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

被引:51
作者
Huang, Z [1 ]
Wang, QP [1 ]
Ly, HD [1 ]
Gorvindarajan, A [1 ]
Scheigetz, J [1 ]
Zamboni, R [1 ]
Desmarais, S [1 ]
Ramachandran, C [1 ]
机构
[1] Merck Frosst Canada Inc, Merck Frosst Ctr Therapeut Res, Pointe Claire, PQ H9R 4P8, Canada
关键词
D O I
10.1177/108705719900400608
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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-1B, and leukocyte 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 fluorescein 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 the substrate of choice for PTPase inhibitor screening in HTS format and assay miniaturization.
引用
收藏
页码:327 / 334
页数:8
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