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

Carbonyl containing compounds can normally be determined by high performance liquid chromatography (HPLC) through 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) was 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 are as follows: 1.5% perchloric acid, reaction temperature of 37 °C, reaction time of 60min, 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 detect limit of 1 mg L−1 for DXP and a linear correlation in the range of 0.005–1 g L−1 (R = 0.999). The relative standard deviation is less than 5.0%. The steady-state kinetic parameters of DXS determined by this method are identical with the reported data.