Enhanced protocol for measuring glutathione peroxidase activity using a new glutathione peroxidase-Tiron assay

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 (Fe²⁺) 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 Fe²⁺ ions, generating Fe³⁺ species that coordinate with Tiron through catechol moieties to form a stable ferri-Tiron complex [Fe(C₆H₄Na₂O₈S₂)]³⁺. 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.