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Abstract
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Conventional methods for measuring glutathione peroxidase (GPx) activity are limited by interference issues, complex protein precipitation
steps, and variable reliability, necessitating the development of improved analytical approaches for both research and clinical
applications. A modified GPx activity assay has been developed utilizing the Tiron reagent system, which eliminates the need for
protein precipitation. The protocol employs a novel termination reagent containing ferrous ion (Fe2þ) and Tiron (C6H4Na2O8S2) to instantly
stop enzymatic decomposition of hydrogen peroxide. Following GPx-mediated H2O2 consumption, residual hydrogen peroxide
undergoes Fenton-type redox reactions with Fe2þ ions, generating Fe3þ species that coordinate with Tiron through catechol moieties
to form a stable ferri-Tiron complex [Fe(C6H4Na2O8S2)]3þ. The assay operates optimally at acidic pH to ensure complex stability
and minimize interference from competing reactions. The modified protocol demonstrates superior performance characteristics
compared to conventional GPx assays, including elimination of interference effects, enhanced accuracy and precision, and improved
reproducibility across diverse sample matrices. The method’s spectrophotometric detection system provides reliable quantification
with minimal matrix effects, while the simplified workflow reduces technical complexity and analysis time. This interference-free
GPx activity assay offers significant advantages for both laboratory research and clinical diagnostics. It achieves this through a combination
of analytical precision, operational simplicity, and broad compatibility with standard laboratory practices and equipment.
The protocol’s robust performance at acidic pH conditions, coupled with its elimination of protein precipitation steps, establishes it
as a valuable alternative to existing methodologies for assessing oxidative stress and evaluating antioxidant capacity.
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