Tissue inhibitors of metalloproteinase 1 (TIMP1) is a member of the family of proteins that regulate the activation and proteolytic activity of the zinc enzymes known as matrix metalloproteinases (MMPs). TIMP-1 is a glycoprotein with a molecular mass of 28?kDa produced by a wide range of cell types. TIMP-1 inhibits active MMP-mediated proteolysis by forming an N-terminal, non-covalent binary complex with the MMP active site. The activity of recombinant human TIMP1 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 rhTIMP1 (MW: 22.2 KD) was diluted to different concentrations with the assay buffer. Mix 8 μl of rhTIMP1 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 human TIMP1 significantly decreased rhMMP2 activity. The inhibition IC50 was <4.4 nM.