title: A generic assay for phosphate-consuming or -releasing enzymes coupled on-line to liquid chromatography for lead finding in natural products.
abstract: A generic continuous-flow assay for phosphate-consuming or -releasing enzymes coupled on-line to liquid chromatography (LC) has been developed. Operating the LC-biochemical assay in combination with mass spectrometry allows the fast detection and identification of inhibitors of these enzymes in complex mixtures. The assay is based on the detection of phosphate, released by the on-line continuous-flow enzymatic reaction, using a fluorescent probe. The probe consists of fluorophore-labeled phosphate-binding protein, which shows a strong fluorescence enhancement upon binding to inorganic phosphate. To detect very small changes of the phosphate concentration in a postcolumn enzymatic reaction medium, the enzymatic removal of phosphate impurities from solvents, reagents, and samples was optimized for application in continuous flow. The potential of the phosphate probe is demonstrated by monitoring the enzymatic activity, i.e., the phosphate release, from alkaline phosphatase. The selectivity of the phosphate readout, necessary to distinguish between phosphate containing substrate or product and free inorganic phosphate released after enzymatic conversion, is shown. The applicability of LC coupled to the enzymatic assay using the phosphate readout was demonstrated by detection of tetramisole in a plant extract as inhibitor of alkaline phosphatase. Parallel mass spectrometry allowed the simultaneous confirmation of the identity of the inhibitor.
authors.affiliation: Kiadis B.V. Niels Bohrweg 11-13, 2333 CA, Leiden, The Netherlands. t.schenk@kiadis.com
authors.forename: T
authors.initials: T
authors.lastname: Schenk
authors.forename: N M G M
authors.initials: NM
authors.lastname: Appels
authors.forename: D A
authors.initials: DA
authors.lastname: van Elswijk
authors.forename: H
authors.initials: H
authors.lastname: Irth
authors.forename: U R
authors.initials: UR
authors.lastname: Tjaden
authors.forename: J
authors.initials: J
authors.lastname: van der Greef
mesh_terms: D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004798:Enzymes / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods; D010710:Phosphates / Q000032:analysis / Q000378:metabolism*; D010936:Plant Extracts / Q000737:chemistry; D013773:Tetramisole / Q000494:pharmacology; D013997:Time Factors
chemical_list: D004791:Enzyme Inhibitors; D004798:Enzymes; D010710:Phosphates; D010936:Plant Extracts; D013773:Tetramisole; D000469:Alkaline Phosphatase