An enzymatic fluorescent assay for the quantification of phosphite in a microtiter plate format

Oliver Berkowitz, Ricarda Jost, Stuart Pearse, Hans Lambers, Patrick Finnegan, G.E.St.J Hardy, P.A. O'Brien

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

A sensitive fluorometric assay for the quantification of phosphite has been developed. The assay uses the enzymatic oxidation of phosphite to phosphate by a recombinant phosphite dehydrogenase with NAD+ as cosubstrate to produce the highly fluorescent reaction product resorufin. The optimized assay can be carried out in a 96-well microtiter plate format for high-throughput screening purposes and has a detection limit of 0.25 nmol phosphite. We used the method to quantify phosphite levels in plant tissue extracts and to determine phosphite dehydrogenase activity in transgenic plants. The assay is suitable for other biological or environmental samples. Because phosphite is a widely used fungicide to protect plants from pathogenic oomycetes, the assay provides a cost-effective and easy-to-use method to monitor the fate of phosphite following application.
Original languageEnglish
Pages (from-to)74-78
JournalAnalytical Biochemistry
Volume412
DOIs
Publication statusPublished - 2011

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