Figure. The binding activity of TIMP3 with MMP2.
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. Besides, Matrix Metalloproteinase 2 (MMP2) has been identified as an interactor of TIMP3, thus a binding ELISA assay was conducted to detect the interaction of recombinant rat TIMP3 and recombinant rat MMP2. Briefly, TIMP3 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100μL were then transferred to MMP2-coated microtiter wells and incubated for 2h at 37℃. Wells were washed with PBST and incubated for 1h with anti-TIMP3 pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody, wells were aspirated and washed 3 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50μL stop solution to the wells and read at 450nm immediately. The binding activity of TIMP3 and MMP2 was shown in Figure 1, and this effect was in a dose dependent manner.

The activity of recombinant rat 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 rrTIMP3 (MW: 23.25 KD) was diluted to different concentrations with the assay buffer. Mix 8 μl of rrTIMP3 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 rat TIMP3 significantly decreased rhMMP2 activity. The inhibition IC50 was <1 nM.