PMID:12597635
| Citation |
Han, YS, Sabbioni, C, van der Heijden, R and Verpoorte, R (2003) High-performance liquid chromatography assay for 1-deoxy-D-xylulose 5-phosphate synthase activity using fluorescence detection. J Chromatogr A 986:291-6 |
|---|---|
| Abstract |
A high-performance liquid chromatography assay for activity of 1-deoxy-D-xylulose 5-phosphate synthase, an early enzyme in the recently discovered 2-C-methyl-D-erythritol-4-phosphate pathway, was developed. In this assay, the enzymatic product 1-deoxy-D-xylulose was first derivatized with a fluorescent reagent 2-anthranilic acid, followed by separation using HPLC on a Nova-Pak phenyl column with a mobile phase containing CH3CN-water-1-butylamine-tetrahydrofuran-H3PO4 (2:97:0.125:0.5:0.25, v/v). The eluate was monitored by fluorescence detection at an excitation wavelength of 320 nm and an emission wavelength of 425 nm for quantitation of the fluorescent derivative. A linear response was obtained between 5 and 200 ng of 1-deoxy-D-xylulose. This assay was successfully applied to measure the 1-deoxy-D-xylulose 5-phosphate synthase activity in a recombinant E. coli overexpressing dxs gene. It demonstrated that this assay is simple, sensitive and selective compared to the methods used at present. |
| Links | |
| Keywords |
Chromatography, High Pressure Liquid/methods; Escherichia coli/enzymology; Fluorescent Dyes; Spectrometry, Fluorescence/methods; Transferases/metabolism; ortho-Aminobenzoates |
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