Homogeneous assays for cellular proteases employing the platinum(II)-coproporphyrin label and time-resolved phosphorescence |
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| Authors: | O'Riordan Tomás C Hynes James Yashunski Dmitri Ponomarev Gelii V Papkovsky Dmitri B |
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| Institution: | Laboratory of Biophysics and Bioanalysis, Department of Biochemistry, University College Cork, Lee Maltings, Prospect Row, Cork, Ireland. |
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| 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. |
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| Keywords: | Time-resolved fluorescence Phosphorescence Homogeneous assays Protease assays Caspase Metalloporphyrin Labels |
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