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VOL. 73 (2), 441-452, 2007 GLUTAMATE DETERMINATIONS USING AMPLEX RED...

in the oxidation of L-glutamic acid by glutamate oxidase to produce
a-ketoglutarate, NH3 and H2O2. In a subsequent reaction H2O2 is
reduced by horseradish peroxidase (HRP) using Amplex red reagent
(10-acetyl-3, 7 dihidroxiphenoxazine) as an electron donor which is
transformed to resorufin, a fluorescent compound that has excitation/
emission maxima of ~540/585 nm.

Following the indications of the protocol, a working solution,
containing 50 µM AR, 0.125 U/ml HRP, 0.04 U/ml L-glutamate
oxidase, 0.25 U/ml L-glutamate pyruvate transaminase and 100 µM
L-alanine in 100 mM Tris-HCl, pH 7.5 buffer, was prepared. Next,
several concentrations of L-Glu (100 nM- 4 µM) were added and
incubated at 37º C for 30 min. Finally, resorufin fluorescence was
detected with a BMG Labtechnologies 96 plate reader using excitation
filter at 544 nM and emission filter at 590 nM. As it can be observed
in Figure 2, the resorufin fluorescence was glutamate concentration-
dependent in the range assayed.

FIGURE 2. Detection of L-glutamic acid using the Amplex® Red reagent-based
assay. Each reaction contained 50 µM Amplex® Red reagent, 0.125 U/mL HRP,
0.04 U/mL L-glutamate oxidase, 0.25 U/mL L-glutamate-pyruvate transaminase,
100 µM L-alanine and the indicated amount of L-glutamic acid in 1X reaction
buffer. Reactions were incubated at 37° C. After 30 minutes, resorfurin
fluorescence was detected with a BMG Labtechnologies 96 plate reader using
excitation filter at 544 nM and emission filter at 590 nM.

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