Fluorometric and Time-Resolved Immunofluorometric Assays for Protein-Tyrosine Phosphatase Activity

Objective: To develop sensitive nonisotopic assays for proteintyrosine phosphatase (PTP) activity. Methods: The fluorometric assay is based on the fact that phosphotyrosine but not tyrosine forms highly fluorescent complexes with Tb3+. Thus, PTP activity can be followed by measuring the decrease of fluorescence due to hydrolysis of phosphotyrosine. The time-resolved immunofluorometric assay employs tyrosine-phosphorylated substrates, immobilized on microtitre wells. After incubation with PTP, the remaining phosphotyrosine residues are reacted with an antiphosphotyrosine antibody. The immunocomplexes formed are detected with an alkaline phosphatase (ALP)-labeled second antibody. The phosphate ester of 5′ fluorosalicylate (FSAP) is used as substrate. The fluorosalicylate produced forms highly fluorescent complexes with Tb3+-EDTA in alkaline solution. The fluorescence is measured with a time-resolved fluorometer. Results: The truncated form of the T-cell protein tyrosine phosphatase (TCΔC11 PTP) was determined in the range of 1100-36,500 U/L by the fluorometric assay and 36-7100 U/L by the time-resolved immunofluorometric assay. Conclusions: The two nonisotopic assays should prove beneficial for the determination and study of various PTP.