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Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns
Published on: April 26, 2016
Overall analyses of the reactions catalyzed by acetohydroxyacid synthase/acetolactate synthase using a precolumn
Xian Hui1, Jin-Meng Tian1, Xin Wang1
1National Engineering Research Center for Miniaturized Detection Systems and College of Life Sciences, Northwest University, 229 North Taibai Road, Xi'an, Shaanxi, 710069, China.
Abstract:
A precolumn derivatization-HPLC method using 2,4-dinitrophenylhydrazine and 4-nitro-o-phenylenediamine as respective labeling reagents for comprehensive analyses of the reactions catalyzed by acetohydroxyacid synthase (AHAS)/acetolactate synthase (ALS) is developed and evaluated in this research. Comparison with the classic Bauerle' UV assay which can analyze the enzymes only through measurement of acetoin production, the HPLC method shows advantages because it can analyze the enzymes not only via determination of consumption of the substrate pyruvate, but also via measurement of formation of the products including acetoin, 2,3-butanedione, and acetaldehyde in the enzymatic reactions. Thus the results deduced from the HPLC method can reflect the trait of each enzyme in a more precise manner. As far as we know, this is the first time that the reactions mediated by AHAS/ALS using pyruvate as a single substrate are globally analyzed and the features of the enzymes are properly discussed.
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