Homogeneous time-resolved fluorescence assays for the detection of activity and inhibition of phosphatase enzymes employing phosphorescently labeled peptide substrates.
A rapid, homogenous, antibody-free assay for phosphatase enzymes was developed using the phosphorescent platinum (II)-coproporphyrin label (PtCP) and time-resolved fluorescent detection. An internally quenched decameric peptide substrate containing a phospho-tyrosine residue, labeled with PtCP-maleimide and dabcyl-NHS at its termini was designed. Phosphatase catalysed dephosphorylation of the substrate resulted in a minor increase in PtCP signal, while subsequent cleavage by chymotrypsin at the dephosphorylated Tyr-Leu site provided a 3.5 fold enhancement of PtCP phosphorescence. This phosphorescence phosphatase enhancement assay was optimized to a 96 well plate format with detection on a commercial TR-F plate reader, and applied to measure the activity and inhibition of alkaline phosphatase, recombinant human CD45, and tyrosine phosphatases in Jurkat cell lysates within 40 min. Parameters of these enzymatic reactions such as Km's, limits of detection (L.O.D's) and IC50 values for the non-specific inhibitor sodium orthovanadate were also determined.
O'Shea
Desmond J
DJ
Biochemistry Department, University College Cork, Cavanagh Pharmacy Building, College Road, Cork, Ireland.
O'Riordan
Tomás C
TC
O'Sullivan
Paul J
PJ
Papkovsky
Dmitri B
DB
D000818:Animals; D002417:Cattle; D004789:Enzyme Activation; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D019169:Jurkat Cells; D049408:Luminescent Agents / Q000032:analysis*; D008163:Luminescent Measurements / Q000379:methods; D010455:Peptides / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000032:analysis* / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013379:Substrate Specificity; D013997:Time Factors
D049408:Luminescent Agents; D010455:Peptides; D010744:Phosphoric Monoester Hydrolases