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QF peptide cleavage assays

SS Salvatore Santamaria
DC Doretta Cuffaro
EN Elisa Nuti
LC Lidia Ciccone
TT Tiziano Tuccinardi
FL Francesca Liva
FD Felicia D’Andrea
RG Rens de Groot
AR Armando Rossello
JA Josefin Ahnström
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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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