Measurement of catalytic activity

1 Preparation and stability of solutions:
· Peptide hydrolysis buffer: 50mM TRIS-HCl, pH 7.5, 150mM NaCl, 5mM CaCl₂, 0.025% Brij 35. Solution is stable for several weeks at 4°C.
· Stock solution of peptide substrate: 100µM solution of Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg in 20% DMSO. Store at -20°C.
· Stock solution of unquenched peptide: 10µM solution of (7-methoxycoumarin-4-yl)acetyl-Pro-Leu-NH₂ (Mca-Pro-Leu) in 20% DMSO. Store at -20°C.

2 Assay protocol:
The activity of MMP-24 is measured fluorimetrically with a synthetic internally quenched fluorescent substrate according to Knight et al.
An excitation wavelength of 328nm and an emission wavelength of 393nm 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 2.5ml. The substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg is diluted in peptide hydrolysis buffer to a concentration of 0.8µM and equilibrated at a temperature of 37°C. Aliquots of 2 to 4µl of the activation mixture are than added and the increase in fluorescence is recorded over a time interval between 2 and 12 minutes. Activity units per ml enzyme solution are calculated according to the following equation:

Activity (U/ml) = (C_Mca-Pro-Leu/F_Mca-Pro-Leu) x (ΔF_{Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg}/V_enzyme) x V_total

C_Mca-Pro-Leu: Concentration of Mca-Pro-Leu used for calibration of the fluorimeter (µmoles/ml).
F_Mca-Pro-Leu: Fluorescence of Mca-Pro-Leu at the concentration C_Mca-Pro-Leu used for fluorimeter calibration.
ΔF_{Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg}: Change in fluorescence during peptide hydrolysis per min.
V_total: Volume of peptide hydrolysis reaction (2.5ml).
V_enzyme: Volume of added enzyme (0.002 to 0.004ml).

3 Inhibitors:
The catalytic domain of MMP-24 is inhibited by tissue inhibitors of MMP-2 (TIMP-2) and by chelators of divalent cations like EDTA or o-phenanthroline.