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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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  • S. Rhee, H. Chae, K. Kim, Peroxiredoxins: a historical overview and speculative preview of novel mechanisms and emerging concepts in cell signaling. Free Radic Biol Med 38 (2005) 1543–1552.

  • L. Li, The Relevance of Mammalian Peroxiredoxins to the Gametogenesis, Embryogenesis, and Pregnancy Outcomes. Reproductive Sciences. 24 (2017) 812-817.

  • Z.A. Wood, L.B. Poole, P.A. Karplus, Peroxiredoxin evolution and the regulation of hydrogen peroxide signaling. Science. 300 (2003) 650-653.

  • G. Leyens, I. Donnay, B. Knoops, Cloning of bovine peroxiredoxins—gene expression in bovine tissues and amino acid sequence comparison with rat, mouse and primate peroxiredoxins. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology. 136 (2003) 943-55.

  • S. Rhee, S. Kang, T. Chang, W. Jeong, K. Kim, Peroxiredoxin, a novel family of peroxidases. IUBMB Life 52 (2001) 35–41.

  • T. Ishii, T. Kawane, S. Taketani, S. Bannai, Inhibition of the thiol-specific antioxidant activity of rat liver MSP23 protein by hemin. Biochem Biophys Res Commun 216 (1995) 970–975.

  • P. Zhang, B. Liu, S. Kang, M. Seo, S. Rhee, L. Obeid, Thioredoxin peroxidase is a novel inhibitor of apoptosis with a mechanism distinct from that of Bcl-2. J Biol Chem. 272 (1997) 30615–30618.

  • B.B. Horta, M.A. de Oliveira, K.F. Discola, J.R. Cussiol, L.E. Netto, Structural and biochemical characterization of peroxiredoxin Qbeta from Xylella fastidiosa: catalytic mechanism and high reactivity. J. Biol. Chem. 285 (2010) 16051–16065.

  • K.S. Yang, S.W. Kang, H.A. Woo, S.C. Hwang, H.Z. Chae, K. Kim, S.G. Rhee, Inactivation of human peroxiredoxin I during catalysis as the result of the oxidation of the catalytic site cysteine to cysteine-sulfinic acid. J. Biol. Chem. 277 (2002) 38029–38036.

  • K.J. Nelson, D. Parsonage, Measurement of peroxiredoxin activity. Current protocols in toxicology. (2011) 17-10.

  • C. Sanchis-Segura, M. Miquel, M. Correa, C.M. Aragon, The catalase inhibitor sodium azide reduces ethanol-induced locomotor activity. Alcohol. 19 (1999) 37-42.

  • Y. Aksoy, M. Balk, H. ÖĞÜŞ, N. Özer, The mechanism of inhibition of human erythrocyte catalase by azide, Turkish Journal of Biology. 28 (2005) 65-70.

  • M.H. Hadwan, S.K. Ali, New spectrophotometric assay for assessments of catalase activity in biological samples, Analytical biochemistry. 542 (2018) 29-33.

  • Y.S. Lim, M.K. Cha, C.H, Yun, H.K. Kim, K.W. Kim, I.H. Kim, Purification and characterization of thiol-specific antioxidant protein from human red blood cell: a new type of antioxidant protein, Biochemical and biophysical research communications. 199 (1994) 199-206.

  • T. Eismann, N. Huber, T. Shin, et al., Peroxiredoxin-6 protects against mitochondrial dysfunction and liver injury during ischemia-reperfusion in mice, American Journal of Physiology-Gastrointestinal and Liver Physiology, 296 (2009) G266-G274.

  • S. Immenschuh, E. Baumgart-Vogt, M. Tan, et al., Differential cellular and subcellular localization of heme-binding protein 23/peroxiredoxin I and heme oxygenase-1 in rat liver, Journal of Histochemistry & Cytochemistry, 51 (2003) 1621-1631.

  • S.H. Bae, S.H. Sung, E.J. Cho, et al., Concerted action of sulfiredoxin and peroxiredoxin I protects against alcohol‐induced oxidative injury in mouse liver, Hepatology, 53 (2011) 945-53.

  • H.I. Choi, S.K. Ma, E.H. Bae, et al., Peroxiredoxin 5 protects TGF-β induced fibrosis by inhibiting Stat3 activation in rat kidney interstitial fibroblast cells, PloS one, 11 (2016) e0149266.

  • S.H. Ahn, H.Y. Yang, G.B. Tran, et al., Interaction of peroxiredoxin V with dihydrolipoamide branched chain transacylase E2 (DBT) in mouse kidney under hypoxia, Proteome science, 13 (2015) 4.

  • M.B. Pascual, A. Mata-Cabana, F.J. Florencio, Lindahl M, F.J. Cejudo, Overoxidation of 2-Cys Peroxiredoxin in Prokaryotes Cyanobacterial 2-Cys Peroxiredoxins Sensitive to Oxidative Stress, J. Biol. Chem., 285 (2010) 34485-34492.

  • S.C. Ferreira, R.E. Bruns, H.S. Ferreira, G.D. Matos, J.M. David, G.C. Brandao, E.P. da Silva, L.A. Portugal, P.S. Dos Reis, A.S. Souza, W.N. Dos Santos, Box-Behnken design: an alternative for the optimization of analytical methods. Analytica chimica acta. 597 (2007) 179-86.

  • T.H. Hou, C.H. Su, W.L. Liu, Parameters optimization of a nano-particle wet milling process using the Taguchi method, response surface method and genetic algorithm, Powder technology, 173 (2007) 153-162.

  • A.I. Khuri, S. Mukhopadhyay, Response surface methodology. Wiley Interdisciplinary Reviews: Computational Statistics, 2 (2010) 128-149.

  • M.A. Bezerra, R.E. Santelli, E.P. Oliveira, L.S. Villar, L.A. Escaleira, Response surface methodology (RSM) as a tool for optimization in analytical chemistry. Talanta. 76 (2008) 965-977.


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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