Concentrations of [ABA3,11]SFTI-1, [ABA3,11]SFTI-1[1,14], and [ABA3,11]SFTI-1[6,5] were determined by amino acid analysis. Trypsin activity was assayed at 25 C in a 96-well plate with 350 uL of total reaction volume per well, with 42 nM bovine -trypsin (Type XIII TPCK treated, EC 18.104.22.168) using N-benzoyl-L-arginine p-nitroanilide (BAPNA) substrate (1.25 mM) and buffer at pH 8 (50 mM Tris-HCl and 25 mM CaCl2). SFTI-1 inhibitors (0-400 nM) and trypsin were incubated together in buffer for 2 h at 25 C. The reaction at 25 C was initiated by addition of the substrate and monitored at 405 nm with readings taken every 60 s for 120 min using a Molecular Devices SpectraMax 250 or a Labsystems Multiskan Ascent. Reactions for each inhibitor concentration were carried out 3 times.
The Ki for inhibition of [ABA3,11]SFTI-1 has previously been reported to be 0.24 nM. Trypsin assays were undertaken for both [ABA3,11]SFTI-1[1,14] and [ABA3,11]SFTI-1[6,5], but both were inactive against trypsin under the same conditions as used for the cyclic peptide.