Serine Protease Inhibition Assay

The inhibitory activities of 8 rCvT-serpins were determined using 4 serine proteases, including trypsin (Sigma, USA), α-chymotrypsin (Sigma, USA), elastase (Sigma, USA), and subtilisin A from B. licheniformis (Sigma, USA), as previously depicted with wispy modifications (Gu et al. 2021 (link)). The residual protease activities were detected at 405 nm on a microplate reader (Thermo Fisher Scientific, USA) by the addition of 1 mM specific chromogenic substrates in 50 mM Tris-HCl buffer containing 50 mM NaCl and 5 mM CaCl₂, pH 7.5, Nα-benzoyl-L-arginine 4-nitroanilide hydrochloride (Sigma B3133, USA) for trypsin, N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide (Sigma S7388, USA) for α-chymotrypsin, N-succinyl-Ala-Ala-Pro-Leu p-nitroanilide (Sigma S8511, USA) for elastase, and Z-Gly-Gly-Leu p-nitroanilide (Sigma C3022, USA) for subtilisin A.