Matrix metalloproteinases are a family of zinc and calcium dependent endopeptidases with the combined ability to degrade all the components of the extracellular matrix. MMP-9 (gelatinase B) can degrade a broad range of substrates including gelatin, collagen types IV and V, elastin and proteoglycan core protein. It is believed to act synergistically with interstitial collagenase (MMP-1) in the degradation of fibrillar collagens as it degrades their denatured gelatin forms. MMP-9 is produced by keratinocytes, monocytes, macrophages and PMN leukocytes. MMP-9 is present in most cases of inflammatory responses. The activity of recombinant human MMP9 is 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 rhMMP9 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 rhMMP9 is diluted to 0.2 ug/mL in assay buffer. Loading into a black well plate 50 µL of 0.2 ug/mL rhMMP9 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 rhMMP9. 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 MMP9 is > 3000 pmol/min/µg.