Amino acids of importance for the antioxidant activity of human serum albumin as revealed by recombinant mutants and genetic variants Makoto Anraku a , Rina Shintomo b , Kazuaki Taguchi a , Ulrich Kragh-Hansen c , Toshiya Kai d , Toru Maruyama b , Masaki Otagiri a,e, ⁎ a Faculty of Pharmaceutical Sciences, Sojo University, 4-22-1 Ikeda, Kumamoto 860-0082, Japan b Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto 862-0973, Japan c Department of Biomedicine, University of Aarhus, DK-8000 Aarhus C, Denmark d Tohoku Nipro Pharmaceutical Corporation, Kagamiishimachi, Iwasegun, Fukushima, Japan e DDS Research Institute, Sojo University, 4-22-1 Ikeda, Kumamoto 860-0082, Japan abstract article info Article history: Received 16 January 2015 Received in revised form 1 May 2015 Accepted 3 May 2015 Available online 30 May 2015 Keywords: Antioxidant Human serum albumin Cysteine-34 Methionine Recombinant mutants Genetic variants Aims: 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 H 2 O 2 as the representative reactive oxygen species and two recombinant mutants and ten genetic variants with single-residue mutations. Main methods: The antioxidant capabilities of the isoforms were registered as their ability to diminish the H 2 O 2 - induced conversion of dihydrorhodamine 123 to rhodamine 123, which can emit fluorescence at 536 nm. Struc- tural properties were examined by circular dichroism and SDS-PAGE. Key findings: Cysteine residues are important for the antioxidant function, but their effect depends on their posi- tion in the protein, with Cys410 N 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, per- haps 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 mu- tants C34S and, in particular, R410C and E505K. Significance: 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. © 2015 Elsevier Inc. All rights reserved. 1. Introduction Primary antioxidant defense systems that consist of low molecular weight antioxidants and antioxidative enzymes such as superoxide dis- mutase, catalase and peroxidases protect living cells and tissues from oxidative damage [10]. However, other proteins can also play an impor- tant role in such protection. In extracellular fluids, a major antioxidant role is performed by serum albumin [4,29]. Specifically, the sulfhydryl group is an important component of the antioxidant potential of albu- min [27,9,26]. However, other amino acids inevitably contribute to the antioxidant potential when exposed to highly reactive radicals, such as the hydroxyl radical, which react with most organic molecules at diffusion-controlled rates [6,24]. Because this aspect of the multifunctional transport protein is not fully understood, we have investigated the antioxidant effect of a se- ries of human serum albumin (HSA) isoforms containing a range of single amino acid substitutions. Because it is a model system, we have studied the quenching effect of the albumins on the oxidation of dihydrorhodamine 123 (DRD) by hydrogen peroxide. The infor- mation gained should be of general relevance to the mechanism of antioxidant protection and should apply to other proteins with anti- oxidant properties that are more difficult to isolate or remain unknown. HSA is synthesized in and secreted from liver cells as a simple pro- tein, i.e., without covalently bound carbohydrate, lipid or prosthetic groups. The single 585 amino acid polypeptide chain forms a heart- shaped molecule (Fig. 1) with three homologous domains (I-III), each of which is composed of two subdomains (A and B) with distinct helical folding patterns connected by flexible loops [7,33]. All but one of the 35 cysteine (Cys) residues are involved in the formation of stabilizing di- sulfide bonds. The remaining residue (Cys34) is often assumed to be Life Sciences 134 (2015) 36–41 ⁎ Corresponding author at: Faculty of Pharmaceutical Sciences, Sojo University, Nishi- ku, 4-22-1 Ikeda, Kumamoto 860-0082, Japan. Tel.: +81 96 326 3887; fax: +81 96 326 5048. E-mail address: otagirim@ph.sojo-u.ac.jp (M. Otagiri). http://dx.doi.org/10.1016/j.lfs.2015.05.010 0024-3205/© 2015 Elsevier Inc. All rights reserved. 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