TAMRA- and Cy5-labeled probe for efficient kinetic characterization of caspase-3

Our objective was to create a novel fluorogenic substrate for efficient in vitro kinetic assays on caspase-3. We designed a TAMRA (5′-tetramethylrhodamine-5(6)-carboxamide)- and Cy5 (cyanine 5)-labeled probe that allowed us to evaluate the caspase-3 activity via the changes in fluorescence intensity...

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Bibliographic Details
Published in:Analytical biochemistry 2014-02, Vol.446, p.22-24
Main Authors: Lee, Gwan-Ho, Lee, Eun Jeong, Hah, Sang Soo
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Apoptosis
Carbocyanines - chemistry
Caspase 3 - chemistry
Caspase 3 - metabolism
Caspase-3
Enzyme Assays - methods
Fluorescence Resonance Energy Transfer
Fluorescent Dyes - chemistry
FRET
Kinetics
Rhodamines - chemistry
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Summary:Our objective was to create a novel fluorogenic substrate for efficient in vitro kinetic assays on caspase-3. We designed a TAMRA (5′-tetramethylrhodamine-5(6)-carboxamide)- and Cy5 (cyanine 5)-labeled probe that allowed us to evaluate the caspase-3 activity via the changes in fluorescence intensity and wavelength. The prepared probe was found to be an efficient and selective substrate of caspase-3, with Vmax of 41.4±3.3nM/min and KM of 1.60±0.23μM. The strategy used in the design of this fluorogenic substrate can be applied in future endeavors to development of substrates for caspase-3 inhibitor screening assays or for real-time detection of apoptosis in living cells.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2013.10.013