Aggrecanases belong to ADAMTS (A disintegrin and metalloprotease with thrombospondin motif) family of proteases. Aggrecanases cleave aggrecan, the major structural component of cartilage. Aggrecanase-1 (ADAMTS-4) is a major aggrecanase in human osteoarthritic cartilage.
The SensoLyteŽ 520 Aggrecanase-1 Assay Kit uses 5-FAM as the fluorophore and TAMRA as the quencher in the FRET peptide substrate. This substrate is used for continuous measurement of the enzyme activity. In an intact FRET peptide, the fluorescence of 5-FAM is quenched by TAMRA. Upon the cleavage of the FRET peptide by ADAMTS-4, the fluorescence of 5-FAM is recovered, and can be continuously monitored at excitation/emission = 490 nm/520 nm. Increase in FAM fluorescence is correlated to aggrecanase-1 activity. 5-FAM/TAMRA based FRET substrate is less interfered by autofluorescence of test compounds due to its longer emission wavelength. The kit can be used to detect the activity of aggrecanase-1 or screen for aggrecanase inhibitors. The assays are performed in a convenient 96-well format.