COMMUNICATION DOI: 10.1002/asia.201301285

Superoxide Dismutase Activity of the Naturally Occurring Human Serum Albumin–Copper Complex without Hydroxyl Radical Formation Ryunosuke Kato,[a] Matofusa Akiyama,[a] Hiroyoshi Kawakami,[b] and Teruyuki Komatsu*[a] Abstract: The superoxide radical anion (O2C) is biologically toxic and contributes to the pathogenesis of various diseases. Here we describe the superoxide dismutase (SOD) activity of human serum albumin (HSA) complexed with a single CuII ion at the N-terminal end (HSA–Cu complex). The structure of this naturally occurring copper-coordinated blood serum protein has been characterized by several physicochemical measurements. The O2C dismutation ability of the HSA–Cu (1:1) complex is almost the same as that of the well-known SOD mimics, such as MnIII-tetrakis(N-methylpyridinium)porphyrin. Interestingly, the HSA–Cu complex does not induce a subsequent Fenton reaction to produce the hydroxyl radical (OHC), which is one of the most harmful reactive oxygen species.

four histidine (His) residues in a distorted square-planar geometry is alternately reduced and oxidized by O2C. CuII þ O2 C  ! CuI þ O2 CuI þ O2 C  þ 2 Hþ ! CuII þ H2 O2 In the human circulatory system, the majority of serum copper is transported by the protein ceruloplasmin (Mw: 151 kDa),[8] and the rest is bound to human serum albumin (HSA, Mw: 66.5 kDa), the most abundant blood plasma protein. HSA consists of three homologous domains I–III, each of which comprises two subdomains, A and B (Figure 1).[9]

Superoxide radical anion (O2C), which is produced in normal metabolic pathways, is one of the main reactive oxygen species (ROS) in biological systems. Superoxide dismutase (SOD) catalyzes the dismutation of O2C with high efficiency[1, 2] and thereby provides an important defense against various diseases in which O2C appears to play a crucial role,[3] such as tissue inflammation and ischemia-reperfusion injury.[4] Furthermore, O2C is known to be a source of highly toxic hydrogen peroxide (H2O2), hydroxyl radical (OHC), and peroxynitrite (ONOO). Excessive production of ROS causes oxidative stress and initiates the pathogenesis of cancers, atherosclerosis, and Alzheimers disease.[5, 6] One of the major forms of SOD is Cu,Zn-SOD, which contains a copper ion as an active site, for example, bovine erythrocyte SOD (Mw: 32.5 kDa).[7] The CuII center coordinated by

Figure 1. Schematic illustrations of the HSA–Cu complex and the CuIIcoordinated N-terminal site (NTS, Asp-Ala-His) (prepared from PDB ID: 1E78 from Ref. [9]).[10]

It is known that HSA possesses two specific binding sites for the copper ion.[11–14] In particular, the N-terminal site (NTS, Asp-Ala-His) captures a single CuII ion with an extremely high binding constant (K  1  1012 m1).[11, 13] Several physicians previously reported that the therapeutic use of HSA brought beneficial outcomes and not only enhanced the copper-ion transport but also reduced myocardial and cerebral ischemia-reperfusion injury.[15, 16] It has therefore been suggested that the tight CuII binding appears to confer HSA some ability to scavenge O2C. Hence, the HSA–Cu complex has been extensively characterized,[11–14] and many copper complexes with similar peptide sequences have been synthesized as SOD mimics.[17, 18] Nevertheless, to the best of our knowledge, the catalytic activity of the HSA–Cu complex has not been evaluated in detail. Here, we report the SOD

[a] R. Kato, Dr. M. Akiyama, Prof. Dr. T. Komatsu Department of Applied Chemistry Faculty of Science and Engineering Chuo University 1-13-27 Kasuga, Bunkyo-ku, Tokyo 112-8551 (Japan) Fax: (+ 81) 3-3817-1910 E-mail: [email protected] [b] Prof. Dr. H. Kawakami Department of Applied Chemistry Tokyo Metropolitan University 1-1 Minami-Osawa, Hachioji-shi, Tokyo 192-0397 (Japan) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/asia.201301285.

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becomes populated by the CuII ion only after saturation of the first site.[11–14] The obtained HSA–Cu (1:1) complex resulted in a single elution peak in the gel permeation chromatogram (Superdex 75 Prep Grade) and only one clear band on a native PAGE gel. Unfortunately, crystal structure analysis could not provide detailed information about the extreme N-terminus of HSA as the terminus is very flexible and does not appear in the electron density map.[9] Next, the SOD activity of the HSA-Cu (1:1) complex was evaluated in phosphate buffer (PB) using the xanthine–(xanthine oxidase)–ferricytochrome c (Cyt c) assay.[19, 20] In the presence of the HSA–Cu complex, the reduction of Cyt c by O2C was significantly inhibited. The IC50 value (the concentration of enzyme necessary to attain 50 % inhibition of Cyt c reduction) of the HSA–Cu complex was determined to be 0.7 mm (Table 1). Under our experimental conditions, the re-

activity of this naturally occurring copper-coordinated blood serum protein. Interestingly, the HSA–Cu complex does not induce a subsequent Fenton reaction to generate OHC, one of the most harmful species in ROS. Quantitative analysis of O2C dismutation activity of the HSA–Cu complex could provide a new insight into the artificial SOD substitute for clinical use. The complexation of CuII ion to HSA was conducted by adding aqueous CuCl2 to a solution of HSA in phosphatebuffered saline (PBS, pH 7.4). The UV/Vis absorption spectral changes in the complexing equilibrium of the CuII ion and HSA are shown in Figure 2. It is remarkable that the

Table 1. Enzymatic activities of SOD mimics. Enzymes[a]

IC50 [mm]

kcat [m1 s1]

Ref.

HSA–Cu Mn-TMPyP

0.7[b] 0.8[b] 0.7[b] 0.52[b] 0.5[d] 0.24[e] 0.03[d]

1.0  107[c] 1.4  107[c] 1.4  107[c] – 1.0  108[d] – 2.3  109[f]

This work This work [19, 25] [23] [21] [20] [21, 24]

Cu2ACHTUNGRE(bpzbiap)Cl3 CuACHTUNGRE(pip)(im)ZnACHTUNGRE(pip)ACHTUNGRE(NO3)3 Cu,ZnACHTUNGRE(bdpi)ACHTUNGRE(CH3CN)2 Cu,Zn-SOD

[a] bpzbiap = 1,5,bis(1-pyrazolyl)-3-[bis(2-imidazolyl)methyl]azapentane; pip = 2-[(2-(pyridyl)ethyliminomethyl]pyridine; im = imidazole; bdpi = 4,5-bis(di(2-pyridylmethyl)aminomethyl)imidazole. [b] In PB solution (pH 7.8, 50 mm) at 25 8C. [c] In HEPES-buffered solution (pH 8.1, 60 mm) at 36 8C. [d] In PB solution (pH 7.8, 45 mm) at 25 8C. [e] In g-collidine-buffered solution (pH 7.77, 50 mm) at 25 8C. [f] In HEPES-buffered solution (pH 7.8, 80 mm) at 21 8C.

Figure 2. Absorbance spectra of HSA upon addition of a solution of CuCl2 in PBS (pH 7.4). Numbers indicate the ratio of [Cu]/ACHTUNGRE[HSA] (mol/ mol). Inset: Absorbance (at 525 nm and 650 nm) as a function of [Cu]/ACHTUNGTRENUG[HSA] (mol/mol).

broad peak (lmax : 525 nm) ascribed to the CuII binding to NTS increased upon the addition of CuII until the [Cu]/ACHTUNGRE[HSA] ratio reached 1.0.[13] At higher molecular ratios ([Cu]/ACHTUNGTRENUG[HSA] = 1.25!2), the absorbance at 525 nm was almost saturated (Figure 2 inset). By contrast, a specific absorbance peak (lmax : 650 nm) of the CuII binding to the multi-metal binding site (MBS), which is suggested to include His-67, His-247, Asn-99, and Asp-249 at the cleft between the subdomain IA and IIA,[12–14] subsequently increased. Valko et al. demonstrated that the CuII ion is bound within the NTS of HSA by the coordination with the initial three amino acid residues involving the a-NH2 terminus, two deprotonated amide nitrogens (Ala-2 and His-3), and the imidazole nitrogen of His-3 (Figure 1).[12] The ESR spectrum of the HSA–Cu complex ([Cu]/ACHTUNGRE[HSA] = 0.5–1.0) recorded by us at 150 8C corresponded to a single paramagnetic species (Figure S1 in the Supporting Information) and is consistent with that of a square planar configuration of four nitrogens around the copper ion. The ESR parameters (g// = 2.178, g ? = 2.052, A// = 216 G) are very close to the values reported by Valko et al. These results supported the notion that the CuII ion first binds strongly to the NTS of HSA, forming a 1:1 complex. The difference in affinities between the two binding sites is so high that the second site

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duction of Cyt c was not suppressed by HSA alone. For that reason, any SOD activity of the albumin protein is excluded. The IC50 of the HSA–Cu complex is still larger than the value of native Cu,Zn-SOD,[21] but of the same order of magnitude as those reported for the best synthetic SOD mimics, for instance, cationic MnIII-tetrakis(N-methylpyridinium)porphyrin (Mn-TMPyP) and imidazolate-bridged dinuclear complexes (Table 1).[19–23] We therefore concluded that the HSA–Cu complex possesses a strong capability to catalyze the dismutation of O2C. In addition to the indirect assay (Cyt c method), a direct measurement of the O2C dismutation kinetics was carried out using stopped-flow spectroscopy.[24, 25] A solution of the HSA–Cu complex in HEPES buffer was rapidly mixed with a solution of O2C in DMSO, and the time-resolved absorption changes at 245 nm accompanying the O2C quenching were monitored. The decays were composed of single-exponential profiles. The determined reaction rate constant (kcat) of 1.0  107 m1 s1 was almost the same as that of MnTMPyP (Table 1).[25] This result also revealed that the HSA– Cu complex is a superior scavenger for O2C. The native Cu,Zn-SOD reacts with O2C at a nearly diffusion-limited rate (2.3  109 m1 s1).[24] Getzoff et al. showed that the positively charged channel above the CuII site in SOD promotes

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the attractive interaction between the enzyme and the negatively charged O2C.[26] This long-range guidance effect induces the enhancement of the diffusion of O2C. Site-directed mutagenesis to insert positively charged amino acids around the NTS of HSA[27] may provide an enhancement of the SOD activity of the HSA–Cu complex. From the viewpoint of developing an artificial SOD substitute as a pharmaceutical agent, the copper complex must fulfill one critical requirement, namely lacking a Fenton reaction-related toxicity. In general, free iron and copper ions catalyze the Fenton reaction to convert H2O2 into OHC. Notably, the CuII ion can generate OHC 60 times faster than FeIII.[28] If a trace amount of CuII ion is released from the NTS of HSA, it must participate in the Fenton reaction. We therefore investigated the OHC formation ability of the HSA–Cu complex using aminophenyl fluorescein (APF), which is useful fluorescence probe to detect OHC selectively.[29] APF immediately reacts with OHC, yielding a strongly fluorescent compound, fluorescein, whereas it does not react with O2C and H2O2. Addition of H2O2 to a solution of CuCl2 in PB containing APF elicited a significant increase in the fluorescence intensity (lem : 515 nm) (Figure 3). Howev-

In conclusion, the HSA–Cu complex shows a high activity of O2C disproportionation without unfavorable OHC formation. It became apparent that its catalytic performance is almost identical to that of a well-known SOD mimic, for instance, cationic Mn-porphyrin. The high current interest in synthesizing SOD mimics as potential therapeutic agents continues to grow, but unresolved issues still remain, such as half-life time, antigenicity, instability, and cost. Elimination of O2C by the HSA-based SOD would be of tremendous medical importance. Our results will serve as a trigger to engender the new field of artificial SOD substitutes. Currently, recombinant HSA is manufactured on an industrial scale,[30] which enables us to produce the synthetic HSA–Cu complex for practical applications.

Experimental Section Preparations of the HSA–Cu Complex Typically, an aqueous solution of CuCl2 (2.0 mm, 0.1 mL) was slowly added to a solution of HSA (0.1 mm), in 1.9 mL phosphate-buffered saline (PBS, pH 7.4), and the mixture was incubated for at least 5 min with rotation in the dark at 25 8C, yielding the HSA–Cu complex (Cu/ HSA = 1.0, mol/mol). By this procedure, several HSA–Cu complexes (Cu/HSA = 0.25–2.5, mol/mol) in different buffer solutions were prepared. The resulting samples were analyzed by native PAGE to confirm the protein integrity. The protein concentration was assayed by using the Pierce 660 nm Protein Assay Kit (Thermo Fisher Scientific K. K.). Xanthine–XOD–Cyt c Assay The O2C was generated in situ by the xanthine–XOD reaction system, and the SOD activity of the HSA–Cu complex was evaluated using the Cyt c reduction technique.[19, 20] To a solution of the HSA–Cu complex in 3.0 mL phosphate buffer (PB, 50 mm, pH 7.8) containing Cyt c (10 mm), xanthine (50 mm), and catalase (500 U mL1) in an optical quartz cuvette with a path length of 10 mm, an amount of XOD sufficient to give an initial rate of DA550 = 0.025 min1 (without HSA–Cu complex; approximately 2.0 mU mL1) was injected at 25 8C. Immediately after the addition of XOD, the absorption at 550 nm was monitored continuously at 25 8C. The increase in the absorbance was almost linear for at least 4 min, and the initial rate constant (vi) at various concentrations of the HSA–Cu complex was determined from the data in this period of time. The IC50 value is defined as the concentration required for 50 % inhibition of Cyt c reduction. Analogous experiments were carried out with Mn-TMPyP.

Figure 3. Time dependence of the fluorescence intensity of fluorescein (lem : 515 nm) generated from APF in a solution of the HSA–Cu complex in 0.1 m PB (pH 7.4) upon addition of H2O2. [APF] = 10 mm, [Cu] = 0.1 mm, [H2O2] = 1 mm, lex : 490 nm.

Stopped-Flow Measurements The rate constant (kcat) of the O2C dismutation was measured by using an SX.18MV stopped-flow spectrometer (Applied Photophysics Ltd., UK).[24, 25] A solution of O2C in DMSO (~ 2 mm, 5 mL), , which was prepared by dissolving KO2 in DMSO,[24, 25] was rapidly mixed with a solution of the HSA–Cu complex (2 mm, Cu/HSA = 1, mol/mol) in 0.1 mL HEPES buffer (60 mm, pH 8.1) at 36 8C. The decay curve accompanying the O2C dismutation was spectrophotometrically monitored at 245 nm and was fitted to a single exponential, giving the apparent rate constant (kapp). The kcat value was then determined from the slope of a linear plot of kapp versus the concentration of the HSA–Cu complex (0–10 mm). Analogous experiments were carried out with Mn-TMPyP.

er, no fluorescence was observed in case of the HSA–Cu complex. We thus reasoned that the CuII ion is not dissociated from the NTS of HSA and also that HSA–Cu itself does not produce OHC via a Fenton chemistry mechanism. In biological processes, free CuII ions can be reduced by various reducing agents such as ascorbate. If the HSA–Cu complex is reduced by ascorbate, the resulting CuI protein may enhance ROS-generating reactions. In fact, upon addition of ascorbate to the solution of CuCl2 containing APF under air, the fluorescence intensity (lem : 515 nm) gradually increased (Figure S2 in the Supporting Information). On the other hand, only negligible fluorescence was observed in case of the HSA–Cu complex. These observations demonstrated that HSA–Cu does not generate OHC from O2 in the presence of ascorbate.

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Assay of Hydroxyl Radical Formation To a solution of the HSA–Cu complex (0.11 mm) and 2-[6-(4’-amino)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (aminophenyl fluorescein; APF, 11 mm) in 0.1 m PB (pH 7.4, 1.35 mL) containing 0.22 % DMF, a solution of H2O2 (10 mm) in 0.1 m PB (pH 7.4, 0.15 mL) was added, and the fluorescence emission at 515 nm was monitored under air at 25 8C (lExc :

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490 nm). APF reacts with OHC, yielding the strongly fluorescent compound fluorescein, but does not react with O2C and H2O2. Thus, the fluorescence intensity depends on the concentration of OHC in the solution.[29] Furthermore, APF is resistant to light-induced autoxidation. As a positive control, the same experiments were carried out using CuCl2.

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In additional experiments, to a solution of the HSA–Cu complex (11 mm) in 0.1 m PB (pH 7.4, 1.35 mL) containing APF (11 mm) and 0.22 % DMF, a solution of ascorbate (3 mm) in 0.1 m PB (pH 7.4, 0.15 mL) was added, and the fluorescence emission at 515 nm was observed under air at 25 8C (lExc : 490 nm). As a positive control, the same experiments were carried out using CuCl2.

Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research on Innovative Area “Coordination Programming” (Area 2107, No. 21108013) from MEXT Japan, A-STEP (No. AS242Z01033P) from JST, and a Joint Research Grant from the Institute of Science and Engineering, Chuo University.

Keywords: copper complex · enzymes · human serum albumin · proteins · superoxide dismutase

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Received: September 21, 2013 Published online: November 12, 2013

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