title: TAMRA- and Cy5-labeled probe for efficient kinetic characterization of caspase-3.
abstract: 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 V(max) of 41.4±3.3 nM/min and K(M) of 1.60±0.23 μM. 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.
authors.affiliation: Department of Chemistry and Research Institute for Basic Sciences, Kyung Hee University, Seoul 130-701, Republic of Korea.
authors.forename: Gwan-Ho
authors.initials: GH
authors.lastname: Lee
authors.affiliation: Department of Chemistry and Research Institute for Basic Sciences, Kyung Hee University, Seoul 130-701, Republic of Korea.
authors.forename: Eun Jeong
authors.initials: EJ
authors.lastname: Lee
authors.affiliation: Department of Chemistry and Research Institute for Basic Sciences, Kyung Hee University, Seoul 130-701, Republic of Korea. Electronic address: sshah@khu.ac.kr.
authors.forename: Sang Soo
authors.initials: SS
authors.lastname: Hah
mesh_terms: D000595:Amino Acid Sequence; D002232:Carbocyanines / Q000737:chemistry*; D053148:Caspase 3 / Q000737:chemistry* / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D007700:Kinetics; D012235:Rhodamines / Q000737:chemistry*
chemical_list: C114557:5-carboxytetramethylrhodamine succinimidyl ester; D002232:Carbocyanines; D005456:Fluorescent Dyes; D012235:Rhodamines; C085321:cyanine dye 5; D053148:Caspase 3
keywords: Apoptosis; Caspase-3; FRET; Kinetics