(34) Assay Card

General Information
Summary SFTI-1(6,5) shown to have trypsin inhibitory activity and is cyclised after incubation with trypsin.
Condition SFTI-1[6,5] (0-150 nM) and trypsin (42 nM) were preincubated together for3h at 303K. The reactions were initiated by addition of substrate and monitored at 405 nm with readings taken every 2 min with shaking between readings using a Molecular Devices SpectraMax 250. All reactions were carried out in duplicate, and the corresponding Ki value was determined using a tight-binding equation.
Result A 1:1 mixture of SFTI-1[6,5] with bovine beta-trypsin was incubated at 303 K (pH 8). Aliquots of the reaction mixture were taken over a 24-h period, and LC-MS analysis confirmed that cyclic SFTI-1 was indeed produced. The ratio of the intact and cleaved form of SFTI-1 was determined by quantitative analysis of the peaks with masses corresponding to SFTI-1[6,5] and cyclic SFTI-1. After 2 min of reaction time, cyclic SFTI-1 could be detected. A steady state was reached after 130 min and resulted in 90% SFTI-1 to 10% SFTI-1[6,5]. Coelution experiments with added synthetic SFTI-1 and SFTI-1[6,5] confirmed that the resulting molecules are indeed the cleaved and intact inhibitor. SFTI-1[6,5] inhibited bovine beta-trypsin with a Ki of 0.54 ± 0.1 nM.
AssayType Protease inhibition

Marx UC, Korsinczky ML, Schirra HJ, Jones A, Condie B, Otvos L Jr, Craik DJ (2003) Enzymatic cyclization of a potent bowman-birk protease inhibitor, sunflower trypsin inhibitor-1, and solution structure of an acyclic precursor peptide. J Biol Chem 278:21782-9

Proteins Assayed SFTI-1(6,5)