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Metalloproteases (MMPs), such as collagenase, and the related tumor necrosis factor converting enzyme (TACE) are involved in degradation of the extracellular matrix during tissue remodeling, cell migration, and processing of signaling molecules like cytokines and cell adhesion molecules. It is thought that there is a link between excess MMP activity and disease states such as tumor metastasis and multiple sclerosis. Fluorescence-quenched peptide substrates have been used to quantitate MMP activity. Fluorescence resonance energy transfer (FRET) occurs when the excitation energy is transferred from an excited fluorescent donor to a quenching acceptor. Cleavage of a scissile peptide bond within a fluorescence quenched substrate leads to the separation of the intramolecular donor-acceptor pair, thus allowing the increase of the fluorescence. The fluorescence increase is then proportional to the amount of peptide hydrolyzed. Most of the known MMP fluorescence-quenched substrates possessed varying activities toward different MMPs and lacked specificity. Recently, Neumann et al. reported a new fluorogenic substrate they termed FS-6 or MOCAc-Lys-Pro-Leu-Gly-Leu-Dap(Dnp)-Ala-Arg-NH2 (code SMO-3670-PI) that possessed an increased specificity constant for collagenases and TACE. This allows for collective determination of activity against total MMP and TACE activity in biological fluids. The cleavage site of this substrate is between Gly and Leu. Lysine was added to the N-terminal position to the popular and widely used substrate MOCAc-Pro-Leu-Gly-Leu-Dap(Dnp)-Ala-Arg-NH2, termed FS-1 ( Code MOC-3163-v). Using HPLC-fluorescence detection, MMP activity was measured in picomolar ranges. The specificity constant kcat/Km for MMP-1, MMP-8, MMP-13 (collagenases), and MMP-14 were all increased between two-fold and nine-fold using the new substrate FS-6, while matrilysin and gelatinases activity was equivalent to the original substrate FS-1. TACE activity was reported by Neumann et al. to be higher than currently used substrates as well. MOCAc-Lys-Pro-Leu-Gly-Leu-Dap(Dnp)-Ala-Arg-NH2 is also water-soluble which allows for in situ applications on viable cell surfaces. Spectral properties (excitationmax = 325 nm and emissionmax = 400 nm) of MOCAc-Lys-Pro-Leu-Gly-Leu-Dap(Dnp)-Ala-Arg-NH2 are similar to FS-1 (excitationmax = 328 nm and emissionmax = 398 nm).This new substrate was used to detect MMPs, Cathepsin D and E, ADAM10 and ADAM17/TACE activity, specifically noting MMP-1 (collagenase 1), MMP-2 (gelatinase A), MMP-7 (matrilysin), MMP-8 (collagenase 2), MMP-9 (gelatinase B), MMP-11 (stromelysin 3), MMP-12 (macrophage elastase), MMP-13 (collagenase 3), MMP-14 (MT1-MMP), and MMP-16 (MT3-MMP). Order this new broad activity MMP substrate today!
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| U. Neumann , H. Kubota, K. Frei, V. Ganu, and D. Leppert, Anal. Biochem., 328, 166 (2004). |
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| U. Neumann , H. Kubota, K. Frei, V. Ganu, and D. Leppert, Anal. Biochem., 328, 166 (2004). | ||||||||||||||||||||
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