title: Conjugated polymer-based real-time fluorescence caspase assays.
abstract: We have developed conjugated polyelectrolyte-based fluorescence turn-on assays for caspase 3 and 8. These assays are composed of a cationic polyphenylene ethynylene polymer PPE4+ and p-nitroaniline modified caspase peptide substrate. The fluorescence of the assay is initially turned-off because of the efficient quenching of the polymer by p-nitroaniline moiety on anionic peptide substrates. A turn-on effect is observed due to the cleavage of the peptide by the enzyme and formation of the neutral p-nitroaniline unit which has no quenching on the polymer. We validated this assay design and obtained kinetic parameters of caspase 3 and caspase 8. These assays demonstrated good sensitivity as in pmol/L (0.1 units/mL) for caspase 3 and nmol/L (0.2 units/mL) for caspase 8. This method also showed high specificity by using caspase 3 assay as a model system and the results demonstrated that other proteases including caspase 8, papain, pepsin, and trypsin did not show observable fluorescence turn-on effect. The dose-response curve of a caspase inhibitor Z-VAD-FMK was evaluated by caspase 3 assay, by which the IC(50) value was determined to be 0.73 μM and was in a good agreement with the literature reported value at 0.62 μM. This design could be applied into the in vitro screening of small molecular inhibitors for drug discovery.
authors: The Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, PR China.
authors: Yonghua
authors: Y
authors: Xie
authors: Rui
authors: R
authors: Zhao
authors: Ying
authors: Y
authors: Tan
authors: Xin
authors: X
authors: Zhang
authors: Feng
authors: F
authors: Liu
authors: Yuyang
authors: Y
authors: Jiang
authors: Chunyan
authors: C
authors: Tan
mesh_terms: D053148:Caspase 3 / Q000737:chemistry*; D053181:Caspase 8 / Q000737:chemistry*; D057075:Enzyme Assays / Q000295:instrumentation / Q000379:methods*; D005453:Fluorescence; D007700:Kinetics; D011108:Polymers / Q000737:chemistry
chemical_list: D011108:Polymers; D053148:Caspase 3; D053181:Caspase 8