Effect of oxidative stress on the structure and function of human serum albumin

Purpose. Human serum albumin (HSA) was mildly oxidized by a metal-catalyzed oxidation system (MCO-HSA), chloramine-T (CT-HSA) or H2O2 (H2O2-HSA), and the effects of these treatments on the structural, drug-binding and esteras e-like properties were studied. Methods. Protein conformation was examined by calorimetric, chromatographic , electrophoretic and spectroscopic techniques. Drug binding was studied by ultrafiltration method, and esterase-like activity was determined using p- nitrophenyl acetate as a substrate. Results. Far-UV and near-UV CD spectra indicated that significant structura l changes had occured as the result of treatment with MCO-HSA and CT-HSA bu t not with H2O2-HSA. However, SDS-PAGE analysis does not provide precise in formation on gross conformational changes such as fragmentation. cross-link ing and SDS-resistant polymerisation. The results of differential scanning calorimetry, the fluorescence of the hydrophobic probe l,1-bis-4-anilino-na phthalene-5,5-sulfonic acid and the elution time from a hydrophobic HPLC co lumn indicated that MCO-HSA and CT-HSA in particular, have a more open stru cture and a higher degree of exposure of hydrophobic areas than unoxidized HSA. In all cases, high-affinity binding of warfarin remained unchanged for all the oxidized HSAs. However, high-affinity binding of ketoprofen to CT- HSA and, especially, MCO-HSA was diminished. In addition, the esterase-like activity of these proteins were all decreased to the same low level. Conclusions. Mild oxidation of HSA has no detectable effect on the binding of drugs to site I in subdomain IIA. In contrast, both the ligand binding p roperty of sire II and the esterase-like activity of oxidized HSAs are decr eased, most probably due to conformational changes in subdomain IIIA.