Validation of the chloramine-T induced oxidation of human serum albumin as a model for oxidative damage in vivo

Abstract

Purpose: The validity of using chloramine-T as a model compound for mimicing oxidative stress was examined using human serum albumin (HSA) as a model. Important sites of oxidation were studied by mild treatment with chloramine-T and by mutating 34Cys for a serine (C34S).

Methods: High-performance liquid chromatography (HPLC) combined with fluorescence detection to confirm the validity of chloramine-T as an oxidizing agent was used. Oxidized amino acid residues were detected by reaction with 5,5'-dithiobis(2-nitro benzoic acid), digestion with cyanogen bromide, followed by capillary electrophoresis. Protein conformation was examined by spectroscopic techniques.

Results: From the HPLC analysis of human serum, the validity of using chloramine-T as an oxidizing agent was confirmed. At low chloramine-T concentrations (CT0.1-HSA, CT1-HSA), 34Cys and Met residues were oxidized, at medium concentrations (CT10-HSA), the tryptophan residue also appeared to be oxidized, and at the highest concentration (CT50-HSA), the net charge of Site II of HSA was found to be more negative. The two highest levels of oxidation of HSA (CT10-HSA, CT50-HSA) resulted in conformational changes with an increased exposure of hydrophobic regions, decreased high-affinity bindings of warfarin and ketoprofen and a reduced esterase-like activity. The latter protein also has a shorter plasma half-life and an increased liver clearance.

Conclusions: We succeeded in imitating oxidative damage to HSA using chloramine-T and the findings show that Site II is more affected than Site I and 34Cys, when HSA is exposed to oxidative stress.