Design of a Novel FRET Substrate with a Long Wavelength Fluorophore for Detection of Matrix Metalloproteinases

摘要

Matrix metalloproteinases (MMPs) belong to a family of secreted or membrane-associated zinc endopeptidases involved in both normal and disease related tissue remodeling. They are capable of degrading extracellular matrix (ECM) proteins and processing a number of bioactive molecules [1,2]. MMPs are key players in normal and pathological processes, including embryogenesis, wound healing, inflammation, arthritis, and cancer [3-5]. The use of FRET technology has facilitated MMP assay development. Previously described FRET substrates include Mca/Dnp, EDANS/DABCYL donor acceptor pairs. The introduction of the longer wavelength 5-FAM/QXL 520 FRET based MMP substrates increased assay sensitivity and signal/background ratio. To further improve MMP FRET assays, i.e. minimize autofluorescence from reaction components, we developed a new series of MMP substrates containing the 5TAMRA/QXL~(TM) 570 FRET pair. In these FRET peptides, the fluorescence of 5-TAMRA is quenched by QXL 570 and recovered upon cleavage of the peptide by active MMP enzyme. The resulting fluorescence is monitored at excitation/emission wavelengths = 540 nm/575 nm. After screening several 5-TAMRA/QXL~R 570 substrates, one sequence was identified to be cleaved bv most of the MMPs. This substrate was chosen for the development of the SensoLyte 570 Generic MMP Assay Kit. This kit provides high sensitivity and accuracy and can detect human MMP-1, 2, 7, 8, 9, 12, 13, and 14. It is ideal for detecting of enzyme activity in the samples containing multiple MMPs or for high throughput screening of MMP inhibitors using purified enzymes.