A reverse-phase high-performance liquid chromatography assay for dihydroxy-acid dehydratase.
A sensitive method for assaying dihydroxy-acid dehydratase activity is described. This enzyme produces alpha-ketoisovaleric and alpha-keto-beta-methylvaleric acids, respectively, in the biosynthesis of valine and isoleucine. These alpha-keto acids, after derivatization with 2,4-dinitrophenyl-hydrazine, were separated and quantified by reverse-phase high-performance liquid chromatography on a Zorbax octadecylsilane C-18 column. As little as 50 pmol of alpha-ketoisovaleric was detected in assays using cell-free extracts from Escherichia coli, whose measured specific activity was 8 mumol of alpha-ketoisovaleric acid produced per hour per milligram protein.
Smyk-Randall
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EM
Dalton Research Center, Columbia, Missouri.
Brown
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OR
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology; D045782:Hemiterpenes; D006835:Hydrazones; D006836:Hydro-Lyases / Q000032:analysis*; D007651:Keto Acids / Q000032:analysis
C001072:2,4-dinitrophenylhydrazone; D045782:Hemiterpenes; D006835:Hydrazones; D007651:Keto Acids; C016211:alpha-keto-beta-methylvaleric acid; C001505:alpha-ketoisovalerate; D006836:Hydro-Lyases; C020052:dihydroxyacid dehydratase