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DEVD‐NucView488: a novel class of enzyme substrates for real‐time detection of caspase‐3 activity in live cells
Author(s) -
Cen Hui,
Mao Fei,
Aronchik Ida,
Fuentes Rholinelle Joy,
Firestone Gary L.
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/fj.07-099234
Subject(s) - intracellular , substrate (aquarium) , caspase 3 , apoptosis , enzyme , cleavage (geology) , fluorescence , cell , dna , caspase , microbiology and biotechnology , chemistry , reagent , biophysics , population , biochemistry , biology , programmed cell death , medicine , physics , ecology , paleontology , environmental health , quantum mechanics , fracture (geology)
Live‐cell detection of intracellular enzyme activity requires that substrates are cell‐permeable and that the generated products are easily detected and retained in cells. Our objective was to create a novel fluorogenic substrate that could be used for real‐time detection of apoptosis in living cells. We have synthesized a highly cell‐permeable caspase‐3 substrate, DEVD‐NucView488, by linking a fluorogenic DNA‐binding dye to the caspase‐3 recognition sequence that renders the dye nonfunctional. On substrate cleavage, the dye is released and becomes highly fluorescent on binding to DNA. DEVD‐NucView488 detected caspase‐3 activation within a live‐cell population much earlier and with higher sensitivity compared with other apoptosis reagents that are currently available. Furthermore, cells incubated with DEVD‐NucView488 exhibited no toxicity and normal apoptotic progression. DEVD‐NucView488 is an ideal substrate for kinetic studies of caspase‐3 activation because it detects caspase‐3 activity in real‐time and also efficiently labels DNA in nuclei of caspase‐3‐activated cells for real‐time fluorescent visualization of apoptotic morphology. The strategy utilized in the design of this fluorogenic substrate can be applied in future endeavors to develop substrates for detecting real‐time intracellular enzyme activity.—Cen, H., Mao, F., Aronchik, I., Fuentes, R. J., Firestone, G. L. DEVD‐NucView488: a novel class of enzyme substrates for real‐time detection of caspase‐3 activity in live cells. FASEB J. 22, 2243–2252 (2008)