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Effect of Oxidative Stress on the Structure and Function of Human Serum Albumin

Abstract

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 esterase–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 structural changes had occured as the result of treatment with MCO–HSA and CT–HSA but not with H2O2–HSA. However, SDS–PAGE analysis does not provide precise information on gross conformational changes such as fragmentation, cross–linking and SDS–resistant polymerisation. The results of differential scanning calorimetry, the fluorescence of the hydrophobic probe 1,1–bis–4–anilino–naphthalene–5,5–sulfonic acid and the elution time from a hydrophobic HPLC column indicated that MCO–HSA and CT–HSA in particular, have a more open structure 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 property of site II and the esterase–like activity of oxidized HSAs are decreased, most probably due to conformational changes in subdomain IIIA.

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Anraku, M., Yamasaki, K., Maruyama, T. et al. Effect of Oxidative Stress on the Structure and Function of Human Serum Albumin. Pharm Res 18, 632–639 (2001). https://doi.org/10.1023/A:1011029226072

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  • human serum albumin
  • oxidative stress
  • structural changes
  • warfarin binding
  • ketoprofen binding
  • esterase–like activity