Serine Protease Inhibition Assay

Protease inhibition assays were performed with purified recombinant NvKSPI-1 and NvKSPI-2 proteins using the method described by Ling et al. [56 (link)]. Three typical serine proteases (bovine pancreatic trypsin, bovine pancreatic chymotrypsin, and proteinase K, 200 ng/mL) (Sigma, Taufkirchen, Germany) and their corresponding substrates (N-benzoyl-Val-Gly-Arg-p-nitroanilide, N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilid, and N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilid) (Sigma, Taufkirchen, Germany) were selected for determination of the spectrum of enzyme inhibition by recombinant NvKSPI-1 and NvKSPI-2 as follows. The recombinant NvKSPI-1 and NvKSPI-2 (1 µg each) were pre-incubated with a reaction buffer (100 mM Tris-HCl, 100 mM NaCl, 1 mM CaCl₂, pH 7.5) containing 200 ng/mL serine protease for 30 min at room temperature, and then 200 μL substrate was added (0.1 mM, 100 mM Tris-HCl, 100 mM NaCl, 1 mM CaCl₂, pH 7.5) before measuring the absorbance at 405 nm every min for 30 min. Substrates alone were used as blanks. Buffer and buffer with bovine serum albumin (BSA) were used as controls. One unit of enzyme activity was defined as an increase of absorbance by 0.001 per min. The experiments were repeated three times as independent biological replicates.