Column 2, 4 - Dinitrobenzene Hydrazine Derivative Hplc Method To Detect Before Triose Phosphate And Armour Hydroxyl Pentanoic Acid Way Important Intermediates 1 - Dna - D - Xylulose - 5 - Phosphoric Acid

Terpenoids are distributed in nature widely, which are one of the biggest families of the natural products. Two pathways for the biosynthesis of this kind of compounds have been found and established to date, one is the classical mevalonate (MVA) pathway, and the other one is the non-mevalonate (MEP) pathway. Studies have shown that MVA pathway, which is present in many animals (including humans), fungi and archaea, whereas MEP pathway is present in many eubacteria (including human pathogens), green algae and lichens. Higher plants can use both biosynthetic pathways to complete the biosynthesis of terpenoids, with theMEP pathway being dominant.In order to study the important intermediates and the key enzymes of the MEP pathway, we established a method in which the carbonyl containing compounds can normally be determined by HPLC through pre-column derivatization with2,4-dinitrophenylhydrazine (DNPH). Using this method,1-deoxy-D-xylulose-5-phosphate (DXP), D-glyceraldehyde-3-phosphate (D-GAP) and dihydroxyacetone phosphate (DHAP) were measured. Firstly, the three compounds were dephosphorylated by alkaline phosphatase, then the products1-deoxy-D-xylulose (DX), D-glyceraldehyde (D-GA) and dihydroxyacetone (DHA) were derived with DNPH in acidic solution to give the corresponding hydrazones which was subsequently determined by HPLC. The optimum derivatization conditions are as follows: reaction temperature37℃, acidity30%perchloric acid, molar ratio of DNPH to carbonyl6:1, reaction time of60min. The HPLC was run with a linear gradient of methanol-water solvent system:0min,40%methanol;17min,80%methanol;18min,40%methanol;20min,40%methanol. The method has a detective limit of1μg/mL for DXP,0.1μg/mL for DHAP and1μg/mL for D-GAP, all the three compounds have good linear correlation in the range of0.001～1mg/mL (R2=0.999). The relative standard deviation of three determinations for each component is less than5.0%, and the recoveries of the three compounds are between98%-102%.In order to verify the usefulness of this method, we determined the contents of DXP, D-GAP and DHAP in the higher plants Mentha haplocalyx, Wrinkled Gianthyssop and Spinacia oleracea. But the results indicated that this method can only be used for qualitative analysis largely because the plant system is too complicated. Subsequently, we detected the three compounds in E.coli BL21strain. The result showed good reproducibility and precision, and recoveries are between70%-80%. Therefore, this method can be applied to detect these physiologically important compounds in bacteria. At last, the steady-state kinetic parameters of DXS were determined with this method and the Km, Vmax and Kcat thus obtained are identical with the previously reported data.