(36) Assay Card

General Information
Summary Bifunctional analogues of SFTI-1 found to be resistant to hydrolysis by trypsin.
Condition The experiments were conducted at 25¬C in 50 mM sodium acetate buffer containing 20 mM CaCl2, pH 3.5. Solutions of peptide and trypsin were mixed together so that the final concentration of the peptide was 200 ¬µM and that of trypsin 2 ¬µM (1 mole%). At discrete intervals, a 195 ¬µl aliquot was taken and the reaction stopped by addition of 5 ¬µl of TFA. The samples were then analysed directly by RP-HPLC.
Result When hydrolysis of the peptides was followed at pH 8 using catalytic quantities of trypsin (1 mol%), none of the peptides showed significant levels of hydrolysis products until at least 30 min of incubation, as judged by HPLC analysis from the still intact peak of the original inhibitors. This shows that all of these synthetic peptides are significantly resistant to hydrolysis by trypsin.
AssayType Enzymatic Digest

Jaulent AM, Leatherbarrow RJ (2004) Design, synthesis and analysis of novel bicyclic and bifunctional protease inhibitors. Protein Eng Des Sel 17:681-7

Proteins Assayed SFTI-1