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Fluorometric Protocol for Estimating Peroxiredoxin Activity in Biological Tissues

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Abstract

This protocol describes a detailed fluorometric method for measuring peroxiredoxin (Prx) enzyme activity in vitro. Peroxide dissociation is the rate-limiting step in the Prx-controlled enzymatic reaction. To prevent interference by the catalase enzyme, we developed a peroxiredoxin assay that measures Prx activity using the substrate tert-Butyl hydroperoxide (t-BOOH). Prx enzyme activity is measured by incubating the enzymatic substrates 1,4-dithio-DL-threitol (DTT) and t-BOOH in a suitable buffer at 37 °C for 10 min in the presence of the desired volume of Prx enzyme. Next, the reagent N-(9-Acridinyl)maleimide (NAM) is used to stop the enzymatic reaction and form a fluorescent end product. Finally, Prx activity is measured by thiol fluorometry using a Box–Behnken design to optimize reaction conditions. This novel protocol was validated by evaluating Prx activity in matched samples against a reference assay. The correlation coefficient between our protocol and the reference assay was 0.9933, demonstrating its precision compared with existing methods. The NAM-Prx protocol instead uses t-BOOH as a substrate to measure Prx activity. Because catalase does not participate in the dissociation of t-BOOH, this approach does not require sodium azide. Furthermore, the method eliminates the need for concentrated acids to terminate the Prx enzymatic reaction since the NAM reagent can inhibit the enzymatic reaction regulated by the Prx enzyme.

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Acknowledgements

We would like to thank all of our colleagues for their continuous support and informative scientific suggestions.

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Authors and Affiliations

  1. Chemistry Department, College of Science, University of Babylon, 51002, Hilla City, Babylon Governorate, PO, Iraq

    Marwah Jaber Hussein & Mahmoud Hussein Hadwan

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  1. Marwah Jaber Hussein
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  2. Mahmoud Hussein Hadwan
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Contributions

All authors contributed to the study’s conception and design. Material preparation, data collection, and analysis were performed by [Mahmoud Hussein Hadwan], and [Marwah Jaber Hussein]. The first draft of the manuscript was written by [Mahmoud Hussein Hadwan] and Marwah Jaber Hussein commented on previous versions of the manuscript. All authors read and approved the final manuscript.”

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Correspondence to Mahmoud Hussein Hadwan.

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Ethics Committee (University of Babylon/ College of Science/ Iraq), Ref. no.: 4122 Date: 11/9/2021. The current study was conducted in accordance with the WSAVA Animal Welfare Recommendations. Also, Declaration of Helsinki depended when dealing with human samples.

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Hussein, M.J., Hadwan, M.H. Fluorometric Protocol for Estimating Peroxiredoxin Activity in Biological Tissues. J Fluoresc 33, 721–730 (2023). https://doi.org/10.1007/s10895-022-03111-0

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