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Comparison of caspase‐8 fluorescent substrates for development of microtiter plate homogeneous cell‐based assay
Author(s) -
Rakhmanova Vera,
Han Xing,
Tong Xiaohe,
Hong Anita L.,
Meyer Rich
Publication year - 2008
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.22.1_supplement.648.19
Subject(s) - caspase , homogeneous , substrate (aquarium) , lysis , chemistry , plate reader , caspase 8 , enzyme , microbiology and biotechnology , apoptosis , fluorescence , cell , biochemistry , chromatography , biophysics , biology , programmed cell death , optics , ecology , physics , thermodynamics
Caspase‐8 is an initiator enzyme accepting signals from death receptors and activating downstream effectors caspases (caspase‐3, ‐7) through proteolytic cleavage. Caspase‐8 has substrate specificity for the amino acid sequence, Ile‐Glu‐Thr‐Asp (IETD). Although there are a number of assays using IETD based caspase‐8 substrates, those protocols are not optimized for 96‐well plate format and include additional steps of cell harvesting, lysate preparation and subsequent aliquoting of prepared lysates into 96‐well plates. To develop homogeneous 96‐well plate caspase‐8 fluorimetric assays, we compared a series of fluorophores (AMC, AFC and Rh110) linked to IETD peptide. AMC and AFC‐linked substrates tested with purified enzyme gave at least a 2‐fold higher signal/background ratio and better kinetic parameters than the Rh110 labeled substrate. When applied to cells grown and lysed in 96‐well plates, the Ac‐IETD‐AMC substrate showed a high background. The longer‐wavelength spectra of the AFC (Ex/Em=380 nm/500 nm) provided greater sensitivity and less interference from cell media. The use of Ac‐IETD‐AFC substrate for detection of caspase‐8 activity in cells enabled the development of a homogeneous assay with a wide linear range and high signal/background ratio. This assay is ideal for measuring enzyme activity in cells grown in 96‐well plates and for screening of apoptosis inducers or inhibitors.