QF peptide cleavage assays were conducted in 96-well black microtiter plates (Scientific Laboratories Supplies Ltd, Wilford, UK) using a Fluostar Omega microplate reader (BMG Labtech, Aylesbury, UK). The activity of ADAMTS-4 (1 nM) and -5 (5 nM) was monitored for 2 h using the fluorescent peptide substrates fluorescein 5(6)-carbonyl-Ala-Glu-Leu-Asn-Gly-Arg-Pro-Ile-Ser-Ile-Ala-Lys-N,N,N0,N0-tetramethyl-6-carboxyrhodamine-NH2 (FAM-AE ~ LQGRPISIAK-TAMRA, ADAMTS-4) and fluorescein 5(6)-carbonyl-Thr-Glu-Ser-Glu ~ Ser-Arg-Gly-Ala-Ile-Tyr-Lys-Lys-N,N,N0,N0-tetramethyl-6-carboxyrhodamine-NH2 (FAM-TESE ~ SRGAIYKK-TAMRA, ADAMTS-5) (custom-synthesized by Bachem, Bubendorf, Switzerland) with an excitation wavelength of 485 nm and an emission wavelength of 538 nm. Final substrate concentrations were 1 μM and 40 μM for ADAMTS-4 and -5, respectively. Fluorescence was expressed in relative fluorescence units (RFU) and normalized against a blank containing only buffer and substrate. Emission spectra were recorded at several concentrations of each compound to exclude auto-fluorescence and maximal inhibitor concentrations were selected accordingly.

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