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

Zheng Huang

Merck Frosst Center for Therapeutic Research, Pointe-Claire, Dorval, Quebec, Canada

Qingping Wang

Merck Frosst Center for Therapeutic Research, Pointe-Claire, Dorval, Quebec, Canada

Hoa D. Ly

Merck Frosst Center for Therapeutic Research, Pointe-Claire, Dorval, Quebec, Canada

Arvind Gorvindarajan

Merck Frosst Center for Therapeutic Research, Pointe-Claire, Dorval, Quebec, Canada

John Scheigetz

Merck Frosst Center for Therapeutic Research, Pointe-Claire, Dorval, Quebec, Canada

Robert Zamboni

Merck Frosst Center for Therapeutic Research, Pointe-Claire, Dorval, Quebec, Canada

Sylvie Desmarais

Merck Frosst Center for Therapeutic Research, Pointe-Claire, Dorval, Quebec, Canada

Chidambaram Ramachandran

Merck Frosst Center for Therapeutic Research, Pointe-Claire, Dorval, Quebec, Canada

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.

Journal of Biomolecular Screening, Vol. 4, No. 6, 327-334 (1999)
DOI: 10.1177/108705719900400608
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