2,4-二硝基苯肼柱前衍生高效液相色谱法测定1-脱氧-D-木酮糖-5-磷酸合酶稳态动力学参数

Determination of Steady-State Kinetic Parameters of 1-Deoxy-D-xylulose-5-phosphate Synthase by Pre-column Derivatization High Performance Liquid Chromatography Using 2,4-Dinitrophenylhydrazine as Derivative Reagent

利用含羰基化合物与2,4-二硝基苯肼在酸性条件下反应得到的产物腙对紫外-可见光有吸收的特性,采用柱前衍生高效液相色谱法测定了1-脱氧-D-木酮糖-5-磷酸合酶稳态动力学参数。酶反应产物1-脱氧-D-木酮糖-5-磷酸在碱性磷酸酶的作用下生成去磷酸化产物1-脱氧-D-木酮糖,然后与2,4-二硝基苯肼衍生成腙。衍生化反应的适宜条件为:HClO4浓度为1.5%,反应温度37℃,反应时间60 min,2,4-二硝基苯肼与1-脱氧-D-木酮糖-5-磷酸的摩尔比为6∶1。色谱条件:0～17 min,40%～80%甲醇;18～20 min,40%甲醇。结果表明,本方法具有较高的灵敏度和良好的线性关系,1-脱氧-D-木酮糖-5-磷酸的检出限为1.0 mg/L,在0.005～1.0 g/L范围内线性相关系数为0.999,相对标准偏差小于5.0%(n=3),标准回收率为102.22%。用本方法测得的1-脱氧-D-木酮糖-5-磷酸合酶稳态动力学参数Km、Vmax和Kcat与文献报道一致。

Carbonyl containing compounds can normally be determined by high performance liquid chromatography（HPLC） using pre-column derivatization with 2,4-dinitrophenylhydrazine（DNPH）.Using this method,the steady-state kinetic parameters of 1-deoxy-D-xylulose-5-phosphate synthase（DXS） were measured.First,the enzymatic product 1-deoxy-D-xylulose-5-phosphate（DXP） was dephosphorylated by alkaline phosphatase,then the product 1-deoxy-D-xylulose（DX） was derived with DNPH in acidic solution to give the corresponding hydrazones which was subsequently determined by HPLC.The optimum derivatization conditions were as follows： acidity 1.5% perchloric acid,reaction temperature 37 ℃,reaction time of 60 min,molar ratio of DNPH to DXP 6： 1,.The HPLC was run with a linear gradient of methanol-water solvent system： 0 min,40% methanol;17 min,80% methanol;18 min,40% methanol;20 min,40% methanol.The method has a detection limit of 1 mg/L for DXP and the linear correlation coefficient in the range of 0.005-1 g/L was 0.999.The relative standard deviation is less than 5.0%.The steady-state kinetic parameters of DXS determined with this method are identical with the reported data.