(31) Assay Card

General Information
Summary SFTI-1 was found to have nano-molar inhibitory effect against matriptase and trypsin.
Condition Inhibitory activity of SFTI-1 to proteases was measured at room temperature in a reaction buffer of 100¬mM Tris-HCl (pH 8.5) containing 100¬mg/mL of bovine serum albumin, using the fluorescent substrate peptides. To a cuvette containing 170¬uL of reaction buffer was added 10¬uL of enzyme solution and 10¬uL of inhibitor solution. After preincubation, 10¬uL of substrate solution was added and the cuvette content mixed thoroughly. The residual enzyme activity was determined by following the change of fluorescence released by hydrolysis of the substrates, using a fluorescent spectrophotometer (Hitachi F4500) with excitation wavelength of 360¬nm and emission at 480¬nm. Fluorescent peptide N-t-Boc Gln-Ala-Arg-AMC was used as substrate for matriptase and trypsin, peptide N-t-Boc-Leu-Gly-Arg-AMC was used as substrate for uPA, and peptide N-t-Boc-Leu-Arg-Arg-AMC was used as substrate for thrombin. Hydrolysis rates were recorded in presence of 6-7 different concentrations of SFTI-1. The Ki values were determined by Dixon plots from two sets of data with different concentrations of substrate.
Result SFTI-1 found to inhibit matriptase with a Ki of 0.92 nM3, trypsin with a Ki of 1.06 nM3, thrombin with a ki of 5050 NM3 and uPA with a Ki of 500 nM3. I was also found that the inhibitory activity of the inhibitory potency of SFTI-1 is 2- to 3-fold higher than that of BBI.
AssayType Protease inhibition

Long YQ, Lee SL, Lin CY, Enyedy IJ, Wang S, Li P, Dickson RB, Roller PP (2001) Synthesis and evaluation of the sunflower derived trypsin inhibitor as a potent inhibitor of the type II transmembrane serine protease, matriptase. Bioorg Med Chem Lett 11:2515-9

Proteins Assayed SFTI-1