Tissue Inhibitors Of Metalloproteinase 3 (TIMP3) is an protein belongs to the tissue inhibitor of metalloproteinases family. They are inhibitors of the?matrix metalloproteinases. TIMP-3 is the only member of the TIMP family which is found exclusively in the extracellular matrix (ECM). It is regulated in a cell cycle-dependent fashion in certain cell types and may serve as a marker for terminal differentiation. The activity of recombinant bovine TIMP3 was measured by its ability to inhibit rhMMP2 cleavage of 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. rhMMP2 was diluted to 100 ug/ml and activated with 1 mM APMA at 37 °C for 1 hour and rbTIMP3 (MW: 23.25 KD) was diluted to different concentrations with the assay buffer. Mix 8 μl of rbTIMP3 curve dilutions, 12.8 μl of activated rhMMP-2, and 59.2 μl of assay buffer, including a control containing assay buffer and the diluted rhMMP-2 and incubate the reactions for 2 hours at 37 °C. Loading 50?μl of the incubated mixtures which were diluted five-fold in assay buffer into empty wells of a plate, and start the reaction by adding 50?μl of 20?μM substrate. Include a substrate blank containing 50?μl of assay buffer and 50?μl of 20?μM substrate. Then read at excitiation and emission wavelengths of 320 nm and 405 nm, respectively, in kinetic mode for 5 minutes. The result was shown in Figure 1 and it was obvious that recombinant bovine TIMP3 significantly decreased rhMMP2 activity. The inhibition IC50 was <15 nM.