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
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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.
KeywordsNon-mevalonate pathway 1-Deoxy-d-xylulose 5-phosphate synthase Activity HPLC 1,2-Diamino-4,5-methylenedioxybenzene
This study was funded by the Natural Science Foundation of China (Grants 21402152, 31770848), and the Scientific Research Project of Shaanxi Province (No. 2017JM2027).
Compliance with Ethical Standards
Conflict of interest
The authors declare that they have no conflict of interest.
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