A Novel WST-8-Based Colorimetric Method for Sensitive and Interference-Free Determination of Glutathione Peroxidase Activity

Abstract Glutathione peroxidase (GPx) is a major antioxidant enzyme that protects cells from oxidative damage, and accurate quantification of GPx is important for biochemical, clinical, and diagnostic research. Current colorimetric and spectrophotometric GPx assays, however, are often limited by low sensitivity, unstable reagents, or interference from biological matrix components. We developed a colorimetric GPx assay based on the tetrazolium salt WST-8. GPx reduces peroxide substrates (ROOH) to water or the corresponding alcohols using reduced glutathione (GSH) as an electron donor, generating oxidized glutathione (GSSG); glutathione reductase then regenerates GSH from GSSG at the expense of NADPH. Unreacted NADPH reduces WST-8 to a stable, water-soluble formazan dye measured at 460 nm, so GPx activity can be calculated indirectly from the extent of NADPH consumption. Assay performance was compared with established DTNB- and UV-based GPx methods. The WST-8-based assay showed high sensitivity, a wide linear range, good reagent stability, and strong resistance to interference from sugars, amino acids, and proteins. Activity values obtained with the new method correlated closely with those from the conventional DTNB assay, confirming its accuracy. The WST-8-GPx assay offers improved sensitivity and interference resistance while remaining operationally simple and compatible with routine microplate instrumentation, making it a practical option for both clinical diagnostics and oxidative-stress research.