Determination of the Activity of 1-Deoxy-d-Xylulose 5-Phosphate Synthase by Pre-column Derivatization-HPLC Using 1,2-Diamino-4,5-Methylenedioxybenzene as a Derivatizing Reagent

• Published in: The Protein Journal 2019-04, Vol.38 (2), p.160-166
• Main Authors: Liang, Yan-Fei, Liu, Hui, Li, Heng, Gao, Wen-Yun
• Format: Article
• Language: English
• Subjects: Animal Anatomy; Aqueous solutions; Bacterial Proteins - chemistry; Biochemistry; Bioorganic Chemistry; Chemical tests and reagents; Chemistry; Chemistry and Materials Science; Chromatography, High Pressure Liquid - methods; D-Xylulose 5-phosphate; Elution; Escherichia coli - enzymology; High performance liquid chromatography; Histology; Kinetics; Liquid chromatography; Monosaccharides; Morphology; Organic Chemistry; Pentosephosphates - chemistry; Phenylenediamines - chemistry; Phosphates; Pyruvic acid; R&D; Reaction time; Reagents; Research & development; Steady state; Substrates; Sugars; Transferases - chemistry; Xylulose
• ISSN: 1572-3887; 1573-4943; 1875-8355
• DOI: 10.1007/s10930-019-09816-9

α-Ketoacids can be determined by HPLC through pre-column derivatization with 1,2-diamino-4,5-methylenedioxybenzene (DMB) as a derivatizing reagent. Using this method, the specific activity and the steady-state kinetic of 1-deoxy- d -xylulose-5-phosphate synthase (DXS) were measured. Firstly, DXS substrate pyruvate was derivatized with DMB in acidic solution; then the corresponding quinoxalinone was elucidated by LC–ESI–MS and quantified by HPLC-UV. The optimum derivatization conditions were as follows: aqueous medium at pH 1.0, reaction temperature 80 °C, reaction time 60 min, molar ratio of DMB to pyruvate 10:1. The HPLC was run with isocratic elution using the mixture of methanol and water (60:40, v/v) as a mobile phase. The detective limit and the linear correlation range of the method were 0.05 µM and 0.002−1.0 mM (R = 0.994), respectively. The relative standard deviation (RSD) of six determinations was 2.48%. The steady-state kinetic parameters of DXS for pyruvate determined with the method were identical to the reported data. The established method is a practical route for evaluation of DXS activity, especially in the research and development of DXS inhibitors.