| Condition |
All solutions for trypsin assays were prepared in 50 mM Tris-HCl buffer, 20 mM CaCl2, pH 8.0. Inhibitor solutions (0-200 µl) of a given peptide ([BiKK] = 0.8 µM, [BIKF] = 1.8 µM, [BBI] = 0.8 µM, [11-mer] = 4 µM, [SFTI-1] = 0.2 µM) were made up to a total volume of 200 µl. To 50 µl of these solutions was added 50 µl of trypsin solution (2 nM). After an incubation time of 5 min, the reaction was started by addition of 100 µl of a solution of Boc-Gln-Ala-Arg-AMC substrate (6 µM). For chymotrypsin assays, solutions were prepared in 144 mM Tris-HCl, pH 7.8. The assays were otherwise conducted in the same fashion as the trypsin assays, using Suc-Ala-Ala-Pro-Phe-AMC as the substrate (20 µM) and a chymotrypsin solution of 8 nM. Inhibitor concentrations were [BBI] = 4 µM and [BiKF] = 8 µM. Initial substrate hydrolysis was monitored by measuring the fluorescence to follow AMC release, with excitation at 360 nm and emission at 460 nm. Initial velocity data were then fitted using the GraFit software package |
Contacts: