Recombinant Human HAI-2A Protein, CF

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

Recombinant Human HAI-2A Protein, CF Summary

• Details of Functionality: Measured by its ability to inhibit trypsin cleavage of a fluorogenic peptide substrate, Mca-RPKPVE-Nval-WRK(Dnp)-NH₂ (Catalog # ES002). The IC₅₀ value is <4.6 nM, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human HAI-2A protein
  Met1-Lys197, with a C-terminal 10-His tag
• Accession #: O43291
• N-terminal Sequence: Ala28
• Protein/Peptide Type: Recombinant Enzymes
• Gene: SPINT2
• 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: 21 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: 27 kDa and 32 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 Tris, NaCl and CaCl₂.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Reconstitution Instructions: Reconstitute at 100 μg/mL in sterile, deionized water.
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
  • Recombinant Human HAI-2A (rhHAI-2A) (Catalog # 1106-PI)
  • Trypsin (Sigma, Catalog # T-1426)
  • Substrate: MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(DNP)-NH₂ (Catalog # ES002) ), 2 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 Trypsin to 0.25 µg/mL with Assay Buffer.
  2. Prepare a curve of rhHAI-2A (MW: 20539 Da) in Assay Buffer. Make the following serial dilutions: 1000, 100, 33.3, 16.7, 8.33, 4.17, 2.08, and 0.417 nM.
  3. Mix equal volumes of the rhHAI-2A curve dilutions and the diluted Trypsin. Include a control (in duplicate) containing Assay Buffer and the diluted Trypsin without any rhHAI-2A.
  4. Incubate reactions for 30 minutes at room temperature.
  5. After incubation, dilute the mixtures by 5 fold in Assay Buffer.
  6. Dilute Substrate to 20 µM in Assay Buffer.
  7. Load 50 µL of the diluted incubated mixtures into a plate, and start the reaction by adding 50 µL of 20 µM Substrate.
  8. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
  9. Derive the 50% inhibition concentration (IC₅₀) by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
  10. The specific activity for Trypsin 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 MCA-Pro-Leu-OH (Bachem, Catalog # M-1975)

  Per Well:
  • Trypsin: 0.00125 µg
  • rhHAI-2A curve: 50, 5, 1.67, 0.83, 0.417, 0.209, 0.104, and 0.0209 nM
  • Substrate: 10 µM

Notes

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

Alternate Names for Recombinant Human HAI-2A Protein, CF

• HAI2A
• HAI-2A
• Placental Bikunin
• SPINT2

Background

Two alternatively spliced forms of HAI-2 have been found in human tissues (1). HAI-2A, the full-length molecule and also known as placental bikunin, is a major form expressed in human tissues. Encoded by the SPINT2 gene, HAI-2A consists of two Kunitz domains, and a C-terminal transmembrane domain (1‑5). Both Kunitz domains can function as inhibitors independent of each other. In addition to HGF activator and trysin, HAI-2A strongly inhibits plasmin, tissue and plasma kallikreins, and factor XIa. In comparison, HAI-2A is a weaker inhibitor of factor VIIa-tissue factor, factors IXa, Xa, and XIIa. Recombinant HAI-2A prolonged the clotting time in an activated partial thromboplastin time assay.

1. Kataoka, H. et al. (2002) Biochem. Biophys. Res. Comm. 290:1096.
2. Kawaguchi, T. et al. (1997) J. Biol. Chem. 272:27558.
3. Marlor. C.W. et al. (1997) J. Biol. Chem. 272:12202.
4. Muller-Pillasch, F. et al. (1998) Biochim. Biophys. Acta 1395:88.
5. Delaria, K.A. et al. (1997) J. Biol. Chem. 272:12209.

Publications for HAI-2A (1106-PI)(3)

Publications using 1106-PI

• R Szabo, TH Bugge Loss of HAI-2 in mice with decreased prostasin activity leads to an early-onset intestinal failure resembling congenital tufting enteropathy PLoS ONE, 2018-04-04;13(4):e0194660. 2018-04-04 [PMID: 29617460] (Bioassay)
• Zoratti G, Tanabe L, Varela F, Murray A, Bergum C, Colombo E, Lang J, Molinolo A, Leduc R, Marsault E, Boerner J, List K Targeting matriptase in breast cancer abrogates tumour progression via impairment of stromal-epithelial growth factor signalling. Nat Commun, 2015-04-15;6(0):6776. 2015-04-15 [PMID: 25873032] (Activation, Human, Mouse)
• Szabo R, Hobson JP, List K, Molinolo A, Lin CY, Bugge TH Potent Inhibition and Global Co-localization Implicate the Transmembrane Kunitz-type Serine Protease Inhibitor Hepatocyte Growth Factor Activator Inhibitor-2 in the Regulation of Epithelial Matriptase Activity. J. Biol. Chem., 2008-08-19;283(43):29495-504. 2008-08-19 [PMID: 18713750] (Bioassay, Degradation, Human)

Bioinformatics

• Gene Symbol: SPINT2
• Uniprot:
  • O43291()