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Acknowledgements
20. Leesnitzer, M. A.; Bickett, D. M.; Moss, M. L.; Becherer,
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21. Recombinant catalytic domains of MMP-1 and full length
active MMP-3 were expressed and purified from Escherichia
coli. Enzymes were refolded in 200 mM NaCl, 50 mM Tris,
5 mM CaCl2, 10 mM ZnSO4 and 0.01% Brij 35, pH 7.6 for 1 h
prior to the assay. The catalytic domains of proMMP-9 were
purified from the media of baculovirus infected T. ni cells.
Assays were run in a total volume of 0.180 mL assay buffer
containing 200 mM NaCl, 50 mM Tris, 5 mM CaCl2, 10 mM
ZnSO4 and 0.01% Brij 35, pH 7.6. MMP-1, MMP-3, and
MMP-9 concentrations were adjusted to 0.5, 0.05, 5, and
0.1 nM, respectively. Enzymes were pre-incubated with inhi-
bitor for 20 min at room temperature and the reactions were
initiated with the addition of the fluorogenic substrate, Dnp-
Pro-Cha-Gly-Cys(Me)-His-Ala-Lys(Nma)–NH2. Dose respon-
ses were generated using 11 point 3-fold dilutions of the inhi-
bitor. Product was measured using excitation and emission
wavelenghts of 343 and 450 nm, respectively.
The authors thankElizabeth Sugg and Stephen Frye for
their advice during the course of this work.
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