Activation of MMP-2 proenzyme and measurement of catalytic activity

1 Preparation and stability of solutions:
∙ APMA-solution: 40mM p-aminophenyl mercuric acetate (APMA) in DMSO. Store at -20°C.
∙ 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-NH₂ in 20%
  DMSO. Store at -20°C.
∙ Stock solution of unquenched peptide: 10µM solution of Mca-Pro-Leu-NH₂ in 20% DMSO. Store at -20°C.

2 Activation:
An aliquot of 19.5μl MMP-2 proenzyme is mixed with 0.5µl APMA solution and is incubated for 60 min. at 37°C.

3 Assay protocol:
The activity of MMP-2 is measured fluorimetrically with a synthetic internally quenched fluorescent substrate according to Knight et al. [1].
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 1 to 2µl 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 (0.001 to 0.002ml).

Due to autoproteolytic activity minor bands of activated enzyme may be visible in the preparation.

Swiss-Prot link P08253: MMP-2 (human)
AfCS Signalling Gateway link A001471: MMP-2 (mouse)