High-performance liquid chromatography assay for 1-deoxy-d-xylulose 5-phosphate synthase activity using fluorescence detection
| Autor: | Robert Verpoorte, Cesare Sabbioni, Robert van der Heijden, Ying-Shan Han |
|---|---|
| Rok vydání: | 2003 |
| Předmět: |
Xylulose
Chromatography Elution Organic Chemistry 1-deoxy-D-xylulose-5-phosphate synthase activity General Medicine Biochemistry High-performance liquid chromatography Fluorescence Fluorescence spectroscopy Analytical Chemistry chemistry.chemical_compound Spectrometry Fluorescence chemistry Transferases Escherichia coli Anthranilic acid ortho-Aminobenzoates Derivatization Chromatography High Pressure Liquid Fluorescent Dyes |
| Zdroj: | Journal of Chromatography A. 986:291-296 |
| ISSN: | 0021-9673 |
| DOI: | 10.1016/s0021-9673(02)02016-2 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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