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Hamza T, Hadwan M H. Accurate and Precise Protocol to Estimate the Activity of Peroxiredoxin Enzyme. rbmb.net 2021; 10 (2) :156-163
URL: http://rbmb.net/article-1-584-en.html
URL: http://rbmb.net/article-1-584-en.html
Pathological Analysis Department, Al-Mustaqbal University College, Hilla City, Babylon Governorate, Iraq.
Abstract: (3639 Views)
Background: Accurate estimation of Peroxiredoxin Enzyme (Prx) activity poses many complications and interferences. The present protocol is free of interference and provides an effective alternative for the assessment of peroxide with high sensitivity. The assay can be used in clinical pathology laboratories since it is simple, rapid, and inexpensive. The systematic reagent consisted of AFS/ASA which acted as a sensitive probe for peroxide.
Methods: Prx activity was estimated by incubating samples in suitable concentrations of 1,4-dithio-DL-threitol (DTT) and hydrogen peroxide (H2O2) or t-Butyl hydroperoxide (t-BOOH), as the substrates. The enzymatic reaction was inhibited after incubation with a working reagent containing ammonium ferrous sulfate (AFS) and aminosalicylic acid (ASA).
Results: Residual peroxide reacted with the working solution to form a brown-colored ferriaminosalicylate (FAS) complex with a maximum absorbance (λmax) of 425 nm. This protocol used sodium azide (NaN3) to eliminate catalase interference and avoided using high concentrations of a strong acid to inhibit the Prx reaction.
Conclusions: We concluded that the new protocol produced the same efficacy as the reference method since a strong correlation coefficient of comparison (r> 0.99) was found between both the FAS and ferrithiocyanate method.
Methods: Prx activity was estimated by incubating samples in suitable concentrations of 1,4-dithio-DL-threitol (DTT) and hydrogen peroxide (H2O2) or t-Butyl hydroperoxide (t-BOOH), as the substrates. The enzymatic reaction was inhibited after incubation with a working reagent containing ammonium ferrous sulfate (AFS) and aminosalicylic acid (ASA).
Results: Residual peroxide reacted with the working solution to form a brown-colored ferriaminosalicylate (FAS) complex with a maximum absorbance (λmax) of 425 nm. This protocol used sodium azide (NaN3) to eliminate catalase interference and avoided using high concentrations of a strong acid to inhibit the Prx reaction.
Conclusions: We concluded that the new protocol produced the same efficacy as the reference method since a strong correlation coefficient of comparison (r> 0.99) was found between both the FAS and ferrithiocyanate method.
Keywords: Amino Salicylic Acid, Ammonium Ferrous Sulfate, Dithiothreitol, Peroxiredoxin, T-Butyl Hydroperoxide.
Type of Article: Original Article |
Subject:
Biochemistry
Received: 2020/10/4 | Accepted: 2020/10/19 | Published: 2021/08/26
Received: 2020/10/4 | Accepted: 2020/10/19 | Published: 2021/08/26
References
1. Rhee SG, Chae HZ, Kim K. Peroxiredoxins: a historical overview and speculative preview of novel mechanisms and emerging concepts in cell signaling. Free Radic Biol Med. 2005;15;38(12):1543-52. [DOI:10.1016/j.freeradbiomed.2005.02.026] [PMID]
2. Hofmann B, Hecht H-J, Flohé L. Peroxiredoxins. Biol Chem. 2002;383(3-4):347-64. [DOI:10.1515/BC.2002.040]
3. Wood ZA, Poole LB, Karplus PA. Peroxiredoxin evolution and the regulation of hydrogen peroxide signaling. Science. 2003;300(5619):650-3 [DOI:10.1126/science.1080405] [PMID]
4. Rhee SG, Kang SW, Chang TS, Jeong W, Kim K. Peroxiredoxin, a novel family of peroxidases. IUBMB life. 2001;52(1-2):35-41. [DOI:10.1080/15216540252774748] [PMID]
5. Winterbourn CC. Reconciling the chemistry and biology of reactive oxygen species. Nat Chem Biol. 2008;4(5):278-86. [DOI:10.1038/nchembio.85] [PMID]
6. Parsonage D, Reeves SA, Karplus PA, Poole LB. Engineering of fluorescent reporters into redox domains to monitor electron transfers. Methods Enzymol. 2010;474:1-21. [DOI:10.1016/S0076-6879(10)74001-5]
7. Veskoukis AS, Margaritelis NV, Kyparos A, Paschalis V, Nikolaidis MG. Spectrophotometric assays for measuring redox biomarkers in blood and tissues: the NADPH network. Redox Rep. 2018;23(1):47-56. [DOI:10.1080/13510002.2017.1392695] [PMID] [PMCID]
8. Nelson KJ, Parsonage D. Measurement of peroxiredoxin activity. Curr Protoc Toxicol. 2011. [DOI:10.1002/0471140856.tx0710s49] [PMID] [PMCID]
9. Lim YS, Cha MK, Yun CH, Kim HK, Kim KW, Kim IH. Purification and characterization of thiol-specific antioxidant protein from human red blood cell: a new type of antioxidant protein. Biochem Biophys Res Commun. 1994;199(1):199-206. [DOI:10.1006/bbrc.1994.1214] [PMID]
10. LE Netto, HZ Chae, SW Kang, SG Rhee, ER Stadtman. Removal of hydrogen peroxide by thiol-specific antioxidant enzyme (TSA) is involved with its antioxidant properties. J Biol Chem. 1996;271(26):15315-21. [DOI:10.1074/jbc.271.26.15315] [PMID]
11. Freitas I, Boncompagni E, Tarantola E, Gruppi C, Bertone V, Ferrigno A, et al. In situ evaluation of oxidative stress in rat fatty liver induced by a methionine-and choline-deficient diet. Oxid Med Cell Longev. 2016;2016:9307064. [DOI:10.1155/2016/9307064] [PMID] [PMCID]
12. Bae SH, Sung SH, Cho EJ, Lee SK, Lee HE, Woo HA, et al. Concerted action of sulfiredoxin and peroxiredoxin I protects against alcohol-induced oxidative injury in mouse liver. Hepatology. 2011;53(3):945-53. [DOI:10.1002/hep.24104] [PMID]
13. Bae SH, Sung SH, Lee HE, Kang HT, Lee SK, Oh SY, et al. Peroxiredoxin III and sulfiredoxin together protect mice from pyrazole-induced oxidative liver injury. Antioxid Redox Signal. 2012;15:17(10):1351-61. [DOI:10.1089/ars.2011.4334] [PMID] [PMCID]
14. Musicco C, Capelli V, Pesce V, Timperio AM, Calvani M, Mosconi L, et al. Accumulation of overoxidized Peroxiredoxin III in aged rat liver mitochondria. Biochim Biophys Acta. 2009;1787(7):890-6. [DOI:10.1016/j.bbabio.2009.03.002] [PMID]
15. Rhee SG, Woo HA, Kil IS, Bae SH. Peroxiredoxin functions as a peroxidase and a regulator and sensor of local peroxides. J Biol Chem. 2012;287(7):4403-10. [DOI:10.1074/jbc.R111.283432] [PMID] [PMCID]
16. Bahar B, Tuncel AF, Holmes EW, Holmes DT. An interactive website for analytical method comparison and bias estimation. Clin Biochem. 2017;50(18):1025-1029. [DOI:10.1016/j.clinbiochem.2017.08.008] [PMID]
17. Khalifa HH, Hadwan MH. Simple Method for the Assessment of Peroxiredoxin Activity in Biological Samples. Chemical Data Collections. 2020;7:100376. [DOI:10.1016/j.cdc.2020.100376]
18. Koel M, Kaljurand M. Application of the principles of green chemistry in analytical chemistry. Pure Appl Chem. 2006;78(11):1993-2002. [DOI:10.1351/pac200678111993]
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