Methylumbelliferyl phosphate

The Abbott IMx , a dedicated commercial immunoassay analyzer that employs FPIAs for small molecules, can also determine larger analytes by a fluorescence-based microparticle capture enzyme immunoassay (MEIA).(44) In this system, antibody-coated0.47- mlatexparticles are used for both sandwich and competitive assays, and alkaline phosphatase conjugates that bind to the particles cleave 4-methylumbelliferyl phosphate to generate the fluorophore. 

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. 

Fig. 3. Hydrolysis of 4-methylumbelliferyl phosphate by calf intestinal alkaline phosphatase. Activities are recorded as turnovers per site per second at 20° and 1 = 0.02, using tris-acetic acid (< pH 8) or ammediol-HCl (> pH 8) buffers.

For the hydrolysis of p-nitrophenyl phosphate by placental phosphatase at pH 10.5 the corresponding figure is 10,380 cal/mole (101a). Taking into account changes in ionization of the enzyme, a value of 9800 cal/mole for 4-methylumbelliferyl phosphate and calf intestinal phosphatase was derived (143). The comparable values for nonenzymic hydrolysis of monoanions of aryl phosphates are 27,000-31,000 cal/mole... 

In another report, Michaelis-Menten parameters of ALP have been obtained using a one-shot Lineweaver-Burk (reciprocal) plot. This can be achieved by simultaneously measuring the conversion of 12 independent concentrations of the substrate (4-methylumbelliferyl phosphate) created on-chip. The enzyme was streptavidin-conjugated ALP that was linked to biotinylated phospholipid bilayers coated inside PDMS microchannels. The blue fluorescence of the enzymatic product, 7-hydroxy-4-methyl coumarin, was measured [ 1043]. The surface-bound enzyme was found to have a lower (sixfold) turnover rate than the free enzyme in solutions. After diffusion mixing between two streams (substrate and buffer)... 

FI, Fernley, H. N., and Walker, P. G., Kinetic behavior of calf-intestinal alkaline phosphatase with 4-methylumbelliferyl phosphate. Biochem. J. 97, 95-103 (1965). 

Fig. 2 Common generic FTP substrates. p-Nitrophenylphosphate (pNPP), 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), and 0-methyl-fluorescein phosphate (OMFP)...

The common fluorogenic substrates used for three widely used enzyme tracers are />hydroxyphenylacetic acid for HRP, 4-methylumbelliferyl phosphate for alkaline phosphatase, and 4-methylumbelliferyl-P-D-galacto-pyranoside for p-galactosidase. 

For each type of assay, the label or tag involved is listed along with typical substrates, type of absorbance, and type of instrument required. RIA radioimmunoassay IRMA immunoradiometric assay CPM counts per minute ELISA enzyme linked immunosorbent assay EIA enzyme immunoassay TMB 5,5 tetramethylbenzidine HPA p hydroxyphenylacetic acid AMPPD 4 methoxy 4 (3 phosphatephenyl) spiro(l,2 dioxetane) 3,2 adamantane p NPP p nitrophenyl phosphate 4 MUP 4 methylumbelliferyl phosphate ONPG o nitrophenyl P galactopyranoside MUG 4 methylumbelliferyl P D galactopyrano side AMPGD 3 (4 methoxyspiro( 1,2 dioxetane 3,2 tricyclo(3.3.1.1(3,7))decan) 4 yl)phenylgalacto pyranoside ECL electrochemiluminescence. 

Alkaline phospha- tase Calf intestine 100000 9-10 p-Nitrophenyl- phosphate 4-Methylumbelliferyl- phosphate Adamantyl-1,2- dioxyethane Phenylphosphate- substituted dioxyethane... 

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). 

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. 

Nitroblue tetrazolium (NBT) in combination with 5-bromo-4-chloro-3-indolylphosphate (BCIP) 4-Methylumbelliferyl phosphate (4-MUP) Insoluble, purple Fluorescent 358 nm excitation, 450 nm... 

Liu and Bonizzoni have reported a supramolecular fluorescent sensing array based on 4-methylumbelliferyl phosphate (170) dye complexed with amine-terminated dendrimer, for qualitative and quantitative analysis of organophosphates in water. The sensor allowed simultaneous differentiation and quantitative analysis of methylphosphonate (171) (a nerve gas byproduct) and glyphosate (172) (a ubiquitous herbicide) over a wide range of concentrations (from 10 pM to 2 mM). ... 

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