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Glutathione Peroxidase Activity of Biological Samples Using A Novel Microplate-Based Method

[ Vol. 14 , Issue. 5 ]

Author(s):

Melek Ugar, Ayse Nur Tufan, Mehmet Altun, Kubilay Guclu and Mustafa Ozyurek*   Pages 512 - 518 ( 7 )

Abstract:


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 microplate method.

Keywords:

GSH-Px activity, microplate based method, modified CUPRAC method, biological samples, neocuproine complex, reduced glutathione (GSH).

Affiliation:

Department of Chemistry, Faculty of Engineering, Istanbul University, Avcilar 34320, Istanbul, Department of Chemistry, Faculty of Engineering, Istanbul University, Avcilar 34320, Istanbul, Department of Chemistry, Faculty of Engineering, Istanbul University, Avcilar 34320, Istanbul, Department of Chemistry, Faculty of Engineering, Istanbul University, Avcilar 34320, Istanbul, Department of Chemistry, Faculty of Engineering, Istanbul University, Avcilar 34320, Istanbul

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