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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Keywords
Oxidase-Based Assay for β Odap in Grass Pea