A high-performance liquid chromatography assay for threonine/serine dehydratase.
Threonine dehydratase, a key enzyme leading to the biosynthesis of isoleucine, catalyzes the production of 2-ketobutyrate from threonine. An uv/fluorometric HPLC assay for threonine dehydratase has been developed that involves derivatization of the 2-ketoacids produced by the enzyme using a specific derivatizing agent, o-phenylenediamine. The derivatized ketoacids can be detected spectrophotometrically or fluorometrically. This novel assay is over 1000-fold more sensitive than the commonly used dinitrophenyl hydrazine derivatization assay. In addition, the HPLC assay allows the identification of the reaction product(s), and can be used to examine the reaction using mixtures of the two substrates, threonine and serine.
Singh
B K
BK
American Cyanamid Company, Princeton, New Jersey 08543-0400.
Szamosi
I
I
Shaner
D
D
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005069:Evaluation Studies as Topic; D007651:Keto Acids / Q000032:analysis; D010655:Phenylenediamines; D010944:Plants / Q000201:enzymology; D012694:Serine; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D013912:Threonine; D013913:Threonine Dehydratase / Q000032:analysis*
D007651:Keto Acids; D010655:Phenylenediamines; D013912:Threonine; D012694:Serine; C034193:1,2-diaminobenzene; D013913:Threonine Dehydratase