Inhibitory activity of SFTI-1 to proteases was measured at room temperature in a reaction buffer of 100Â¬mM Tris-HCl (pH 8.5) containing 100Â¬mg/mL of bovine serum albumin, using the fluorescent substrate peptides. To a cuvette containing 170Â¬uL of reaction buffer was added 10Â¬uL of enzyme solution and 10Â¬uL of inhibitor solution. After preincubation, 10Â¬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 360Â¬nm and emission at 480Â¬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.