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Precise Spectrophotometric Method for Measurement of Peroxiredoxin Activity in Biological Samples


Affiliations
1 Chemistry Dept., Kufa University, Najaf City, Najaf Governorate, Iraq
2 Chemistry Dept., College of Science, University of Babylon, Hilla City, Babylon Governorate, P.O. 51002, Iraq
     

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Herein, we describe a simple spectrophotometric method for the measurement of peroxiredoxin activity and demonstrate reproducibility, accuracy, and precision. In these experiments, peroxiredoxin activity was measured by incubating enzyme samples with phosphate buffer solution containing suitable concentrations of the substrates 1,4-dithio-DL-threitol (DTT) and hydrogen peroxide. Titanium sulfate was added to stop enzyme reactions, and subsequent reactions with residual hydrogen peroxide produced pertitanic acid, which was spectrophotometrically measured at 405 nm. Advantages of this method are including the elimination of catalase interference and allowing application of this method to all types of biological tissues. The peroxiredoxin assay is simple and can be completed with few additions. The method is precise, with coefficients of variation of 2.93% within runs and 5.4% between runs. Data from the present peroxiredoxin assay were strongly correlated with those from the ferrous oxidation–xylenol orange (FOX) method (r = 0.9835).

Keywords

Peroxiredoxin, Pertitanic Acid, FOX Reagent, Dithiothreitol, H2O2.
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  • Precise Spectrophotometric Method for Measurement of Peroxiredoxin Activity in Biological Samples

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Authors

Seenaa Kadhum Ali
Chemistry Dept., Kufa University, Najaf City, Najaf Governorate, Iraq
Mahmoud Hussein Hadwan
Chemistry Dept., College of Science, University of Babylon, Hilla City, Babylon Governorate, P.O. 51002, Iraq

Abstract


Herein, we describe a simple spectrophotometric method for the measurement of peroxiredoxin activity and demonstrate reproducibility, accuracy, and precision. In these experiments, peroxiredoxin activity was measured by incubating enzyme samples with phosphate buffer solution containing suitable concentrations of the substrates 1,4-dithio-DL-threitol (DTT) and hydrogen peroxide. Titanium sulfate was added to stop enzyme reactions, and subsequent reactions with residual hydrogen peroxide produced pertitanic acid, which was spectrophotometrically measured at 405 nm. Advantages of this method are including the elimination of catalase interference and allowing application of this method to all types of biological tissues. The peroxiredoxin assay is simple and can be completed with few additions. The method is precise, with coefficients of variation of 2.93% within runs and 5.4% between runs. Data from the present peroxiredoxin assay were strongly correlated with those from the ferrous oxidation–xylenol orange (FOX) method (r = 0.9835).

Keywords


Peroxiredoxin, Pertitanic Acid, FOX Reagent, Dithiothreitol, H2O2.

References