3,6-Fluorescein Diphosphate: A Sensitive Fluorogenic and Chromogenic Substrate for Protein Tyrosine Phosphatases

Abstract

A highly sensitive and continuous protein tyrosine phosphatase (PTPase) assay using 3,6-fluorescein diphosphate (FDP) is described. Leukocyte phosphatase CD45 (leukocyte common antigen), protein tyrosine phosphatase-lB, and leukocyte common antigen-related protein LAR preferentially hydrolyze FDP to fluorescein monophosphate (FMP) with Vmax and Km values comparable with those of phosphotyrosine peptide substrates. Further hydrolysis of FMP to fluorescein was less efficient because of increased Km 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 the substrate of choice for PTPase inhibitor screening in HTS format and assay miniaturization.

Picomolar Concentrations of Free Zinc(II) Ions Regulate Receptor Protein-tyrosine Phosphatase β Activity

“…Measuring zinc in the reagents used in the assay demonstrated that all the chemicals contained zinc at concentrations greater than 1 ppb. Notably, reagents, such as the substrate FDP, are usually not thought to be a source of zinc contamination and thought to be free of inhibitors (26). However, FDP has zinc concentrations far exceeding the concentrations that would cause 50% inhibition of PTP-1B (27.5 ppb at 0.42 M FDP).…”

“…The enzyme was added to this buffer to yield final concentrations of 0.5-5 nM. The reaction was initiated by adding FDP, a fluorogenic phosphatase substrate (26), to a final concentration of 0.8 M. Assays were performed in triplicate in a total volume of 100 l in 96-well black optical bottom plates (p-clear plate black, reference number 655090, Greiner Bio-One Ltd., Stonehouse, UK). The increase in fluorescence due to the hydrolysis of FDP to fluorescein was measured at 485 Ϯ 20 nm excitation and 530 Ϯ 20 nm emission for up to 1 h, using a fluorescence plate reader (Synergy HT, BioTek, Winooski, VT).…”

Nucleotide Activation of the Ca-ATPase

“…Time-resolved Fluorescence Spectroscopy-The free dyes fluorescein monophosphate and fluorescein are reported to have a "very high" quantum yield (56). However, the absolute value for the quantum yield of fluorescein monophosphate has not been reported, and fluorescein monophosphate is not commercially available.…”

Structural Studies of a Stabilized Phosphoenzyme Intermediate of Ca2+-ATPase

“…Obviously, the cytoplasmic domains are also able to exert some influence over the membrane domain, but this appears to be confined to relatively minor structural changes that open and close gates that control access of Ca 2ϩ to the binding sites, as well as to some as yet undefined mechanism of altering Ca 351 to a phenolic oxygen of the covalently attached fluorescein. Because the pH sensitivity of the spectral properties of fluorescein is due to protonation or deprotonation of one of the phenolic oxygens, covalent attachment of phosphate at the same site on fluorescein would no doubt also alter its spectral properties: phosphorylation of fluorescein is already known to result in low fluorescence (54), and 3-methylfluorescein monophosphate is a commonly used non-fluorescent but fluorogenic substrate for phosphatases as well as for Ca 2ϩ -ATPase (55). Furthermore, phosphate transfer from Asp 351 to FITC would provide an elegant explanation for our findings that high fluorescence and low fluorescence forms of FITC-ATPase have similar proteolytic patterns and cross-linking properties.…”