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: 100mM solution of Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg in 20% dimethylsulfoxide. Store solution at -20°C.
Stock solution of unquenched peptide: 10mM solution of (7-methoxycoumarin-4-yl)acetyl-Pro-Leu-NH₂ (Mca-Pro-Leu) in 20% dimethylsulfoxide. Store solution at -20°C.

Assay protocol
The activity of MT1-MMP catalytic domain 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 2 to 4µl of matrix metalloproteinase 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:

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 (mmoles/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 :  Volume of peptide hydrolysis reaction (2.5 ml)
  v :  Volume of added enzyme (0.002 ml to 0.004 ml)

Sequence
Y⁸⁹ A I Q G L K W Q H N E I T F C I Q N Y T P K V G E Y A T Y E A I R K A F R V W E S A T P L R F R E V P Y A Y I R E G H E K Q A D I M I F F A E G F H G D S T P F D G E G G F L A H A Y F P G P N I G G D T H F D S A E P W T V R N E D L N G N D I F L V A V H E L G H A L G L E H S S D P S A I M A P F Y Q W M D T E N F V L P D D D R R G I Q Q L Y G G E S G²⁶⁵

Inhibitors
The catalytic domain of MMP-14 is inhibited by tissue inhibitors of MMP-2 and -3 (TIMP-2 and TIMP-3) and by chelators of divalent cations like EDTA or o-phenanthroline.

Swiss-Prot link P50281: MMP-14 (human) (precursor)
AfCS Signalling Gateway link A001466: MMP-14 (mouse)