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.
Keywords: 1,2-Diamino-4,5-methylenedioxybenzene; 1-Deoxy-D-xylulose 5-phosphate synthase; Activity; HPLC; Non-mevalonate pathway.