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Preparation and stability of solutions: Activation buffer: 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM CaCl 2. Trypsin solution: 0.50 mg TPCK-trypsin / mL activation buffer. The solution is stored in aliquots at -20 °C. Aprotinin solution: 1 mg aprotinin / mL activation buffer. The solution is stored at - 20 °C. Peptide hydrolysis buffer: 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM CaCl 2 , 0.025 % Brij 35. The solution is stable for several weeks at 4 °C. Stock solution of peptide substrate: 100 µM solution of Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg in 20 % dimethylsulfoxide. The solution is stored at -20 °C. Stock solution of unquenched peptide: 10 µM solution of (7-methoxycoumarin-4-yl)acetyl-Pro-Leu-NH 2 (Mca-Pro-Leu) in 20 % dimethylsulfoxide. The solution is stored at -20 °C. 4.2 Activation: Aliquots of 10 µL progelatinase B monomer are mixed with 20 µL trypsin solution and activation buffer in a total volume of 100 ul. The mixture is incubated for 20 min at 37 °C. Thereafter trypsin is inhibited by addition of 10 µL aprotinin solution.
The activity of gelatinase B is measured fluorimetrically with a synthetic internally quenched fluorescent substrate according to Knight et al. . An excitation wavelength of 328 nm and an emission wavelength of 393 nm are set in an appropriate fluorimeter. The instrument is calibrated with the unquenched peptide Mca-Pro-Leu at a concentration corresponding to between 2 and 10 % hydrolysis of the protease substrate. Kinetic reactions are conveniently carried out in a constant volume of 2.5 mL . The substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg is diluted in peptide hydrolysis buffer to a concentration of 0.8 µM and equilibrated at a temperature of 37 °C. Aliquots of 5 µL to 10 µL of the activation mixture are than added and the increase in fluorescence is recorded over a time interval between 2 and 12 min. Activity units per mL enzyme solution are calculated according to the following equation: c Mca-Pro-Leu deltaF Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg Activity U/mL = ?????.. V total F Mca-Pro-Leu v enzyme c Mca-Pro-Leu : Concentration of Mca-Pro-Leu used for calibration of the fluorimeter ( umoles/mL) F Mca-Pro-Leu : Fluorescence of Mca-Pro-Leu at the concentration c Mca-Pro-Leu used for fluorimeter calibration deltaF Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg : Change in fluorescence during peptide hydrolysis per min V : Volume of peptide hydrolysis reaction (2.5 mL ) v : Volume of added enzyme (0.005 mL to 0.010 mL ) .