Fluorescent Peptide Probes for High-Throughput Measurement of Protein Phosphatases

• Published in: Analytical chemistry (Washington) 2003-05, Vol.75 (9), p.2042-2047
• Main Authors: Noble, James E, Ganju, Pam, Cass, Anthony E. G
• Format: Article
• Language: English
• Subjects: Analytical chemistry; Chemistry; Enzyme Inhibitors - analysis; Enzyme Inhibitors - pharmacology; Exact sciences and technology; Fluorescence; Fluorescent Dyes - chemistry; Kinetics; Measurement; Miscellaneous; Oxadiazoles - chemistry; Peptides; Peptides - chemistry; Phosphates; Phosphoprotein Phosphatases - antagonists & inhibitors; Phosphoprotein Phosphatases - chemistry; Proteins; Reverse Transcriptase Polymerase Chain Reaction; Rosaniline Dyes - chemistry
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac025838e

A homogeneous microplate assay for the serine/threonine protein phosphatases PP1 and PP2A, employing fluorescent-labeled phosphopeptides, has been developed. Phosphopeptides derived from a phosphoacceptor site in myelin basic protein were designed with a cysteine adjacent to the phosphoresidue, allowing site-selective labeling with dyes. The fluorescence emission from the environmentally sensitive fluorophore 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide was found to be sensitive to the phosphorylation status of an adjacent threonine residue. Upon complete dephosphorylation of the dye-labeled phosphopeptide, a 56% decrease in fluorescence intensity was observed. The change in fluorescence was correlated with the release of inorganic phosphate from the phosphopeptide as measured using the malachite green assay. Conjugation of the fluorophore to the phosphopeptide was found to have no adverse effect on catalysis. A series of four phosphopeptide substrates were developed and characterized to probe PP1 and PP2A activity. The optimum phosphopeptides were then used to determine inhibition parameters for three natural protein phosphatase inhibitors. The use of a peptide-based approach has introduced a degree of specificity not observed with many conventional phosphatase substrates, while retaining the advantages of a real-time homogeneous fluorescence-based format, making the assay ideal for high-density screening.