Serpin A1/Alpha-1-antitrypsin His Tag Protein, Human

SerpinA1, also known as Alpha-1 Antitrypsin (AAT), is a prototype member of the Serpin superfamily, which are serine protease inhibitors. This inhibitor specifically targets the protease neutrophil elastase. Primarily produced in the liver, Alpha-1 Antitrypsin functions as an antiprotease, with its main role being the inactivation of neutrophil elastase to prevent tissue damage. SerpinA1 (Alpha-1 Antitrypsin), an acute phase protein and a classical inhibitor of neutrophil elastase, is localized within lipid rafts in primary human monocytes in vitro. Its association with monocytes is modulated by cholesterol levels; it is inhibited by cholesterol-depleting agents such as nystatin, filipin, methyl-beta-cyclodextrin (MbetaCD), and oxidized low-density lipoprotein (oxLDL), and conversely, enhanced by the presence of free cholesterol.

Assay protocol

Principle: Measured by its ability to inhibit trypsin cleavage of a fluorogenic peptide substrate, Mca-RPKPVE-Nval-WRK(Dnp)-NH2.

Materials

1. Assay Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij 35, pH 7.5 (TCNB)

2. Serpin A1/Alpha-1-antitrypsin His Tag Protein, Human

3. Trypsin (Sigma, Catalog # T1426)

4. Fluorogenic Peptide Substrate: Mca-RPKPVE-Nval-WRK(Dnp)-NH2 (R&D, ES001)

5. 96 ELISA Removable Plate, Black, High binding (GENEVER, Catalog # GMO2-96H)

6. Plate Reader (PerkinElmer, excitation 320 nm, and emission 405 nm)

Produce

1. Dilute Trypsin to 0.25 μg/mL in Assay Buffer.

2. Prepare a curve of Serpin A1 in Assay Buffer. Make the following serial dilutions: 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.13 nM, 1.56 nM, 0.78 nM, 0.39 nM, 0.20 nM.

3. Combine 25 μL of 0.25 μg/mL Trypsin with 25 μL of Serpin A1 serial curve dilutions. Include two controls of 25 μL Assay Buffer with 25 μL of 0.25 μg/mL Trypsin.

4. Incubate at room temperature for 30 minutes.

5. After incubation, add 200 μL of Assay Buffer to each serial curve dilution.

6. Dilute Substrate to 20 μM in Assay Buffer.

7. In a plate, load 50 μL of the diluted Serpin A1 curve, and start the reaction by adding 50 μL of 20 μM Substrate to wells.

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) value for Serpin A1 by plotting RFU/min (or specific activity) vs. concentration with 4PL fitting.

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

*Adjusted for Substrate Blank

**Derived using calibration standard MCA-Pro-Leu-OH (Shyuanye, T77046).