Validation of the chloramine-T induced oxidation of human serum albumin as a model for oxidative damage in vivo.
OBJECTIVE
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.
Faculty of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto 862-0973, Japan.
Makoto
M
Anraku
Ulrich
U
Kragh-Hansen
Keiichi
K
Kawai
Toru
T
Maruyama
Yasuomi
Y
Yamasaki
Yoshinobu
Y
Takakura
Masaki
M
Otagiri
D000818:Animals; D002700:Chloramines / Q000737:chemistry / Q000633:toxicity*; D004195:Disease Models, Animal; D006801:Humans; D051379:Mice; D009154:Mutation; D018384:Oxidative Stress / Q000187:drug effects* / Q000235:genetics; D012709:Serum Albumin / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics; D014105:Tosyl Compounds / Q000737:chemistry / Q000633:toxicity*
D002700:Chloramines; D012709:Serum Albumin; D014105:Tosyl Compounds; C016300:chloramine-T