Fl winlab software package

Activation of proMMP-9 from THP-1 cells was achieved by limited proteolysis with trypsin as described previously, and the activation was stopped by adding soybean trypsin inhibitor (SBTI) [37 (link),48 (link),62 (link)]. The enzyme activity of un-activated and trypsin-activated MMP-9 was determined with the fluorescence-quenched substrate Mca-PLGLDpaAR-NH₂ (10 μM) in 0.1 M HEPES pH 7.5 (containing 10 mM CaCl₂, 0.005% Brij-35) at 37 °C in a total volume of 100 μL. The initial rate of the reaction was determined on a Perkin Elmer LS 50 Luminescence spectrometer using the FL WinLab Software Package (Perkin Elmer). The reactions were followed for one minute, and during that time, 600 data points were collected. The excitation and emission wavelengths were λ_ex = 320 nm and λ_em = 405 nm and a slit width = 10 nm at both wavelengths.

To determine the kinetic and inhibitor kinetic coefficients K_m, and K_i, initial rate experiments were performed using a Perkin Elmer LS 50 Luminescence spectrometer and the FL WinLab Software Package (Perkin Elmer). The reactions were followed for one minute and during that time 600 data points were collected. The excitation and emission wavelengths for the two fluorescence quenched MMP peptide substrates, McaPLGLDpaAR-NH₂ and Mca-RPPGFSAFK(Dpn)-OH were; λ_ex = 320 nm, λ_em = 405 nm and a slit width = 10 nm at both wavelengths. All assays were performed at 37°C in an assay buffer of 0.1 M Hepes pH 7.5, 0.005% Brij-35, 10 mM CaCl₂ and a total assay volume of 100 μl.