An enzymatic fluorescent assay for the quantification of phosphite in a microtiter plate format.
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.
Centre for Phytophthora Science and Management, School of Biological Sciences and Biotechnology, Murdoch University, Murdoch, WA 6150, Australia. o.berkowitz@murdoch.edu.au
Oliver
O
Berkowitz
Ricarda
R
Jost
Stuart J
SJ
Pearse
Hans
H
Lambers
Patrick M
PM
Finnegan
Giles E St J
GE
Hardy
Philip A
PA
O'Brien
D017360:Arabidopsis / Q000737:chemistry; D057075:Enzyme Assays / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D009243:NAD; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000378:metabolism; D010078:Oxazines / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D017905:Phosphites / Q000032:analysis*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D005456:Fluorescent Dyes; D010078:Oxazines; D017905:Phosphites; D011994:Recombinant Proteins; D009243:NAD; C014180:resorufin; D009247:NADH, NADPH Oxidoreductases; C429539:NAD phosphite oxidoreductase