• Purity

  >95%, by SDS-PAGE under reducing conditions and visualized by silver stain

• Endotoxin Level

  <1.0 EU per 1 μg of the protein by the LAL method.  

• Activity

  Measured by its ability to inhibit Recombinant Human Coagulation Factor II/Thrombin (Catalog # ) cleavage of a fluorogenic peptide substrate Boc-VPR-AMC (Catalog # ). The IC    ₅₀ value is<1.5 nM, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com  

• Source

  Spodoptera frugiperda,     Sf 21 (stably transfected)-derived Gly20-Ser499, with a C-terminal 10-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Gly20

• Predicted Molecular Mass

  56 kDa

• SDS-PAGE

  62 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% (v/v) Bri-35, pH 7.5 (TCNB)

• Recombinant Human Serpin D1/Heparin Cofactor II (rhSerpin D1) (Catalog # 3198-PI)

• Recombinant Human Coagulation Factor II/Thrombin (Thrombin) (Catalog # )

• Heparin (Sigma, Catalog # H3393), 20 mg/mL in deionized water

• Substrate: Boc-Val-Pro-Arg-AMC (R&D Systems, Catalog # ES011)

• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)

• Fluorescent Plate Reader (Model: Spectramax Gemini EM by Molecular Devices) or equivalent

1. Dilute Thrombin to 0.4 µg/mL with 48.6 µg/mL Heparin in Assay Buffer.

2. Prepare a curve of rhSerpin D1 (MW: 56,300 Da) in Assay Buffer. Make the following serial dilutions: 2000, 500, 250, 125, 50, 25, 10, 5, and 1 nM.

3. Mix equal volumes of the rhSerpin D1 curve dilutions and the diluted Thrombin/Heparin mixture. Include a control containing Assay Buffer and the diluted Thrombin/Heparin mixture.

4. Incubate mixtures at room temperature for 30 minutes.

5. After incubation, dilute the mixtures by five fold in Assay Buffer.

6. Dilute Substrate to 200 µM in Assay Buffer.

7. Load 50 µL of the diluted incubated mixtures in a plate, and start the reaction by adding 50 µL of 200 µM Substrate.

8. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.

9. Derive the 50% inhibiting concentration (IC₅₀) value for rhSerpin D1 by plotting RFU/min (or specific activity) vs. concentration with 4‑PL fitting.

10. The specific activity for Thrombin at each point may be determined using the following formula (if needed):

     *Adjusted for Substrate Blank
     **Derived using calibration standard 7-amino, 4-Methyl Coumarin (Sigma, Catalog # A-9891).

Per Well:

• Thrombin: 0.002 µg

• rhSerpin D1 curve: 100, 25, 12.5, 6.25, 2.5, 1.25, 0.5, 0.25, and 0.05 nM

• Substrate: 100 µM

Background: Serpin D1/Heparin Cofactor II

Serpin D1, also known as Heparin Cofactor II, 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). The full‑length human cDNA was expressed and the purified protein corresponded to the secreted form with anti‑thrombin activity.

• References:

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.

• Entrez Gene IDs:

  3053 (Human); 15160 (Mouse)

• Alternate Names:

  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