Further Improvements of the Flow Injection Glutamate Oxidase-Based Assay for β Odap in Grass Pea

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Date

1996-06

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Addis Ababa Universty

Abstract

The content of p-N-oxalyl-L-a,p-diaminopropionic acid (P-ODAP) in thirty grass pea seed samples were determined by a FI system incorporating a glutamate oxidase reactor. The assay of p-ODAP was made via the oxidation product, hydrogen peroxide, which was detected spectrophotometrically at 512 nm using Trinder chromogenic reagent. The same samples were assayed by the Rao's modified colorimetric procedure at 476 nm. The concentrations obtained by the enzymatic method was 10-50% higher than that obtained by the colorimetric method. The results of another variation of Rao's method (Campbell procedure) was 53-66% lower than that of the FI method. Separations of proteins from p-ODAP in grass pea extracts were made by two precipitation methods with trichloroacetic acid (1 %) and perchloric acid (0.46%) and by ultrafiltration. Sample injections of glucose spiked extracts after the three sample clean-up procedures enhanced stability of glucose oxidase reactors in a FI system. The response of a FI system with a small GOD reactor to glucose-spiked crude extract gradually decreased to zero after 36 injections. Synthesis and characterization of sulphonated 2,4-dichloronaphthol, as substitute for 2,4-dichlorophenol-6-sulphonate in Trinder reagent, was studied. The molar absorptivities for hydrogen peroxide at 512 nm P.max) were 15,220 and 11,380 M·j cm·j at pH 4.2-5 and at pH 7 respectively. This reagent was used in the determination of hydrogen peroxide and glucose in both batch and flow injection systems

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Oxidase-Based Assay for β Odap in Grass Pea

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