title: Sensitive fluorogenic substrate for alkaline phosphatase.
abstract: Alkaline phosphatase serves both as a model enzyme for studies on the mechanism and kinetics of phosphomonoesterases and as a reporter in enzyme-linked immunosorbent assays (ELISAs) and other biochemical methods. The tight binding of the enzyme to its inorganic phosphate product leads to strong inhibition of catalysis and confounds measurements of alkaline phosphatase activity. We have developed an alkaline phosphatase substrate in which the fluorescence of rhodamine is triggered on P-O bond cleavage in a process mediated by a "trimethyl lock." Although this substrate requires a nonenzymatic second step to manifest fluorescence, we demonstrated that the enzymatic first step limits the rate of fluorogenesis. The substrate enables the catalytic activity of alkaline phosphatase to be measured with high sensitivity and accuracy. Its attributes are ideal for enzymatic assays of alkaline phosphatase for both basic research and biotechnological applications.
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
authors.forename: Michael N
authors.initials: MN
authors.lastname: Levine
authors.forename: Ronald T
authors.initials: RT
authors.lastname: Raines
mesh_terms: D000469:Alkaline Phosphatase / Q000032:analysis* / Q000378:metabolism; D002384:Catalysis; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D012235:Rhodamines / Q000737:chemistry; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes; D012235:Rhodamines; D000469:Alkaline Phosphatase