Homogeneous assays for cellular proteases employing the platinum(II)-coproporphyrin label and time-resolved phosphorescence

Tomás C. O'Riordan; James Hynes; Dmitri Yashunski; Gelii V. Ponomarev; Dmitri B. Papkovsky

doi:10.1016/j.ab.2005.04.016

Homogeneous assays for cellular proteases employing the platinum(II)-coproporphyrin label and time-resolved phosphorescence

Tomás C. O'Riordan
, James Hynes
, Dmitri Yashunski
, Gelii V. Ponomarev
, Dmitri B. Papkovsky

School of Biochemistry and Cell Biology

Research output: Contribution to journal › Article › peer-review

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.

Original language	English
Pages (from-to)	111-119
Number of pages	9
Journal	Analytical Biochemistry
Volume	342
Issue number	1
DOIs	https://doi.org/10.1016/j.ab.2005.04.016
Publication status	Published - 1 Jul 2005

Keywords

Caspase
Homogeneous assays
Labels
Metalloporphyrin
Phosphorescence
Protease assays
Time-resolved fluorescence

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10.1016/j.ab.2005.04.016

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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.",

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year = "2005",

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doi = "10.1016/j.ab.2005.04.016",

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T1 - Homogeneous assays for cellular proteases employing the platinum(II)-coproporphyrin label and time-resolved phosphorescence

AU - O'Riordan, Tomás C.

AU - Hynes, James

AU - Yashunski, Dmitri

AU - Ponomarev, Gelii V.

AU - Papkovsky, Dmitri B.

PY - 2005/7/1

Y1 - 2005/7/1

N2 - 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.

AB - 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.

KW - Caspase

KW - Homogeneous assays

KW - Labels

KW - Metalloporphyrin

KW - Phosphorescence

KW - Protease assays

KW - Time-resolved fluorescence

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DO - 10.1016/j.ab.2005.04.016

M3 - Article

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AN - SCOPUS:20444371561

SN - 0003-2697

VL - 342

SP - 111

EP - 119

JO - Analytical Biochemistry

JF - Analytical Biochemistry

IS - 1

ER -

Homogeneous assays for cellular proteases employing the platinum(II)-coproporphyrin label and time-resolved phosphorescence

Abstract

Keywords

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