FLOW-INJECTION ASSAY FOR THE NEUROTOXIN BETA-ODAP USING AN IMMOBILIZED GLUTAMATE OXIDASE REACTOR WITH PREREACTORS TO ELIMINATE GLUTAMATE INTERFERENCES

The neurotoxic amino acid, beta-N-oxalyl-L-alpha,beta-diaminopropionic acid (beta-ODAP,ODAP) was oxidized by immobilized glutamate oxidase ( GlOD) to produce hydrogen peroxide. The peroxide reacts with Trinder r eagent in a reactor with immobilized horseradish peroxidase to form a red-colored quinone imine dye, which was detected spectrophotometrical ly at 512 nm. Determinations were made in a flow injection (FI) setup consisting of four packed-bed enzyme reactors containing GlOD (20 mu L ), catalase (20 mu L), GlOD (250 mu L), and peroxidase (50 mu L) in se ries. Glutamate is oxidized quantitatively in the first reactor, but t he hydrogen peroxide is destroyed in the second so that interference f rom this substrate are removed. This step destroys only a few percent of the ODAP in the sample. Most of the remaining ODAP is oxidized in t he third reactor. Injections of 20-mu L ODAP standards gave a response curve which was linear within the range 10-650 mu M. Phosphate buffer extracts of grass peas (lathyrus sativus) were purified by centrifuga tion and membrane filtration. Samples were injected into the FI setup to assay the toxin at a rate of 20 samples per hour. The beta-ODAP con tent of a batch of dry seed corresponded to 0.74% (w/w) with a relativ e standard deviation of 2.8%. Thermal treatment of ODAP standards at 8 0-90 degrees C reduced the response to 62% of that before heating. The decrease is due to beta reversible arrow alpha isomerization, and the experiment thus confirms that the method is selective for the toxic b eta-isomer.