Assembly and selective "in synthesis" labeling of quenched fluorogenic protease substrates |
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Authors: | Chersi Alberto Ferracuti Silvia Falasca Giuliana Butler Richard H Fruci Doriana |
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Affiliation: | Laboratory of Biochemistry, Regina Elena Institute CRS, 00158 Rome, Italy. |
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Abstract: | Because impaired cellular protease activities are linked to many diseases, such as cancer, inflammation, neurodegeneration, and infection, internally quenched fluorescent peptides have recently been developed as tools for analyzing the specificities of these enzymes. Here we report convenient and cost-effective approaches for the selective "in synthesis" assembly of such substrate peptides for protease assays. Fluorescein and Dabcyl groups were covalently and selectively attached during synthesis to epsilon-amino groups of internal lysines. Functionality was then tested by digestion with leucine aminopeptidase, chymotrypsin, and microsomal vesicles. All peptides proved to be appropriate substrates of the enzymes tested and of the endogenous peptidases in the microsomal vesicles. In summary, we describe an innovative and cheap method to develop completely functional quenched fluorescent peptides that are usable in specific detection of individual proteases, in particular aminopeptidases, in both in vitro and in vivo systems. |
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Keywords: | Fluorescence Quenching Selective labeling Peptide synthesis |
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