Amperometric sensor for glutathione reductase activity determination in erythrocyte hemolysate.
The development of an amperometric sensor for glutathione reductase (GR) activity in erythrocyte hemolysate to contribute to oxidative stress evaluation is presented. In this assay, the reduced form of glutathione, the product of the GR reaction, reacts with 5,5(')-dithiobis(2-nitrobenzoic acid), producing GSTNB, which is easily reduced in the electrode surface. The current was recorded during 180 s after the sample addition, applying a potential of -300 mV. The sensor presented a suitable sensitivity, a good operational range, and precision. The effects of pH variations and specific uncompetitive inhibitor (safranin-O) in the enzyme activity were also evaluated. The GR activity determination in human erythrocyte hemolysate using this method has provided results that are statistically equal to those obtained by the classical spectrophotometric method, with 95% of confidence. The advantages of this method are the saved time, reagents, and samples and the possibility of its use in the field.
Alves
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AA
Labex, Laboratory of Biochemistry of Exercise, Departamento Bioquímica, IB-UNICAMP, Campinas, 13083-971 Sao Paulo, Brazil.
Pereira da Silva
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L
Macedo
D V
DV
Kubota
L T
LT
D002457:Cell Extracts; D004563:Electrochemistry; D004566:Electrodes; D004912:Erythrocytes / Q000201:enzymology*; D005978:Glutathione / Q000032:analysis / Q000737:chemistry; D005980:Glutathione Reductase / Q000032:analysis* / Q000378:metabolism*; D006801:Humans; D009579:Nitrobenzoates / Q000737:chemistry; D010084:Oxidation-Reduction; D015203:Reproducibility of Results
D002457:Cell Extracts; D009579:Nitrobenzoates; D005980:Glutathione Reductase; D005978:Glutathione