title: Homogeneous assays for cellular proteases employing the platinum(II)-coproporphyrin label and time-resolved phosphorescence.
abstract: Phosphorescent platinum(II) coproporphyrin label (PtCP) is evaluated for the detection of cellular proteases by time-resolved fluorescence in homogeneous format. An octameric peptide containing the recognition motif for the caspase-3 enzyme was dual labeled with a new maleimide derivative of PtCP and with the dark quencher dabcyl. Following photophysical characterization, the quenched substrate was employed in cleavage assays for caspase-3 using Jurkat and HL60 cell lines treated with proapoptotic stimuli performed on a commercial plate reader. Dose-response and time course assays for the drug camptothecin were obtained for comparison with conventional fluorometric detection.
authors.affiliation: Laboratory of Biophysics and Bioanalysis, Department of Biochemistry, University College Cork, Lee Maltings, Prospect Row, Cork, Ireland.
authors.forename: Tomás C
authors.initials: TC
authors.lastname: O'Riordan
authors.forename: James
authors.initials: J
authors.lastname: Hynes
authors.forename: Dmitri
authors.initials: D
authors.lastname: Yashunski
authors.forename: Gelii V
authors.initials: GV
authors.lastname: Ponomarev
authors.forename: Dmitri B
authors.initials: DB
authors.lastname: Papkovsky
mesh_terms: D002166:Camptothecin / Q000494:pharmacology; D053148:Caspase 3; D020169:Caspases / Q000032:analysis* / Q000096:biosynthesis; D003306:Coproporphyrins / Q000737:chemistry*; D004790:Enzyme Induction; D005456:Fluorescent Dyes; D006801:Humans; D019169:Jurkat Cells; D008163:Luminescent Measurements / Q000379:methods*; D008301:Maleimides / Q000737:chemistry*; D009944:Organoplatinum Compounds / Q000737:chemistry*; D010447:Peptide Hydrolases / Q000032:analysis*
chemical_list: D003306:Coproporphyrins; D005456:Fluorescent Dyes; D008301:Maleimides; D009944:Organoplatinum Compounds; C107920:platinum 3,8,13,18-tetramethyl-21H,23H-porphine-2,7,12,18-tetrapropionic acid; C504440:platinum(II) coproporphyrin maleimide; D010447:Peptide Hydrolases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D002166:Camptothecin