3,4-fluorescein diphosphate: A sensitive fluorogenic and chromogenic substrate for protein tyrosine phosphatases

A highly sensitive and continuous protein tyrosine phosphatase (PTPase) ass ay using 3,6-fluorescein diphosphate (FDP) is described. Leukocyte phosphat ase CD45 (leukocyte common antigen), protein tyrosine phosphatase-1B, and l eukocyte common antigen-related protein LAR preferentially:hydrolyze FDP to fluorescein monophosphate (FMP) with V-max and K-m values comparable with those of phosphotyrosine peptide substrates. Further hydrolysis of FMP to f luorescein was less efficient because of increased K-m values compared with those of FDP, FMP absorbs strongly at 445 nm and fluoresces intensely near 515 nm, both of which are insensitive to pH perturbations above pH 6. Its high catalytic efficiency, coupled with the highly-sensitive dual detection in the visible wavelength region and wider pH operating range, make FDP th e substrate of choice for PTPase inhibitor screening in HTS format and assa y miniaturization.