Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
1171381 | Analytica Chimica Acta | 2007 | 8 Pages |
Abstract
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 40Â 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.
Related Topics
Physical Sciences and Engineering
Chemistry
Analytical Chemistry
Authors
Desmond J. O'Shea, Tomás C. O'Riordan, Paul J. O'Sullivan, Dmitri B. Papkovsky,