Recombinant Mouse Serpin D1/Heparin Cofactor II Protein, CF

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

Recombinant Mouse Serpin D1/Heparin Cofactor II Protein, CF Summary

• Details of Functionality: Measured by its ability to inhibit Recombinant Human Coagulation Factor II/Thrombin (Catalog # 1473-SE) cleavage of a fluorogenic peptide substrate Boc-VPR-AMC (Catalog # ES011). The IC 50 value is <1.2 nM, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived mouse Serpin D1/Heparin Cofactor II protein Glu24-Ser478, with a C-terminal 10-His tag
• Accession #: P49182
• N-terminal Sequence: Glu24
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Serpind1
• 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: 53 kDa.
  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: 70-80 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 degreesC as supplied. 3 months, -20 to -70 degreesC under sterile conditions after reconstitution.
• Buffer: Lyophilized from a 0.2 μm filtered solution in HEPES 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 HEPES and 150 mM NaCl, pH 7.4.
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
  • Recombinant Mouse Serpin D1/Heparin Cofactor II (rmSerpin D1) (Catalog # 1265-PI)
  • Recombinant Human Coagulation Factor II/Thrombin (Catalog # 1473-SE)
  • Heparin (Sigma, Catalog # H3393), 20 mg/mL stock in deionized water
  • Substrate: BOC-Val-Pro-Arg-AMC (Catalog # ES011) , 100 mM stock in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute Thrombin to 2 µg/mL and Heparin to 0.1 mg/mL each in Assay Buffer.
  2. Mix equal portions of Thrombin and Heparin for final concentrations of 1 µg/mL and 0.05 mg/mL respectively.
  3. Prepare a curve of rmSerpin D1 (MW: 53,374 Da) in Assay Buffer. Make the following serial dilutions: 200, 100, 50, 25, 15, 10, 5, 2.5, 1.25 and 0.625 nM.
  4. Mix equal volumes of the rmSerpin D1 curve dilutions and the Thrombin/Heparin mixture. Include a control (in duplicate) containing Assay Buffer and the Thrombin/Heparin mixture.
  5. Incubate mixtures at room temperature for 30 minutes.
  6. After incubation, dilute the mixtures 5 fold in Assay Buffer.
  7. Dilute Substrate to 200 µM in Assay Buffer.
  8. Load 50 µL of the diluted incubated mixtures in a plate, and start the reaction by adding 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. Derive the 50% inhibition concentration (IC₅₀) for rmSerpin D1 by plotting RFU/min (or specific activity) vs. concentration with

      4-PL fitting.

  11. The specific activity for Thrombin 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 (Sigma, Catalog # A-9891).

  Per Well:
  • Thrombin: 0.005 µg
  • Heparin: 2.5 μg/mL
  • rmSerpin D1 curve: 10, 5, 2.5, 1.5, 0.75, 0.5, 0.25, 0.125, 0.0625, and 0.03125 nM
  • Substrate: 100 µM

Notes

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

Alternate Names for Recombinant Mouse Serpin D1/Heparin Cofactor II Protein, CF

• HC2
• HC2LS2
• HCF2
• HCF2clade D (heparin cofactor), member 1
• HC-II
• Heparin Cofactor II
• HLS2HCII
• leuserpin 2
• LS2
• Protease inhibitor leuserpin-2
• Serpin D1
• serpin peptidase inhibitor, clade D (heparin cofactor), member 1

Background

Serpin D1 is a member of the Serpin superfamily of the serine protease inhibitors (1). Similar to Serpins A5 and C1, it inhibits thrombin and this activity is enhanced by heparin. Interestingly, a C-terminal His-tagged recombinant Serpin D1 had enhanced heparin effect, which was maintained in a plasma-based thrombin inhibition assay (2). Congenital Serpin D1 deficiency is a potential risk factor for thrombosis (3). Mouse Serpin D1 is synthesized as a 478 amino acid precursor with a 23 amino acid signal peptide (4). The mature chain (residues 24 to 478) is secreted.

1. Silverman, G.A. et al. (2001) J. Biol. Chem. 276:33293.
2. Bauman, S.J. et al. (1999) J. Biol. Chem. 274:34556.
3. Tollefsen, D.M. (2002) Arch. Pathol. Lab. Med. 126:1394.
4. Zhang, G.S. et al. (1994) Biochemistry 33:3632.

Bioinformatics

• Gene Symbol: Serpind1
• Uniprot:
  • P49182()