Overall analyses of the reactions catalyzed by acetohydroxyacid synthase/acetolactate synthase using a precolumn derivatization-HPLC method.

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.