Assay for aliphatic amino acid decarboxylases by high-performance liquid chromatography.
A sensitive and rapid assay for aliphatic amino acid decarboxylases based on separation of the product from the substrate by ion-pairing reversed-phase high-performance liquid chromatography and subsequent fluorometric detection has been developed. The resolution of substrates and products of seven amino acid decarboxylases, namely, arginine, aspartate, 2,6-diaminopimelate, histidine, glutamate, lysine, and ornithine decarboxylase, is complete within 15 to 35 min of isocratic elution. The limit of detection for the product is 40 pmol. The applicability of the procedure was assessed with glutamate decarboxylase. The formation of the product 4-aminobutyrate proved to be linear with time and protein concentration. The method allows the time course of the reaction to be followed in a single assay and works well with crude extracts of bacteria or tissues.
Kochhar
S
S
Biochemisches Institut der Universität Zürich, Switzerland.
Mehta
P K
PK
Christen
P
P
D002262:Carboxy-Lyases / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000201:enzymology; D005968:Glutamate Decarboxylase / Q000032:analysis; D013050:Spectrometry, Fluorescence
D002262:Carboxy-Lyases; D005968:Glutamate Decarboxylase