Activity:
Specific activity can be assayed with the synthetic substrate N-(2,4)-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg (Dnp-peptide) (Masui et al.). Substrate concentration should be 0.5mg/ml in 50mM TRIS-HCl, pH 7.0, 200mM NaCl, 5mM CaCl₂, 1µM ZnCl₂, 0.05% sodium azide, 0.05% BRIJ35, containing 0.05mg/ml albumine. One unit MMP catalyzes the hydrolysis of 1µmol Dnp-peptide/min. at 37°C and pH 7.0.
Alternatively the fluorogenic substrate Substrat (7-Methoxycoumarin-4-yl) acetyl-Pro-Leu-Gly-Leu-N-ß-Dnp-L-(α,ß-diaminopropionyl)Ala-Arg-NH₂ (Knight et al. 1992) can be used. Hydrolysis of the Gly-Leu bond separates the highly fluorescent (7-Methoxycoumarin-4-yl)acetyl group from the 2,4-dinitrophenyl resulting in an increase of fluorogenic intensity. The K_m value for the gelatinase A is 7.0x10⁵M^-1s^-1. Substrate should be kept as a  9.15mM stock solution in DMSO (10mg/ml). In the assay the substrate concentration should be ~25µM. The assay can be performed in a 96-well microtiter plate (100/200µl per well) suitable for fluorogenic measurements (Ex 328 nm; Em 393 nm).

Activation:
Requires activation by 2mM (final concentration) AMPA or 1mM mersalic acid for 60-120 min. at 37°C. We do not recommend to use trypsin for activation! Do not dilute enzyme for activation!

Inhibitors:
Activated enzyme is inhibited by tissue inhibitors of matrix metalloproteinase-2 (TIMP-2) and by chelators of divalent cations like EDTA or o-phenanthroline.

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