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首页> 外文期刊>The protein journal >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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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

机译：用1,2-二氨基-4,5-亚甲基二苯苯苯苯苯苯苯苯苯苯并衍生化衍生物预衍生化-HPLC的1-脱氧-D-木糖糖5-磷酸合酶的活性的测定

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摘要

alpha-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 degrees C, reaction time 60min, 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 mu M and 0.002-1.0mM (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.
机译：α-酮酸可以通过HPLC通过用1,2-二氨基-4,5-亚甲基二苯苯苯（DMB）作为衍生试剂的柱子衍生化。使用该方法，测量了1-脱氧-D-木糖-5-磷酸合酶（DXS）的特定活性和稳态动力学。首先，DXS底物丙酮酸用DMB在酸性溶液中衍生化;然后通过LC-ESI-MS阐明相应的喹喔啉酮并通过HPLC-UV定量。最佳衍生条件如下：pH 1.0的水性介质，反应温度80℃，反应时间60min，DMB的摩尔比10：1。使用甲醇和水（60:40，V / V）作为流动相，HPLC与等异构洗脱。该方法的侦探极限和线性相关范围分别为0.05μm和0.002-1.0mm（r = 0.994）。六个测定的相对标准偏差（RSD）为2.48％。用该方法测定的丙酮酸DXS的稳态动力学参数与报告的数据相同。建立的方法是评估DXS活性的实际途径，特别是在DXS抑制剂的研究和开发中。

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