- Email: [email protected]
Study on the interaction of copper–zinc superoxide dismutase with aluminum ions by electrochemical and fluorescent method
Spectrochimica Acta Part A 65 (2006) 896–900
Study on the interaction of copper–zinc superoxide dismutase with aluminum ions by electrochemical and fluorescent method Junwei Di a,b , Kaian Yao c , Weiying Han a , Shuping Bi a,∗ a
Department of Chemistry, State Key Laboratory of Coordination Chemistry of China, Key Laboratory of MOE for Life Science, Nanjing University, Nanjing 210093, PR China b Department of Chemistry, Suzhou University, Suzhou 215006, PR China c Department of Environmental Science, Nanjing University, Nanjing 210093, PR China Received 19 October 2005; accepted 20 January 2006
Abstract The interaction of superoxide dismutase (SOD) with aluminum (Al) ions was investigated by cyclic voltammetry, fluorescence spectroscopy and synchronous fluorescence spectroscopy. The electrochemical activity of the SOD enzyme electrode was inhibited irreversibly by the addition of Al. Meanwhile, the static fluorescence quenching mechanism further revealed the existing of molecular complex of SOD with Al3+ . The association constant was obtained from Lineweaver-Burk plot. The experimental results of voltammetry and fluorescence spectroscopy indicated that the conformation of SOD molecule was altered by the formation of Al–SOD complex. It may influence the activity of SOD enzyme since the optimum action of SOD depends upon a particular configuration of electrostatic charges in the enzyme molecule. © 2006 Elsevier B.V. All rights reserved. Keywords: Aluminum ions; Superoxide dismutase (SOD); Cyclic voltammetry; Fluorescence spectroscopy
1. Introduction Aluminum (Al) is an abundant metal found in the earth’s crust and has been attributed no role by nature in living processes. Over decades, due to the advent of technology and the introduction of a variety of chemicals into the atmosphere, acid rains have led to the mobilization of this metal. Therefore, the toxicity of Al has been the subject of much research in the past few decades [1–3]. It has been suggested that elevated levels of Al in brain tissue correlate with occurrence of neurological brain disorders such as dialysis dementia, Parkinson’s disease and Alzheimer’s disease. The exact mechanism of Al toxicity is not known but there is considerable evidence that shows the metal’s capacity to exacerbate the generation of reactive oxygen species (ROS) despite the fact that Al is a trivalent cation incapable of redox changes [4–7]. We have reported that aluminum ions may increase oxidative stress by means of enhancing the melanin formation [8,9]. ROS such as superoxide anion (O2 •− ), hydrogen peroxide (H2 O2 ) and the hydroxyl radical (• OH) are generated in
∗
Corresponding author. Tel.: +86 25 86205840; fax: +86 25 83317761. E-mail address: [email protected] (S. Bi).
1386-1425/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2006.01.026
vivo from the incomplete reaction of oxygen during aerobic metabolism or from exposure to environmental agents such as radiation, redox cycling agents or stimulated host phagocytes [10–14]. These oxygen species can cause widespread damage to biological macromolecules leading to lipid peroxidation, protein oxidation, enzyme inactivation, DNA base modifications and DNA strand breaks [13]. In man, an antioxidant system is present which protects cell from those reactive species. This system consists of the antioxidant enzymes, such as superoxide dismutases (SOD), which catalyze the dismutation of O2 •− to H2 O2 and O2 , catalase (CAT) and glutathione peroxidase (GSHPx) which remove hydrogen peroxide [5]. The damage brought about by oxidative stress is expected to be exacerbated if the antioxidant enzymes themselves are damaged and inactivated by such events. CAT and GSH-Px have been shown to be inhibited by Al3+ [4,5,14]. However, Al has been controversially reported to alter SOD activity, such as no significant inhibitory effect, by inhibiting in various experimental models, as well as in uremic subjects in experimental animals or activating enzyme activity by aluminum phosphide [5,14–17]. Therefore, it is necessary to investigate the interaction of Al with SOD, which will unveil the mechanism of Al-induced alteration of SOD activity. Investigation of direct electron transfer between enzyme and electrode surface can not only establish a foundation
J. Di et al. / Spectrochimica Acta Part A 65 (2006) 896–900
897
for fabrication biosensors but also provide a good model for mechanistic studies of electron transfer between enzymes in biological systems [18–20]. Recently, we have realized the direct electron transfer of SOD in silica sol–gel thin film on gold electrode surface without any mediators or promoters [21]. Fluorescence spectroscopy is another effective method of study on protein conformation in aqueous solution [22]. In the present work, the interaction of SOD with aluminum ions was investigated by electrochemical method based on SOD enzyme electrode and fluorescence spectrum techniques. 2. Experimental 2.1. Reagents SOD 3200 U mg−1 (EC 1.15.11) was obtained from Suzhou University (Suzhou, China). An 1000 U ml−1 SOD stock solution was stored at 4 ◦ C. Aluminum stock solution (0.02 mol l−1 ) was prepared by KAl(SO4 )2 ·12H2 O and adjusted to pH 3. A 0.1% PVA stock solution was prepared by dissolving 0.1 g polyvinyl alcohol (PVA-124, average degrees of polymerization were 2400–2500, Shanghai Chemical Reagent Plant imported from Japan) in 100 ml water under heating to near boiling. NaAc and HAc buffer solution (pH 5.0) was used. All other chemicals were of analytical grade and the twice-distilled water was used.
Fig. 1. The cyclic voltammograms at the SOD enzyme electrode in NaAc–HAc (pH 5.0) buffer solution. Addition of Al3+ concentration in the buffer solution: (a) 0, (b) 0.02, (c) 0.05, (d) 0.1, (e) 0.2 and (f) 0.5 mmol l−1 . Scan rate: 100 mV s−1 .
2.2. Preparation of the SOD enzyme electrode and voltammetric experiments
The direct electron transfer of SOD at a bare gold electrode is very slow and thus it has not been observed actually. It could be greatly enhanced by promoters, such as cysteine, 3-mercaptopropionic acid and bis(4-pyridyl)disulfide [23–25]. Recently, we have realized the direct electron transfer of SOD embedded in the thin silica sol–gel film at the gold electrode without any mediators or promoters and maintained good SOD activity [21]. On the other hand, the catalytic rate of native SOD is independent of pH over the range from 5 to 9.5 [26]. When SOD was embedded by silica sol–gel on the gold electrode surface, one pair of well-shaped voltammetric peaks was
The electrochemical measurements were made with standard three-electrode potentiostats (Model 820a Electrochemical Workstation, CH Instruments Inc., Shanghai, China). A threeelectrode voltammetric system consisted of a SOD enzyme electrode, a platinum counter electrode and a saturated calomel reference electrode (SCE). The preparation of silica colloidal sol and the SOD enzyme electrode were described previously [21]. The silicic acid solution was prepared by Na2 SiO3 ·9H2 O and the cation exchange process. The gold electrode (3 mm in diameter, made in our laboratory) was polished on abrasive paper, sonicated in ethanol and twice-distilled water and pretreated electrochemically in 0.05 mol l−1 H2 SO4 solution, respectively. The gold electrode was then dipped in the mixed solution containing 0.25 ml silica colloidal sol, 0.15 ml 0.1% PVA, 0.25 ml 1000 U ml−1 SOD per ml. Finally, the electrode was stored for 2–3 h at room temperature. The enzyme electrodes were stored at room temperature (25–35 ◦ C) with moisture-conditioned in a sealed container using a cup of water when not in use. The voltammetric experiments were carried out at SOD enzyme electrode in 10 ml 0.1 mol l−1 NaAc–HAc buffer solution. The next measurement was carried out after the solution was stirred by magnetic stirring for 1 min.
400 V. The excitation and emission slits were 10 nm. All experiments were carried out at room temperature. 3. Results and discussion 3.1. The effect of Al3+ on the electrochemical process of SOD
2.3. Fluorometric experiments Fluorescence spectrums were carried out by means of a F2500 fluorophotometer (Hitachi). The xenon lamp was used at
Fig. 2. The relationship of cyclic voltammetric currents and Al3+ concentration in buffer solution. Curve a: anodic current; curve c: cathodic current. Other experimental conditions as in Fig. 1.
898
J. Di et al. / Spectrochimica Acta Part A 65 (2006) 896–900
Fig. 3. The excitation fluorescence spectrum of SOD (A) and effect of Al3+ on fluorescence spectra of SOD (λex = 280 nm) (B) in NaAc–HAc (pH 5.0) buffer solution. The concentration of SOD: 4 mol l−1 . The concentration of Al3+ : (a) 0, (b) 0.02, (c) 0.04, (d) 0.08, (e) 0.2, (f) 0.4, (g) 0.8 and (h) 1.0 mmol l−1 .
observed in 0.1 mol l−1 NaAc–HAc buffer solution (Fig. 1, curve a). The formal potential E◦ , estimated as (Epa + Epc )/2, was about 0.20 V versus SCE. The current ratio of the cathodic peak current to the anodic one (Ipc /Ipa = 0.7 at 100 mV s−1 ) and the separation between the anodic and cathodic peak potentials (Ep = Epa − Epc = 0.17 V at 100 mV s−1 ) indicated that the electrode process of SOD was quasi-reversible. The electrochemical behavior of SOD changed when Al3+ ions were added in the buffer solution. With the increase of the concentration of Al3+ , both the cathodic and anodic peak currents were decreasing (Fig. 2) and the difference of the anodic and cathodic peak potential was increasing (Fig. 1). These indicated that Al3+ inhibited the electrochemical activity of SOD. If the enzyme electrode was pretreated by EDTA or citrate solution, the electrochemical
Fig. 4. Lineweaver-Burk plot of Al3+ on SOD.
activity of the SOD enzyme electrode did not get back. This indicates that the effect of Al3+ on the electrochemical activity is irreversible and permanent, which may result from the combination of the SOD molecular residues with Al ions and the conformational changes of SOD molecules. 3.2. The effect of Al3+ on the fluorescence spectrum of SOD Fluorescence spectroscopy is an effective method of study on protein conformation in aqueous solution [22]. Fig. 3 shows the fluorescence spectrum of SOD. The maximum excitation wavelength was 280 nm. The emission spectrum exhibited a peak at 314 nm and a shoulder-peak at about 340 nm. As shown in Fig. 3B, addition of Al3+ ions in SOD solution resulted in quenching of the fluorescence of SOD although the maximum emission wavelength did not alter in the emission spectrum.
Fig. 5. Double-log plot of Al3+ on SOD.
J. Di et al. / Spectrochimica Acta Part A 65 (2006) 896–900
899
Fig. 6. Synchronous fluorescence spectra of SOD in NaAc–HAc (pH 5.0) buffer solution at the wavelength interval of 20 nm (A) and 80 nm (B). (—) Before; (···) after addition of 1 mmol l−1 Al3+ .
Considering the electrochemical investigation, the fluorescence quenching effect resulted from the formation of a nonfluorescent Al–SOD complex, which was static quenching. According to Lineweaver-Burk equation [27,28]: (F0 − F )−1 = F0−1 + KD F0−1 [Al]−1 , the disassociation constant KD was obtained from the slope and intercept of the line (R = 0.9954) in Fig. 4. KD was 1.1 × 10−4 and KA was 9.1 × 103 (KA = 1/KD ). Supposing that there have n sites for Al3+ in the Al–SOD complex, this can be written as: nAl3+ + SOD = Aln SOD. The complex is non-fluorescent. Therefore, we obtained the following equation [28,29]: log(F0 − F)/F = log K + nlog[Al]. From double-log plot of Al3+ on SOD (Fig. 5), we obtained that n was 0.47 from the linear slope. This indicates that one molecule combines with Al3+ at least two ligand sites since Al(III) has generally six ligand sites. 3.3. The effect of Al3+ on the synchronous fluorescence spectrum of SOD Fig. 6A is synchronous fluorescence spectra of SOD at wavelength interval 20 nm at which the spectra are mainly contributed by tyrosine residues in SOD molecules [30,31]. There are two peaks in the spectrum. The main peak is located at 281 nm and the second peak is at 307 nm. After addition of Al3+ in the SOD solution, the peak positions did not shift but the fluorescence intensity was decreased (Fig. 6A, curve b). Fig. 6B is the synchronous fluorescence of SOD at wavelength interval 80 nm at which the spectra are contributed mainly by tryptophan residues in SOD molecules [30,31]. The main peak is located at 286 nm and the small shoulder-peak is at about 334 nm. After addition of Al3+ in the SOD solution, the fluorescence intensity decreased for the former peak and increase for the latter (Fig. 6B, curve b). The above results demonstrated that the tyrosine and tryptophan residues were influenced and
the conformation of SOD was altered because of Al–SOD complex formation. 4. Conclusion Getzoff et al. have proposed that the optimum action of Cu–Zn SOD depends upon a particular configuration of electrostatic changes in the enzyme molecule through substrate (O2 •− ) guidance and charge complementary [32]. Sequenceconserved residues create an extensive electrostatic field that directs the negatively charged O2 •− to the highly positive catalytic binding site at the bottom of the active-site channel. Swain and Chainy [14] proposed that Al3+ possibly alters SOD activity by disturbing the required electrostatic field because it has small ionic radius and strong electric charges. Our experimental results demonstrated that Al3+ ions could combine with SOD to form complex whose associative constant KA is 9.1 × 103 . The changes of electrochemical activity and fluorescent characteristic reflected a conformational change of the SOD molecule. Therefore, it can be concluded that Al3+ ions may induce the irreversible conformational changes of SOD molecules and influence the activity of the enzyme. Acknowledgements This project is supported by the National Natural Science Foundation of China (No. 20575025), Research Founding from MOE for Ph.D. Program (20050284030), Jiangsu Natural Science Foundation (BK2005209) and Analytical Measurement Funding of Nanjing University. The authors would like to thank Professors H.Y. Chen and X.Z. You for their help and encourages. References [1] G. Berthon, Coordin. Chem. Rev. 149 (1996) 241.
900
J. Di et al. / Spectrochimica Acta Part A 65 (2006) 896–900
[2] T.E. Lewis, Chemistry and Toxicology of Aluminum, Lewis Publishers Inc., Michigan, 1989, pp. 290–298. [3] R.J.P. Williams, Coordin. Chem. Rev. 228 (2002) 93. [4] C. Exley, Aluminum and Alzheimer’s Disease: The Science that Describes the Link, Elsevier Press, London, 2001, pp. 311–321. [5] P. Zatta, T. Kiss, M. Suwalsky, G. Berthon, Coordin. Chem. Rev. 228 (2002) 271. [6] C. Exley, J. Inorg. Biochem. 76 (1999) 133. [7] J.M.C. Gutteridge, G.J. Quinlan, I. Clark, B. Halliwell, Biochim. Biophys. Acta 835 (1985) 441. [8] J. Di, S. Bi, Spctrochim. Acta A 59 (2003) 1689. [9] J. Di, S. Bi, Spctrochim. Acta A 59 (2003) 3075. [10] B. Halliwell, J.M.C. Gutteridge, Biochem. J. 219 (1984) 1. [11] F. Auch`ere, F. Rusnak, J. Biol. Inorg. Chem. 7 (2002) 664. [12] J.P. Kehrer, Toxicology 149 (2000) 43. [13] S.Y. Kim, O.J. Kwon, J. Park, Biochimie 83 (2001) 437. [14] C. Swain, G.B.N. Chainy, Mol. Cell. Biochem. 187 (1998) 163. [15] F. Atienzar, D. Desor, D. Burnel, J.M. Keller, P. Lehr, P. Vasseur, Biol. Trace Element Res. 65 (1) (1998) 19. [16] C. Swain, G.B.N. Chainy, J. Trace Elements Med. Biol. 11 (2) (1997) 77. [17] M.A. Serra, V. Barassi, C. Canavese, E. Sabbioni, Biol. Trace Element Res. 31 (1991) 79.
[18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32]
F.A. Armstrong, G.S. Wilson, Electrochim. Acta 45 (2000) 2623. W. Zhang, G. Li, Anal. Sci. 20 (2004) 603. H. Liu, D. Pang, Prog. Chem. 11 (2002) 425. J. Di, S. Bi, M. Zhang, Biosens. Bioelectron. 19 (2004) 1479. S. Wang, X. Xu, Q. Liu, Y. Xie, Prog. Chem. (Chinese) 13 (2001) 257. Y. Tian, M. Shioda, S. Kasahara, T. Okajima, L. Mao, T. Hisabori, T. Ohsaka, Biochim. Biophys. Acta 1569 (2002) 151. B. Ge, F.W. Scheller, F. Lisdat, Biosens. Bioelectron. 18 (2003) 295. X. Wu, X. Meng, Z. Wang, Z. Zhang, Bioelectrochem. Bioenerg. 48 (1999) 227. J.A. Tainer, E.D. Getzoff, J.S. Richardson, D.C. Richardson, Nature 306 (1983) 284. M. Yang, P. Yang, L. Zhang, Chin. Sci. Bull. 39 (1994) 31. L. Sheng, S. Liu, J. Ye, X. Xu, Y. Xie, Q. Liu, Chin. J. Inorg. Chem. 19 (2003) 191. Y. Zhang, G. Zhang, Y. Wang, J. Lu, J. Anal. Sci. 16 (2000) 445. X. Du, R. Li, K. Wang, Chin. Sci. Bull. 46 (2001) 394. J. Chou, X. Qu, T. Lu, Y. Wu, Spectrosc. Spectr. Anal. 17 (1997) 108. E.D. Getzoff, J.A. Tainer, P.K. Weiner, P.A. Kollman, J.S. Rchardson, D.C. Richardson, Nature 306 (1983) 287.
Study on the interaction of copper–zinc superoxide dismutase with aluminum ions by electrochemical and fluorescent method Junwei Di a,b , Kaian Yao c , Weiying Han a , Shuping Bi a,∗ a
Department of Chemistry, State Key Laboratory of Coordination Chemistry of China, Key Laboratory of MOE for Life Science, Nanjing University, Nanjing 210093, PR China b Department of Chemistry, Suzhou University, Suzhou 215006, PR China c Department of Environmental Science, Nanjing University, Nanjing 210093, PR China Received 19 October 2005; accepted 20 January 2006
Abstract The interaction of superoxide dismutase (SOD) with aluminum (Al) ions was investigated by cyclic voltammetry, fluorescence spectroscopy and synchronous fluorescence spectroscopy. The electrochemical activity of the SOD enzyme electrode was inhibited irreversibly by the addition of Al. Meanwhile, the static fluorescence quenching mechanism further revealed the existing of molecular complex of SOD with Al3+ . The association constant was obtained from Lineweaver-Burk plot. The experimental results of voltammetry and fluorescence spectroscopy indicated that the conformation of SOD molecule was altered by the formation of Al–SOD complex. It may influence the activity of SOD enzyme since the optimum action of SOD depends upon a particular configuration of electrostatic charges in the enzyme molecule. © 2006 Elsevier B.V. All rights reserved. Keywords: Aluminum ions; Superoxide dismutase (SOD); Cyclic voltammetry; Fluorescence spectroscopy
1. Introduction Aluminum (Al) is an abundant metal found in the earth’s crust and has been attributed no role by nature in living processes. Over decades, due to the advent of technology and the introduction of a variety of chemicals into the atmosphere, acid rains have led to the mobilization of this metal. Therefore, the toxicity of Al has been the subject of much research in the past few decades [1–3]. It has been suggested that elevated levels of Al in brain tissue correlate with occurrence of neurological brain disorders such as dialysis dementia, Parkinson’s disease and Alzheimer’s disease. The exact mechanism of Al toxicity is not known but there is considerable evidence that shows the metal’s capacity to exacerbate the generation of reactive oxygen species (ROS) despite the fact that Al is a trivalent cation incapable of redox changes [4–7]. We have reported that aluminum ions may increase oxidative stress by means of enhancing the melanin formation [8,9]. ROS such as superoxide anion (O2 •− ), hydrogen peroxide (H2 O2 ) and the hydroxyl radical (• OH) are generated in
∗
Corresponding author. Tel.: +86 25 86205840; fax: +86 25 83317761. E-mail address: [email protected] (S. Bi).
1386-1425/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2006.01.026
vivo from the incomplete reaction of oxygen during aerobic metabolism or from exposure to environmental agents such as radiation, redox cycling agents or stimulated host phagocytes [10–14]. These oxygen species can cause widespread damage to biological macromolecules leading to lipid peroxidation, protein oxidation, enzyme inactivation, DNA base modifications and DNA strand breaks [13]. In man, an antioxidant system is present which protects cell from those reactive species. This system consists of the antioxidant enzymes, such as superoxide dismutases (SOD), which catalyze the dismutation of O2 •− to H2 O2 and O2 , catalase (CAT) and glutathione peroxidase (GSHPx) which remove hydrogen peroxide [5]. The damage brought about by oxidative stress is expected to be exacerbated if the antioxidant enzymes themselves are damaged and inactivated by such events. CAT and GSH-Px have been shown to be inhibited by Al3+ [4,5,14]. However, Al has been controversially reported to alter SOD activity, such as no significant inhibitory effect, by inhibiting in various experimental models, as well as in uremic subjects in experimental animals or activating enzyme activity by aluminum phosphide [5,14–17]. Therefore, it is necessary to investigate the interaction of Al with SOD, which will unveil the mechanism of Al-induced alteration of SOD activity. Investigation of direct electron transfer between enzyme and electrode surface can not only establish a foundation
J. Di et al. / Spectrochimica Acta Part A 65 (2006) 896–900
897
for fabrication biosensors but also provide a good model for mechanistic studies of electron transfer between enzymes in biological systems [18–20]. Recently, we have realized the direct electron transfer of SOD in silica sol–gel thin film on gold electrode surface without any mediators or promoters [21]. Fluorescence spectroscopy is another effective method of study on protein conformation in aqueous solution [22]. In the present work, the interaction of SOD with aluminum ions was investigated by electrochemical method based on SOD enzyme electrode and fluorescence spectrum techniques. 2. Experimental 2.1. Reagents SOD 3200 U mg−1 (EC 1.15.11) was obtained from Suzhou University (Suzhou, China). An 1000 U ml−1 SOD stock solution was stored at 4 ◦ C. Aluminum stock solution (0.02 mol l−1 ) was prepared by KAl(SO4 )2 ·12H2 O and adjusted to pH 3. A 0.1% PVA stock solution was prepared by dissolving 0.1 g polyvinyl alcohol (PVA-124, average degrees of polymerization were 2400–2500, Shanghai Chemical Reagent Plant imported from Japan) in 100 ml water under heating to near boiling. NaAc and HAc buffer solution (pH 5.0) was used. All other chemicals were of analytical grade and the twice-distilled water was used.
Fig. 1. The cyclic voltammograms at the SOD enzyme electrode in NaAc–HAc (pH 5.0) buffer solution. Addition of Al3+ concentration in the buffer solution: (a) 0, (b) 0.02, (c) 0.05, (d) 0.1, (e) 0.2 and (f) 0.5 mmol l−1 . Scan rate: 100 mV s−1 .
2.2. Preparation of the SOD enzyme electrode and voltammetric experiments
The direct electron transfer of SOD at a bare gold electrode is very slow and thus it has not been observed actually. It could be greatly enhanced by promoters, such as cysteine, 3-mercaptopropionic acid and bis(4-pyridyl)disulfide [23–25]. Recently, we have realized the direct electron transfer of SOD embedded in the thin silica sol–gel film at the gold electrode without any mediators or promoters and maintained good SOD activity [21]. On the other hand, the catalytic rate of native SOD is independent of pH over the range from 5 to 9.5 [26]. When SOD was embedded by silica sol–gel on the gold electrode surface, one pair of well-shaped voltammetric peaks was
The electrochemical measurements were made with standard three-electrode potentiostats (Model 820a Electrochemical Workstation, CH Instruments Inc., Shanghai, China). A threeelectrode voltammetric system consisted of a SOD enzyme electrode, a platinum counter electrode and a saturated calomel reference electrode (SCE). The preparation of silica colloidal sol and the SOD enzyme electrode were described previously [21]. The silicic acid solution was prepared by Na2 SiO3 ·9H2 O and the cation exchange process. The gold electrode (3 mm in diameter, made in our laboratory) was polished on abrasive paper, sonicated in ethanol and twice-distilled water and pretreated electrochemically in 0.05 mol l−1 H2 SO4 solution, respectively. The gold electrode was then dipped in the mixed solution containing 0.25 ml silica colloidal sol, 0.15 ml 0.1% PVA, 0.25 ml 1000 U ml−1 SOD per ml. Finally, the electrode was stored for 2–3 h at room temperature. The enzyme electrodes were stored at room temperature (25–35 ◦ C) with moisture-conditioned in a sealed container using a cup of water when not in use. The voltammetric experiments were carried out at SOD enzyme electrode in 10 ml 0.1 mol l−1 NaAc–HAc buffer solution. The next measurement was carried out after the solution was stirred by magnetic stirring for 1 min.
400 V. The excitation and emission slits were 10 nm. All experiments were carried out at room temperature. 3. Results and discussion 3.1. The effect of Al3+ on the electrochemical process of SOD
2.3. Fluorometric experiments Fluorescence spectrums were carried out by means of a F2500 fluorophotometer (Hitachi). The xenon lamp was used at
Fig. 2. The relationship of cyclic voltammetric currents and Al3+ concentration in buffer solution. Curve a: anodic current; curve c: cathodic current. Other experimental conditions as in Fig. 1.
898
J. Di et al. / Spectrochimica Acta Part A 65 (2006) 896–900
Fig. 3. The excitation fluorescence spectrum of SOD (A) and effect of Al3+ on fluorescence spectra of SOD (λex = 280 nm) (B) in NaAc–HAc (pH 5.0) buffer solution. The concentration of SOD: 4 mol l−1 . The concentration of Al3+ : (a) 0, (b) 0.02, (c) 0.04, (d) 0.08, (e) 0.2, (f) 0.4, (g) 0.8 and (h) 1.0 mmol l−1 .
observed in 0.1 mol l−1 NaAc–HAc buffer solution (Fig. 1, curve a). The formal potential E◦ , estimated as (Epa + Epc )/2, was about 0.20 V versus SCE. The current ratio of the cathodic peak current to the anodic one (Ipc /Ipa = 0.7 at 100 mV s−1 ) and the separation between the anodic and cathodic peak potentials (Ep = Epa − Epc = 0.17 V at 100 mV s−1 ) indicated that the electrode process of SOD was quasi-reversible. The electrochemical behavior of SOD changed when Al3+ ions were added in the buffer solution. With the increase of the concentration of Al3+ , both the cathodic and anodic peak currents were decreasing (Fig. 2) and the difference of the anodic and cathodic peak potential was increasing (Fig. 1). These indicated that Al3+ inhibited the electrochemical activity of SOD. If the enzyme electrode was pretreated by EDTA or citrate solution, the electrochemical
Fig. 4. Lineweaver-Burk plot of Al3+ on SOD.
activity of the SOD enzyme electrode did not get back. This indicates that the effect of Al3+ on the electrochemical activity is irreversible and permanent, which may result from the combination of the SOD molecular residues with Al ions and the conformational changes of SOD molecules. 3.2. The effect of Al3+ on the fluorescence spectrum of SOD Fluorescence spectroscopy is an effective method of study on protein conformation in aqueous solution [22]. Fig. 3 shows the fluorescence spectrum of SOD. The maximum excitation wavelength was 280 nm. The emission spectrum exhibited a peak at 314 nm and a shoulder-peak at about 340 nm. As shown in Fig. 3B, addition of Al3+ ions in SOD solution resulted in quenching of the fluorescence of SOD although the maximum emission wavelength did not alter in the emission spectrum.
Fig. 5. Double-log plot of Al3+ on SOD.
J. Di et al. / Spectrochimica Acta Part A 65 (2006) 896–900
899
Fig. 6. Synchronous fluorescence spectra of SOD in NaAc–HAc (pH 5.0) buffer solution at the wavelength interval of 20 nm (A) and 80 nm (B). (—) Before; (···) after addition of 1 mmol l−1 Al3+ .
Considering the electrochemical investigation, the fluorescence quenching effect resulted from the formation of a nonfluorescent Al–SOD complex, which was static quenching. According to Lineweaver-Burk equation [27,28]: (F0 − F )−1 = F0−1 + KD F0−1 [Al]−1 , the disassociation constant KD was obtained from the slope and intercept of the line (R = 0.9954) in Fig. 4. KD was 1.1 × 10−4 and KA was 9.1 × 103 (KA = 1/KD ). Supposing that there have n sites for Al3+ in the Al–SOD complex, this can be written as: nAl3+ + SOD = Aln SOD. The complex is non-fluorescent. Therefore, we obtained the following equation [28,29]: log(F0 − F)/F = log K + nlog[Al]. From double-log plot of Al3+ on SOD (Fig. 5), we obtained that n was 0.47 from the linear slope. This indicates that one molecule combines with Al3+ at least two ligand sites since Al(III) has generally six ligand sites. 3.3. The effect of Al3+ on the synchronous fluorescence spectrum of SOD Fig. 6A is synchronous fluorescence spectra of SOD at wavelength interval 20 nm at which the spectra are mainly contributed by tyrosine residues in SOD molecules [30,31]. There are two peaks in the spectrum. The main peak is located at 281 nm and the second peak is at 307 nm. After addition of Al3+ in the SOD solution, the peak positions did not shift but the fluorescence intensity was decreased (Fig. 6A, curve b). Fig. 6B is the synchronous fluorescence of SOD at wavelength interval 80 nm at which the spectra are contributed mainly by tryptophan residues in SOD molecules [30,31]. The main peak is located at 286 nm and the small shoulder-peak is at about 334 nm. After addition of Al3+ in the SOD solution, the fluorescence intensity decreased for the former peak and increase for the latter (Fig. 6B, curve b). The above results demonstrated that the tyrosine and tryptophan residues were influenced and
the conformation of SOD was altered because of Al–SOD complex formation. 4. Conclusion Getzoff et al. have proposed that the optimum action of Cu–Zn SOD depends upon a particular configuration of electrostatic changes in the enzyme molecule through substrate (O2 •− ) guidance and charge complementary [32]. Sequenceconserved residues create an extensive electrostatic field that directs the negatively charged O2 •− to the highly positive catalytic binding site at the bottom of the active-site channel. Swain and Chainy [14] proposed that Al3+ possibly alters SOD activity by disturbing the required electrostatic field because it has small ionic radius and strong electric charges. Our experimental results demonstrated that Al3+ ions could combine with SOD to form complex whose associative constant KA is 9.1 × 103 . The changes of electrochemical activity and fluorescent characteristic reflected a conformational change of the SOD molecule. Therefore, it can be concluded that Al3+ ions may induce the irreversible conformational changes of SOD molecules and influence the activity of the enzyme. Acknowledgements This project is supported by the National Natural Science Foundation of China (No. 20575025), Research Founding from MOE for Ph.D. Program (20050284030), Jiangsu Natural Science Foundation (BK2005209) and Analytical Measurement Funding of Nanjing University. The authors would like to thank Professors H.Y. Chen and X.Z. You for their help and encourages. References [1] G. Berthon, Coordin. Chem. Rev. 149 (1996) 241.
900
J. Di et al. / Spectrochimica Acta Part A 65 (2006) 896–900
[2] T.E. Lewis, Chemistry and Toxicology of Aluminum, Lewis Publishers Inc., Michigan, 1989, pp. 290–298. [3] R.J.P. Williams, Coordin. Chem. Rev. 228 (2002) 93. [4] C. Exley, Aluminum and Alzheimer’s Disease: The Science that Describes the Link, Elsevier Press, London, 2001, pp. 311–321. [5] P. Zatta, T. Kiss, M. Suwalsky, G. Berthon, Coordin. Chem. Rev. 228 (2002) 271. [6] C. Exley, J. Inorg. Biochem. 76 (1999) 133. [7] J.M.C. Gutteridge, G.J. Quinlan, I. Clark, B. Halliwell, Biochim. Biophys. Acta 835 (1985) 441. [8] J. Di, S. Bi, Spctrochim. Acta A 59 (2003) 1689. [9] J. Di, S. Bi, Spctrochim. Acta A 59 (2003) 3075. [10] B. Halliwell, J.M.C. Gutteridge, Biochem. J. 219 (1984) 1. [11] F. Auch`ere, F. Rusnak, J. Biol. Inorg. Chem. 7 (2002) 664. [12] J.P. Kehrer, Toxicology 149 (2000) 43. [13] S.Y. Kim, O.J. Kwon, J. Park, Biochimie 83 (2001) 437. [14] C. Swain, G.B.N. Chainy, Mol. Cell. Biochem. 187 (1998) 163. [15] F. Atienzar, D. Desor, D. Burnel, J.M. Keller, P. Lehr, P. Vasseur, Biol. Trace Element Res. 65 (1) (1998) 19. [16] C. Swain, G.B.N. Chainy, J. Trace Elements Med. Biol. 11 (2) (1997) 77. [17] M.A. Serra, V. Barassi, C. Canavese, E. Sabbioni, Biol. Trace Element Res. 31 (1991) 79.
[18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32]
F.A. Armstrong, G.S. Wilson, Electrochim. Acta 45 (2000) 2623. W. Zhang, G. Li, Anal. Sci. 20 (2004) 603. H. Liu, D. Pang, Prog. Chem. 11 (2002) 425. J. Di, S. Bi, M. Zhang, Biosens. Bioelectron. 19 (2004) 1479. S. Wang, X. Xu, Q. Liu, Y. Xie, Prog. Chem. (Chinese) 13 (2001) 257. Y. Tian, M. Shioda, S. Kasahara, T. Okajima, L. Mao, T. Hisabori, T. Ohsaka, Biochim. Biophys. Acta 1569 (2002) 151. B. Ge, F.W. Scheller, F. Lisdat, Biosens. Bioelectron. 18 (2003) 295. X. Wu, X. Meng, Z. Wang, Z. Zhang, Bioelectrochem. Bioenerg. 48 (1999) 227. J.A. Tainer, E.D. Getzoff, J.S. Richardson, D.C. Richardson, Nature 306 (1983) 284. M. Yang, P. Yang, L. Zhang, Chin. Sci. Bull. 39 (1994) 31. L. Sheng, S. Liu, J. Ye, X. Xu, Y. Xie, Q. Liu, Chin. J. Inorg. Chem. 19 (2003) 191. Y. Zhang, G. Zhang, Y. Wang, J. Lu, J. Anal. Sci. 16 (2000) 445. X. Du, R. Li, K. Wang, Chin. Sci. Bull. 46 (2001) 394. J. Chou, X. Qu, T. Lu, Y. Wu, Spectrosc. Spectr. Anal. 17 (1997) 108. E.D. Getzoff, J.A. Tainer, P.K. Weiner, P.A. Kollman, J.S. Rchardson, D.C. Richardson, Nature 306 (1983) 287.