(37) Assay Card

General Information
Summary SFTI-1 analogues assayed for trypsin inhibitory activity.
Condition 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 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.
Result 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.
AssayType Protease inhibition

Korsinczky ML, Clark RJ, Craik DJ (2005) Disulfide bond mutagenesis and the structure and function of the head-to-tail macrocyclic trypsin inhibitor SFTI-1. Biochemistry 44:1145-53

Proteins Assayed [ABA3,11]SFTI-1