Further Applications of a Glutamate Oxidase Reactor for the Determination of the Neurotoxin P-Odap in a Flow Injection System
No Thumbnail Available
Date
1995-06
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Addis Ababa University
Abstract
A flow injection system involving immobilized GIOD reactor was employed to
study the kinetics of the isomerization of ,8-0DAP to a-ODAP. The amino acid was
oxidized by dissolved oxygen to produce hydrogen peroxide which subsequently
reacted with T rinder chromogenic reagent (catalyzed by horseradish peroxidase in
solution, HRP) to form red-coloured quinone imine dye for spectrophotometric
detection at 512 nm. Injections of 20 III ,8-0DAP standards resulted in a linear range
of 10 - 300 11M. Thermal treatment (at 80 DC) of ,8-0DAP standards and grass pea
extracts reduced the FI response to 62 - 63 % of that before heating, because of the
isomerization of the,8-isomer to the non-toxic a-isomer. This reconfirms the reported
selectivity of GIOD to the toxin. The experimental convenience of the ,8-selective
flow system has been exploited to study the effects of the neurotoxin concentration
and temperature on the rates of ,8-a conversion. The results of these studies showed
that the isomerization of the toxin to the nontoxic isomer followed zero order kinetics
with an initial apparent rate constant equal to 15.26 lIM1min. The equilibrium time
varied linearly with the initial concentration of ,8-0DAP. Values for the initial
apparent rate constant of isomerization (at pH 7) were 3.56 lIM1min, 6.2811M1min,
15.2611M1min and 30.8911M1min at 60,70,80 and 90DC, respectively. Isomerization
at pH 2 proceeded at a faster rate (24.5 lIM1min) than at pH 7.
The complete flow injection set-up for the determination of ,8-0DAP in grass
pea samples consisted of two packed-bed enzyme reactors in series, namely a mixture
of GIOD (25 II\) and catalase (25 II\), and GIOD (250 III). The major interference from
glutamate was destroyed by its quantitative oxidation in the pre-reactor. The product,
hydrogen peroxide, was destroyed in the first reactor which contained GIOD and
catalase. Most of the P-ODAP was oxidized in the big (250 III) reactor, because only
a few percent of this compound is destroyed in the first reactor. The P-ODAP
concentrations of five grass pea samples (0.599 - 0.749%) were determined from flow
injection peaks of filtered extracts in phosphate buffer (pH 7). These results were
found to be in good agreement with the values obtained using the OPT method
Description
Keywords
Glutamate Oxidase