Background: Glutathione peroxidase (GSH-Px) is one of the major enzyme in the antioxidative
defense mechanism present in the cells.
Methods: In the present study a novel and simple microplate-based method was developed, using the
cupric neocuproine complex (Cu(Nc)2
2+) as a chromogenic oxidizing reagent, for the assessment of
GSH-Px activity of biological samples for the first time. In this GSH-Px enzyme activity measurement,
the GSH-Px catalyzed oxidation of Reduced Glutathione (GSH) gives rise to oxidized form of GSH
(GSSG). The recommended method was based on the reduction of Cu(Nc)2
2+ to highly colored Cu(I)-
neocuproine complex (Cu(Nc)2
+) by the unconsumed GSH, and measurement spectrophotometrically at
450 nm, the difference being correlated to GSH-Px activity of the analytes.
Results: Under the optimum conditions, a linear calibration graph was obtained in the range of 18.5-
92.5 µM of GSH with Limit Of Detection (LOD) of 1.03 µM. Application of developed method to tissue
homogenates (n = 9) provided GSH-Px activity values in agreement with those of the reference
GSH-Px-5,5’-dithio-bis(2-nitrobenzoic acid) (DTNB) method.
Conclusion: Cupric Ion Reducing Antioxidant Capacity (CUPRAC) antioxidant method was implemented
in a microformat (96 well plates) and the reaction time of original CUPRAC method was significantly
shortened from 30 to 4 min. Multi-sample can be simultaneously detected thanks to a novel