Active Aprotinin (AP)

Aprotinin (AP) is a competitive serine protease inhibitor. Reversibly binds to and blocks the enzymatic active site. Inhibits a range of serine proteases including trypsin, chymotrypsin, kallikrein and plasmin. Inhibits cytopathogenic effect of SARS-CoV-2 and double-stranded RNA formation in SARS-CoV-2-infected cells. The activity of recombinant bovine AP was measured by its ability to inhibit trypsin cleavage of a peptide substrate BAPNA in the assay buffer 200 mM Triethanolamine hydrochloride, 20 mM CaCl2, pH 7.8. The reaction was performed in adding 20 μl 4 mg/mL trypsin diluted by 1mM HCl to 160 μl assay buffer and 20 ul 0.85% (w/v) NaCl and start the reaction by adding 100 µl of 1mg/ml BAPNA. Include a substrate blank containing 160 μl assay buffer, 20 μl 1mM HCl, 20 ul 0.85% (w/v) NaCl and 100 µL of 1mg/ml substrate. Rapidly mixing at 25 ℃, then read at 405 nm in kinetic mode for 5 minutes using a microplate reader controlling the ∆A405nm/min=0.08-0.12. The 20 ul different concentrations of recombinant bovine AP was incubated with 20 ul 4 mg/mL trypsin in 160 ul assay buffer at 25℃ for 10 minutes followed by adding 100 ul substrate, then read at 405 nm in kinetic mode for 5 minutes using a microplate reader. Under these conditions, the enzyme amount of 50% inhibition of trypsin activity per minute is defined as a unit. The specific activity of recombinant bovine AP is >3000 U/mg.