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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

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:

Dithiothreitol Fluorometry N-(9-Acridinyl)maleimide Peroxiredoxin Tert-Butyl hydroperoxide

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Assessment of Cellular Oxidation using a Subcellular Compartment-Specific Redox-Sensitive Green Fluorescent Protein

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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.