MMP-2 proenzyme (human fibroblasts)

≥850mU/mg protein (Y. Masui, et al.; Biochem. Med. 17 , 215 (1977) ) . One unit is defined as the amount of enzyme that hydrolyzes 1μmol Dnp-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg-OH per min. at 37°C, pH 7.0.

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 CaCl 2 , 1μM ZnCl 2 , 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 (7-Methoxycoumarin-4-yl) acetyl-Pro-Leu-Gly-Leu-N-β-Dnp-L- (α, β-diaminopropionyl) Ala-Arg-NH 2 (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 7.0x10 5 M -1 s -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) APMA or 1mM mersalyl 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.