MMP2 is a zinc-dependent enzymes capable of cleaving components of the extracellular matrix, which belongs to the matrix metalloproteinase (MMP) family .It is a gelatinase A, 72 kDa type IV collagenase which can hydrolyze gelatin under certain conditions. Gelatin zymography is mainly used for the detection of the gelatinases, MMP-2 and MMP-9 and It is extremely sensitive because levels of 10 pg of MMP-2 can already be detected .Briefly，various concentrations of MMP2 (50ng, 25ng, 13ng, 6.5ng, 3.3ng, 1.7ng) were denatured by SDS loading buffer, electrophoresed through sodium dodecylsulphate–polyacrylamide gel (SDS–PAGE; 10% gels) containing gelatin (1 mg/ml) with nonreducing conditions. After renaturation, incubation and CCB-stained, active MMP2 would hydrolyze gelatin nearby, which was indicated by the white binds on the gel. In this experiment we use trypsin as positive control. The result was shown in figure 1.

The activity of recombinant human MMP2 is also measured by its ability to cleave a fluorogenic peptide substrate MCA-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH2 in the assay buffer 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5. The rhMMP2 is diluted to 100 ug/ml in assay buffer, then activated by p-aminophenylmercuric acetate (APMA) in a final concentration of 1 mM incubated at 37 °C for 1 hours. The activated rhMMP2 is diluted to 2.5 ug/mL in assay buffer. Loading into a black well plate 50 µL of 2.5 ug/mL rhMMP2 and start the reaction by adding 50 µL of 20 µM substrate, with a substrate blank containing 50 µL assay buffer, 50 µL substrate, and no rhMMP2. Then read at excitiation and emission wavelengths of 320 nm and 405 nm, respectively, in kinetic mode for 5 minutes. The specific activity of recombinant human MMP2 is > 170 pmol/min/µg.