Citation: HU Yue, WANG Xue-Jiao, LI Heng, GAO Wen-Yun. 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[J]. Chinese Journal of Analytical Chemistry, ;2012, 40(12): 1859-1864. doi: 10.3724/SP.J.1096.2012.20447 shu

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

1.
College of Life Sciences, Northwest University, Xi'an 710069, China

Corresponding author: GAO Wen-Yun,
Received Date: 27 April 2012
Available Online: 3 July 2012
Fund Project: 本文系国家自然科学基金项目(No.21172179) (No.21172179)西北大学研究生创新基金(No.10YYB02)资助 (No.10YYB02)

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.
- Non-mevalonate pathway,
- 1-Deoxy-D-xylulose-5-phosphate synthase,
- Kinetic parameters,
- 2,4-Dinitrophenylhydrazine,
- High performance liquid chromatography
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