3- 0-methylfluorescein

Fig. 4 Structures of fluorogenic phosphatase substrates (a) 6,8-difluoro-4-methylumelliferyl phosphate (b) 3-0-methylfluorescein phosphate...

Applied are fluorogenic compounds such as 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) or compounds changing the absorption spectra after dephosphorylation, such phospho-tyrosine, paro-nitrophenol phosphate (pNPP) or 3-0-methylfluorescein phosphate (OMFP) [22, 26] (Figure 3). In a phosphatase inhibition assay, the phosphatase is first incubated with the inhibitor and then the phosphatase activity assay is carried out. Reduced activity due to inhibition is measured by one of the above-mentioned assay types, which are chosen depending on the phosphatase. A parameter often obtained in inhibition assays is the inhibitor concentration 50 (ICjq) (inhibition constant), which is the concentration where 50% of the enzyme is inhibited. 

In practice it is often more convenient to measure the release of a phenol from an aryl phosphomonoester. Standard serum phosphatase methods employ phenyl phosphate (188), p-nitrophenyl phosphate (189), phenolphthalein monophosphate (140), or thymolphthalein monophosphate (141) where the phenol released can be determined spectrophoto-metrically [only the Bodansky method (13) uses a Pi determination]. A number of fluorogenic substrates have been used for phosphatase studies, e.g., jS-naphthyl phosphate (30, 148), 4-methylumbelliferyl phosphate (143), and 3-O-methylfluorescein phosphate (144) The main advantage here is the much greater sensitivity of fluorescence as compared with spectrophotometric assays as little as 1 pmole of 4-methyl-umbelliferone can be detected in continuous assay. 

Hfll HD, Summer GK, Waters MD (1968) An automated fluorometric assay for alkaline phosphatase using 3-O-methylfluorescein phosphate. Anal Biochem 24 9—17... 

Alkaline phosphatase sodium-/5-glycero phosphate 3-O-methylfluorescein phosphate... 

Other chromogenic substrates can also be exploited, including phenolphthalein monophosphate, thymophthalein monophosphate, P glycerophosphate, and uridine phosphate. Fluorigenic substrates can also be used such as P-naphthyl phosphate, 4-methylumbelliferyl phosphate, and 3-o-methylfluorescein monophosphate (21). 

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