title: Amino acids of importance for the antioxidant activity of human serum albumin as revealed by recombinant mutants and genetic variants.
abstract: OBJECTIVE
  To determine molecular information about the antioxidant properties of human serum albumin, which is an important extracellular antioxidant. To obtain this information, we studied this function of the protein by using H2O2 as the representative reactive oxygen species and two recombinant mutants and ten genetic variants with single-residue mutations.
  
  
  METHODS
  The antioxidant capabilities of the isoforms were registered as their ability to diminish the H2O2-induced conversion of dihydrorhodamine 123 to rhodamine 123, which can emit fluorescence at 536 nm. Structural properties were examined by circular dichroism and SDS-PAGE.
  
  
  RESULTS
  Cysteine residues are important for the antioxidant function, but their effect depends on their position in the protein, with Cys410 > Cys34 ~ Cys169 (when not involved in forming a disulfide bond). Likewise, the substitution of a glutamic acid at position 122 or 541, but not at 240 or 560, improves the antioxidant effect, perhaps by making the methionine residues in their vicinity, Met123 and Met548, respectively, more accessible for the oxidant. A lysine at position 505, but not at 82 or 570, decreases the oxidative effect. Finally, the mutations D269G and K276N had no effect. In certain cases, albumin acts as a sacrificial antioxidant, as in the case of the mutants C34S and, in particular, R410C and E505K.
  
  
  CONCLUSIONS
  The information gained is of protein chemical relevance, but it may also be helpful in understanding the function of proteins that act as antioxidants in biological systems subjected to oxidative stress in conditions such as inflammation and aging.
authors.affiliation: Faculty of Pharmaceutical Sciences, Sojo University, 4-22-1 Ikeda, Kumamoto 860-0082, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Anraku
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto 862-0973, Japan.
authors.forename: Rina
authors.initials: R
authors.lastname: Shintomo
authors.affiliation: Faculty of Pharmaceutical Sciences, Sojo University, 4-22-1 Ikeda, Kumamoto 860-0082, Japan.
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Taguchi
authors.affiliation: Department of Biomedicine, University of Aarhus, DK-8000 Aarhus C, Denmark.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Kragh-Hansen
authors.affiliation: Tohoku Nipro Pharmaceutical Corporation, Kagamiishimachi, Iwasegun, Fukushima, Japan.
authors.forename: Toshiya
authors.initials: T
authors.lastname: Kai
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto 862-0973, Japan.
authors.forename: Toru
authors.initials: T
authors.lastname: Maruyama
authors.affiliation: Faculty of Pharmaceutical Sciences, Sojo University, 4-22-1 Ikeda, Kumamoto 860-0082, Japan; DDS Research Institute, Sojo University, 4-22-1 Ikeda, Kumamoto 860-0082, Japan. Electronic address: otagirim@ph.sojo-u.ac.jp.
authors.forename: Masaki
authors.initials: M
authors.lastname: Otagiri
mesh_terms: D019943:Amino Acid Substitution; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D020125:Mutation, Missense; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012235:Rhodamines / Q000737:chemistry; D012709:Serum Albumin / Q000737:chemistry* / Q000235:genetics; D013050:Spectrometry, Fluorescence
chemical_list: D011994:Recombinant Proteins; D012235:Rhodamines; D012709:Serum Albumin; C058319:dihydrorhodamine 123; D006861:Hydrogen Peroxide
keywords: Antioxidant; Cysteine-34; Genetic variants; Human serum albumin; Methionine; Recombinant mutants