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P. 109
An. R. Acad. Nac. Farm., 2007, 73: 441-452
Artículo original
Glutamate determinations using Amplex Red
Glutamic Acid Assay are affected by P2X
agonist BzATP
Recibido el 23 de abril de 2007
PATRICIA MARÍN-GARCÍA, JESÚS SÁNCHEZ-NOGUEIRO,
DAVID LEÓN *
Department of Biochemistry, Veterinary Faculty,
Universidad Complutense de Madrid. Spain
ABSTRACT
Amplex Red (AR) reagent is the most stable and sensitive fluorogenic substrate
known for horseradish peroxidase (HRP). Thus, in the presence of hydrogen
peroxide (H2O2), AR (a nonfluorescent compound) is converted to resorufin (a
strongly fluorescing product) by the action of HRP. In the last years, multiple
assays have been developed using this reagent to quantify a diverse assortment of
analyses and also to detect the activity of many different enzymes. We recently
showed that BzATP, agonist of several ionotropic nucleotide receptors (P2X),
interfered with Amplex Red oxidation catalyzed by HRP. In the present work we
reported that glutamate determinations using Amplex Red Glutamic Acid/Glutamate
Oxidase Assay Kit are also strongly interfered by BzATP. These data are really
interesting because demonstrate that resorufin fluorescence, in the presence of
BzATP, is not proportional to glutamate concentration and, therefore, it must not
be used as method to measure glutamate concentration.
Key words: Amplex Red.—Glutamate.—P2X.—BzATP.—Interferences.
* Contact information: David León Navarro.
Department of Biochemistry, Veterinary Faculty, Universidad Complutense de
Madrid.
Avda. Puerta de Hierro, s/n. 28040 Madrid, Spain.
Telf.: 34-91-394-3894. Fax: 34-91-394-3909.
E-mail: daleon@vet.ucm.es
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