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Fluorometric Protocol for Estimating Peroxiredoxin Activity in Biological Tissues.

Marwah Jaber Hussein1, Mahmoud Hussein Hadwan2

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

Journal of Fluorescence
|December 12, 2022
PubMed
Summary

This study introduces a novel fluorometric assay for measuring peroxiredoxin (Prx) enzyme activity using tert-Butyl hydroperoxide (t-BOOH) as a substrate. The assay is precise and avoids interference from catalase, offering a reliable method for Prx quantification.

Keywords:
DithiothreitolFluorometryN-(9-Acridinyl)maleimidePeroxiredoxinTert-Butyl hydroperoxide

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Area of Science:

  • Biochemistry
  • Enzymology
  • Analytical Chemistry

Background:

  • Peroxiredoxins (Prx) are crucial enzymes involved in redox homeostasis.
  • Existing methods for measuring Prx activity can be affected by catalase interference or require harsh reaction termination steps.

Purpose of the Study:

  • To develop a novel, sensitive, and reliable fluorometric assay for quantifying peroxiredoxin (Prx) enzyme activity in vitro.
  • To establish a method that circumvents interference from catalase and avoids the need for strong acids for reaction termination.

Main Methods:

  • A fluorometric assay was developed using tert-Butyl hydroperoxide (t-BOOH) as the substrate for Prx.
  • The reaction was terminated and a fluorescent product formed using N-(9-Acridinyl)maleimide (NAM).
  • Reaction conditions were optimized using a Box-Behnken design, and the assay was validated against a reference method.

Main Results:

  • The novel assay demonstrated high precision, with a correlation coefficient of 0.9933 compared to a reference assay.
  • The use of t-BOOH as a substrate eliminated the need for sodium azide, as catalase does not interfere with t-BOOH dissociation.
  • The NAM reagent effectively inhibited the Prx enzymatic reaction, negating the requirement for concentrated acids.

Conclusions:

  • The developed NAM-Prx assay provides a robust and sensitive method for measuring Prx activity.
  • This protocol offers advantages over existing methods, including improved specificity and simplified termination procedures.
  • The assay is suitable for various applications requiring accurate quantification of Prx enzyme activity.