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A novel fluorimetric assay for detection of aggrecanase‐1 (ADAMTS‐4) activity using a long wavelength FRET peptide substrate
Author(s) -
Rakhmanova Vera A,
Lui Siu Kei,
Fan Weizhen,
Bai Yuehong,
He Jianjun,
Hong Anita,
Tong Xiaohe
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.523.2
Subject(s) - aggrecanase , adamts , förster resonance energy transfer , aggrecan , chemistry , metalloproteinase , disintegrin , cleavage (geology) , peptide , biochemistry , microbiology and biotechnology , biophysics , fluorescence , thrombospondin , matrix metalloproteinase , osteoarthritis , biology , medicine , articular cartilage , physics , alternative medicine , pathology , quantum mechanics , fracture (geology) , paleontology
Aggrecanase‐1, also called ADAMTS‐4, is a member of the ADAMTS (A disintegrin and metalloprotease with thrombospondin motif) family of proteases. Aggrecanase cleaves specific peptide bonds in aggrecan, the main proteoglycan of articular cartilage. The high activity of aggrecanase results in cartilage degradation in diseases such as arthritis. To develop homogeneous high throughput screening of ADAMTS‐4 inhibitors, we synthesized a novel internally quenched peptide substrate for aggrecanase‐1 consisting of 5‐FAM/TAMRA as the FRET pair. Active ADAMTS‐4 cleaves this FRET substrate into two separate fragments resulting in an increase of 5‐FAM fluorescence, monitored at excitation/emission = 490 nm/520 nm. The new FRET substrate is more sensitive to aggrecanase‐1 cleavage than the previously described Abz/Dnp substrate and can detect subnanogram amounts of ADAMTS‐4. Its cleavage by other members of aggrecanase family, such as ADAMTS‐1 and ADAMTS‐5, is negligible. Inhibitor screening assays with several known metalloprotease inhibitors were validated using this substrate. This novel FRET substrate provides a convenient homogeneous format for aggrecanase‐1 activity assay where signal is less interfered by the autofluorescence of test compounds due to the long wavelength excitation and emission of 5‐FAM, resulting in an overall increase of signal to background ratio.