Introduction: Human serum albumin (HSA) is a critical protein in human blood plasma, which can be highly damaged by oxidative stress. The aim of this study was to analyze modifications of this protein after oxidation using a Fenton system.
Methods: In this 2015 experiment, different ratios of Fenton reagent (Fe2+/H2O2) was incubated with one concentration of human serum albumin (1mg/ml). Hence, HSA was incubated 30 min with various combinations of a Fenton system and quantified oxidation products such as carbonyl groups, fragmentations, degradations, and oxidized free thiol group using reliable techniques. Image and data analysis were carried out using ImageJ software and Excel (version 2007), respectively.
Results: An SDS-PAGE profile showed no cross link and aggregation. However, protein band intensity has decreased to 50% in the highest ratio of H2O2/Fe. Carbonylation assay indicated carbonyl/protein (molc/molp) ratio increased linearly in lower ratios and the values plateau at higher levels of H2O2/Fe 2+. The only free sulfhydryl group on HSA was oxidized in all ratios of the Fenton system.
Conclusion: To sum, the structure of HSA has been changed following treatment with Hydroxyl Radical as the main product of Fenton reaction. These data confirm the antioxidant activity of HSA.
Keywords: Carbonylation assay; Fenton system; HSA; SDS-PAGE.