value is <3 nM, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human Kininogen protein
  Gln19-Ser644, with an N-terminal signal sequence and a C-terminal 10-His tag
• Accession #: P01042
• N-terminal Sequence: Ser390 (light chain) & Lys438 (minor species) with Gln19 predicted to be present and blocked
• Structure / Form: High molecular weight form consisting of the mature chain, the heavy and light chains, and a minor species
• Protein/Peptide Type: Recombinant Enzymes
• Gene: KNG1
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Inhibition Activity
• Theoretical MW: 71 kDa (mature), 41 kDa (heavy) & 30 kDa (light).
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 120 kDa, 60 kDa and 48 kDa, reducing conditions

Packaging, Storage & Formulations

• 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 reconstitution.
• Buffer: Lyophilized from a 0.2 μm filtered solution in Sodium Acetate and NaCl.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Reconstitution Instructions: Reconstitute at 100 μg/mL in sterile 25 mM Tris and 100 mM NaCl, pH 7.5.
• Assay Procedure:
  • Activation Buffer: 50 mM Tris, 5 mM DTT, pH 7.0
  • Assay Buffer: 50 mM Tris, pH 7.0
  • Recombinant Human Kininogen High Molecular Weight (HKa) (rhKininogen) (Catalog # 1569-PI)
  • Papain (Sigma, Catalog # P4762)
  • Substrate: Z-Phe-Arg-AMC (R&D Systems, Catalog # ES009), 10 mM stock in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Chill Activation Buffer on ice.
  2. Dilute Papain to 100 µg/mL in Activation Buffer.
  3. Incubate at room temperature for 10 minutes.
  4. Prepare a dilution curve of rhKininogen (MW: 71,211Da) in Assay Buffer. Make the following serial dilutions: 400, 200, 100, 50, 25, 12.5, 6.25, and 3.125 nM.
  5. Dilute activated Papain to 2 µg/mL in Assay Buffer.
  6. Mix equal volumes of the rhKininogen curve dilutions and the diluted active Papain. Include a control (in duplicate) containing Assay Buffer and the diluted active Papain.
  7. Incubate mixtures at room temperature for 10 minutes.
  8. Dilute Substrate to 200 µM in Assay Buffer.
  9. Perform a 1/5 dilution with Assay Buffer to the incubated mixture of rhKininogen and Papain.
  10. Load 50 µL of diluted incubated mixture into the plate, and start the reaction by adding 50 µL of 200 µM Substrate.
  11. Read at excitation and emission wavelengths of 380 nm and 460 nm, respectively, for 5 minutes in kinetic mode.
  12. Derive the 50% inhibition concentration (IC₅₀) for rhKininogen by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
  13. The specific activity for Papain at each point may be determined 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 (AMC) (Sigma, Catalog # A-9891).

  Per Well:
  • Papain: 0.010 µg
  • Substrate: 100 µM
  • rhKininogen curve: 20, 10, 5, 2.5, 1.25, 0.625, 0.3125, and 0.15625 nM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human Kininogen HMW (HKa) Protein, CF

• alpha 2-thiol Proteinase Inhibitor
• BDK
• BDK;BK;HAE6;HK;HMWK;Kininogen-1;KNG;KNG1
• Kininogen
• KNG1

Background

Kininogen, also known as alpha 2-thiol proteinase inhibitor, is a multi-function protein. There are two alternatively spliced forms, designated as the high molecular weight (HMW) and low MW (LMW) forms (1). The HMW form is synthesized as a 644 amino acid residue precursor with a signal peptide (residues 1‑18). The mature chain (residues 19‑644) is further processed into the heavy (residues 19‑380) and the light (residues 390‑644) chains. The active peptide bradykinin (residues 381‑389) is released, which has a variety of functions including muscle contraction, hypotension and inflammation. The heavy chain consists of three cystatin‑like domains, which are responsible for inhibiting cysteine proteases. The light chain consists of a His-rich domain, which is associated with the clotting activity. In comparison to the HMW form, the LMW Kininogen (427 residues) has the same sequence in its heavy chain and bradykinin, but a different sequence in its light chain (residues 402‑427). The LMW form is not involved in blood clotting.

1. Takagaki, Y. et al. (1985) J. Biol. Chem. 260:8601.

Publications for Kininogen (1569-PI)(1)

Publication using 1569-PI

• Ludvigsen M, Jacobsen C, Maunsbach AB, Honore B Identification and characterization of novel ERC-55 interacting proteins: evidence for the existence of several ERC-55 splicing variants; including the cytosolic ERC-55-C. Proteomics, 2009-12-01;9(23):5267-87. 2009-12-01 [PMID: 19927312] (Surface Plasmon Resonance, Human)

Bioinformatics

• Gene Symbol: KNG1
• Uniprot:
  • P01042()