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Rapid label-free detection of metal-induced Alzheimer’s amyloid β peptide aggregation by electrochemical method
Electrochemistry Communications 10 (2008) 1797–1800
Contents lists available at ScienceDirect
Electrochemistry Communications journal homepage: www.elsevier.com/locate/elecom
Rapid label-free detection of metal-induced Alzheimer’s amyloid b peptide aggregation by electrochemical method Jie Geng, Haijia Yu, Jinsong Ren, Xiaogang Qu * Division of Biological Inorganic Chemistry, State Key Laboratory of Rare Earth Resource Utilization, Graduate School of the Chinese Academy of Sciences, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun, Jilin 130022, China
a r t i c l e
i n f o
Article history: Received 15 August 2008 Received in revised form 11 September 2008 Accepted 12 September 2008 Available online 20 September 2008 Keywords: Ab aggregation Metal Electrochemistry Tyrosine
a b s t r a c t Amyloid b peptide plays a critical role in the pathogenesis of Alzheimer’s disease (AD). Metal ions are highly enriched in cerebral amyloid deposits in AD and are proposed to be able to mediate Ab conformation. Therefore, a rapid, low-cost, and sensitive detection of metal-induced Ab aggregation and their relation to AD is clearly needed for the clinical diagnosis and treatment. In this report, we study metal-induced Ab aggregation by a rapid, label-free electrochemical method and monitor both the aggregation kinetics and the morphology in the absence or presence of Zn (II) and Cu (II). Different electrochemical behaviors of Ab in the absence or presence of metal ions have been observed both in the aggregation kinetics and final aggregates. Our results provide new insights into the application of the rapid, low-cost and sensitive electrochemical device for clinical diagnosis of metal effects on AD or other neurodegenerative diseases which are related to protein misfolding. Ó 2008 Elsevier B.V. All rights reserved.
1. Introduction Alzheimer’s disease (AD) is the most common form of neurodegenerative disorders among the elderly, which is characterized by numerous senile plaques and neurofibrillary tangles in specific regions of brain [1]. The major component of these plaques is the b-amyloid peptide (Ab). The mechanisms underlying the pathogenesis of AD remain poorly understood. Recent studies have suggested that amyloid b-aggregation is an essential event in the pathogenesis of AD [2–5]. Factors influencing Ab aggregation have attracted much attention. Transition metals such as copper and zinc are present at high concentrations in Ab plaques in AD patients [6]. Ab peptides have high affinity for metal ions and exposure to certain metal ions has been proposed as a risk factor for developing AD. Effects of metal ions, in particularly copper and zinc, on the aggregation of Ab have been widely studied [7–13]. We have reported that Cu (II) and Zn (II) can inhibit Ab induced DNA condensation [14] and Cu (II) or Zn (II) binding to Ab [15] can release water molecules. Further studies indicate that Zn (II) binding causes more water molecules released [15] demonstrating that hydration changes can be different upon different metal binding. Since metal ions are highly enriched in cerebral amyloid deposits in AD and are proposed to be able to mediate Ab conformation, a rapid, low-cost, and sensitive detection of metal-induced
* Corresponding author. Tel./fax: +86 431 85262656. E-mail address: [email protected] (X. Qu). 1388-2481/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.elecom.2008.09.020
Ab aggregation and their relation to AD is clearly needed for the clinical diagnosis and treatment. Electrochemical methods have been widely used to study the electron transfer process of protein and DNA [16–25]. Recently, Vestergaard and co-workers reported a rapid label-free electrochemical detection of Ab aggregation [26]. The electrochemical oxidation of tyrosine residue can be used as a probe to detect Ab aggregation. In this report, we study metal-induced Ab aggregation by electrochemical method and monitor both the aggregation kinetics and the morphology in the absence or presence of Zn (II) and Cu (II). 2. Experimental procedures 2.1. Reagents Ab1-40 (lot no. U10012) was purchased from American peptide cooperation. The synthetic Ab peptide was characterized and prepared as previously described [14,15]. All metal chloride salts were obtained from Sigma. HFIP was obtained from Acros organics. Solutions were all prepared in ultrapure water purified through a MilliQ system (Millipore, USA). 2.2. Electrochemical measurement Square wave voltammetry (SWV) was carried out with a CHI660B electrochemistry workstation (CHI, USA). All electrochemical experiments employed a conventional three-electrode
J. Geng et al. / Electrochemistry Communications 10 (2008) 1797–1800
2.3. Electron microscopy Ab1-40 (50 lM) was incubated at 37 °C in the absence or presence of two equivalents of Cu (II) or Zn (II) for 6 days. Samples (10 ll) of aggregated peptide were spotted onto carbon-coated copper grids for 30 min. The grids were blotted with filter paper to remove excess buffer and the sample was stained with 1.5% (w/v) phosphotungstic acid (pH 7.0). Grids were blotted again and air-dried before analysis on a transmission electron microscope (JEOL JEM-1011), operating with a voltage of 100 kV.
3. Results and discussion A well-defined oxidation peak of tyrosine residue in the square wave voltammogram (SWV) was observed at 0.71 V (Fig. 1A) on GCE in detection of Ab and its peak potential is in agreement with the previously reported value [26]. The current intensity decreased dramatically in the presence of Zn (II) or Cu (II) (Fig. 1) indicating that metal binding can cause Ab conformational change [11–15] and influence the microenvironment of tyrosine residue. On the contrary to Zn (II) or Cu (II), Mg (II) did not change the current intensity within experimental errors (Fig. 1B). This is consistent with the fact that Mg (II) hardly alters the peptide conformation. We also found that Ab aggregation induced by Zn (II) or Cu (II) was reversible upon EDTA chelation (Fig. 1A) in accordance with the previous results [8]. The influence of Zn (II)/Cu (II) on Ab aggregation is dose-dependent (Fig. 1B). The peak current decreased gradually as the ratio of [metal]/[Ab] increased. The decrease reached a plateau at 4:1 ratio, where the current intensity remained 45% for Zn (II) and 65% for Cu (II) to their individual initial values. Therefore, the ratio 4:1 might be the highest binding ratio when Zn (II) or Cu (II) binding to Ab [8,27]. The peak current of Ab-Zn (II) was more decreased than that of Ab-Cu (II) at the same binding ratio indicating that Zn (II) was more efficient to induce Ab aggregation than Cu (II) under physiological conditions [9]. We further studied time dependence of Ab aggregations in the absence or presence of metal ions by the electrochemical method. Ab or Ab-metal complex was incubated at 37 °C for indicated period of time before measurements. In the absence of metal ions, the aggregation profile was sigmoidal (Fig. 1C), which indicated the aggregation was a nucleation-dependent process [28]. The current signal of Ab nearly disappeared when incubated for 6 days due to formation of ordered fibrils (Fig. 1C), while in the presence of Zn (II) or Cu (II) (Fig. 1C), the current signal decreased dramatically
A
600
Current(nA)
500 400 300 200 100 0 0.5
0.6
0.7
0.8
0.9
Potential(V)
B Normalized Current
cell (1 ml, single electrolyte compartment) with a glassy carbon electrode (CHI, USA) used as the working electrode, a platinum wire (CHI, USA) as the auxiliary electrode, and an Ag/AgCl electrode (CHI, USA) as the reference electrode. The glassy carbon electrode (GCE) (Ø = 3.0 mm) was pretreated as previously reported [16]. To prepare the analytes, 50 lM Ab was incubated at 37 °C in the absence or presence of different concentration of metal ions for a defined period of time. For chelation experiment, EDTA was added to the Ab-metal complexes to a final concentration of 1 mM and incubated for 1 h. Unless otherwise stated, all buffers used were 20 mM Tris buffer, pH 7.0. For preparation of peptide-modified electrodes, aliquots of incubated Ab or Ab-metal complex were removed and diluted to the required concentrations (10 lM for Ab). A droplet of 10 ll of sample was dropped onto an inverted GCE and allowed to adsorb for 10 min. Analyses were carried out using the same buffer as the supporting electrolyte. SWV was carried out with initial potential 0.4 V, end potential l.0 V, step potential 4 mV, amplitude 25 mV, and frequency 15 Hz.
1.1 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0
2
4
6
8
10
[Metal Ions]/ A β
C Normalized Current
1798
1.0 0.8 0.6 0.4 0.2 0.0 0
20
40 60 80 100 120 140 Incubation Time (h)
Fig. 1. (A) Square wave voltammograms of Ab1-40 in the absence (black) or presence of two equivalents of Cu (II) (blue) or Zn (II) (red) and after EDTA chelation with Cu (II) (cyan) or Zn (II) (green); (B) dependence of current intensity on the molar ratio of metal ions to Ab:Cu (II), black squares; Zn (II), red circles; Mg (II), green triangles. Ab was incubated in the absence or presence of various concentrations of metals at 37 °C for 1 h; (C) kinetics of Ab1-40 aggregations in Tris buffer (20 mM Tris, pH7.0). Ab1-40 was incubated in the absence (black squares) or presence of two equivalents of Cu (II) (red circles) or Zn (II) (green triangles) at 37 °C for a defined period of time and measured by SWV.
in the first few days. Unlike Ab alone, the Ab-metal complexes did not show significant lag phase in the process of aggregation. We inferred that these two metal ions accelerated Ab aggregation, consistent with the earlier reports [8,9]. In the presence of Zn (II)/ Cu (II), the current decrease can reach a plateau in a short time where the current was about 35% for Zn (II) and 60% for Cu (II) of their individual initial values (Fig. 1C). However, for Ab alone, the current signals decreased almost to zero after a lag phase of 2 days. Previous studies have shown that Ab forms non-fibrillar aggregates in the presence of Zn (II) or Cu (II) [29–30] and metal binding can inhibit the transition to the more compact fibrillar conformation. Therefore, tyrosine residues in metal-induced non-fibrillar aggregates are more exposed than those in the ordered fibrils, and the
1799
J. Geng et al. / Electrochemistry Communications 10 (2008) 1797–1800
ET n
Monomer
l eta m o
Zn2+
ET
Cu 2+
ET
Scheme 1. Illustration of Ab aggregation in the absence or presence of Zn2+ or Cu2+.
dration [15] by osmotic stress method. Furthermore, zinc binding even causes water molecules released more than twice as much as copper binding [15]. Based on kinetic studies on Ab aggregation (Fig. 1C), it can be seen that the profile of Ab aggregation in the absence of metal ions was sigmoidal, in accordance with a nucleation process of Ab self-aggregation examined by Th T fluorescence [28]; while in the presence of metal ions, the lag phase almost disappeared due to a rapid metal-induced aggregation. However, the final current signals of Ab-metal complexes were significantly higher than that of Ab incubated alone. All these results indicate that metal ions can affect both the kinetics and the morphology of Ab aggregation. Different metal ions can have different effects and the difference can be detected by a rapid label-free electrochemical method (Scheme 1). Metal binding can induce Ab conformational transition and release a large amount of water molecules by forming Ab-metal intermediate [15], which can be followed by rapid Ab amorphous aggregations, thus preventing fibrillogenesis. 4. Conclusions In summary, different electrochemical behaviors of Ab in the absence or presence of metal ions and the influences of Zn (II) and Cu (II) can be used to detect metal-induced Ab aggregation and electrochemical method can monitor the aggregation kinetics and the morphology. This will be helpful for the potential application of the rapid, low-cost and sensitive electrochemical device for the clinical diagnosis of metal effects on AD or other neurodegenerative diseases which are related to protein misfolding. Acknowledgements Fig. 2. Transmission electron microscopy images of aggregated Ab1-40 in the absence (A) or presence of two equivalents of Cu (II) (B) or Zn (II) (C).
redox current can be detected in non-fibrillar aggregates. The aggregates induced by Zn (II) are more compact than Cu (II) because the current decrease is more significantly. This is also supported by our transmission electron microscopy (TEM) results (Fig. 2). In the absence of metal ions, Ab forms ordered fibrils (Fig. 2A). In the presence of Cu (II), TEM analysis shows that Ab forms amorphous aggregates which were approximately 10– 20 nm in size (Fig. 2B). Moreover, in the presence of Zn (II), Ab forms even larger amorphous aggregates (Fig. 2C) which are different from those induced by Cu (II), further supporting our electrochemical results. Metal ions play important roles in the pathogenesis of AD. We have reported that both copper and zinc binding to Ab cause dehy-
This project was supported by NSFC and Funds from the Chinese Academy of Sciences and Jilin Province. References [1] [2] [3] [4] [5] [6]
K.S. Kosik, Science 256 (1992) 780. J. Hardy, D.J. Selkoe, Science 297 (2002) 353. W.L. Klein, W.B. Stine Jr., D.B. Teplow, Neurobiol. Aging 25 (2004) 569. A. Lorenzo, B.A. Yankner, Proc. Natl. Acad. Sci. USA 91 (1994) 12243. Y.H. Suh, F. Checler, Pharmacol. Rev. 54 (2002) 469. M.A. Lovell, J.D. Robertson, W.J. Teesdale, J.L. Campbell, W.R. Markesbery, J. Neurol. Sci. 158 (1998) 47. [7] A.I. Bush, Trends Neurosci. 26 (2003) 207. [8] C.S. Atwood, R.D. Moir, X. Huang, R.C. Scarpa, N.M. Bacarra, D.M. Romano, M.A. Hartshorn, R.E. Anzi, A.I. Bush, J. Biol. Chem. 273 (1998) 12817. [9] A.I. Bush, W.H. Pettingell, G. Multhaup, M.d. Paradis, J.P. Vonsattel, J.F. Gusella, K. Beyreuther, C.L. Masters, R.E. Tanzi, Science 265 (1994) 1464.
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[10] J. Dong, C.S. Atwood, V.E. Anderson, S.L. Siedlak, M.A. Smith, G. Perry, P.R. Carey, Biochemistry 42 (2003) 2768. [11] T. Miura, K. Suzuki, N. Kohata, H. Takeuchi, Biochemistry 39 (2000) 7024. [12] E. Gaggelli, A. Janicka-Klos, E. Jankowska, H. Kozlowski, C. Migliorini, E. Molteni, D. Valensin, G. Valensin, E. Wieczerzak, J. Phys. Chem. B 112 (2008) 100. [13] E. Gaggelli, Z. Grzonka, H. Kozłowski, C. Migliorini, E. Molteni, D. Valensin, G. Valensin, Chem. Commun. (2008) 341–343. [14] H. Yu, J. Ren, X. Qu, Biophys. J. 92 (2007) 185. [15] H. Yu, J. Ren, X. Qu, ChemBioChem 9 (2008) 879. [16] X. Li, Y. Peng, J. Ren, X. Qu, Biochemistry 45 (2006) 13543. [17] X. Qu, J. Chou, T. Lu, S. Dong, C. Zhou, T.M. Cotton, J. Electroanal. Chem. 381 (1995) 81. [18] X. Qu, T. Lu, S. Dong, C. Zhou, T.M. Cotton, Bioelectrochem. Bioenerg. 34 (1994) 153. [19] Y. Xiao, X. Qu, K.W. Plaxco, A.J. Heeger, J. Am. Chem. Soc. 129 (2007) 11896. [20] L. Guilloreau, S. Combalbert, A. Sournia-Saquet, H. Mazarguil, P. Faller, Chem. Biochem. 8 (2007) 1317.
[21] X. Huang, M.P. Cuajungco, C.S. Atwood, M.A. Hartshorn, J.D. Tyndall, G.R. Hanson, K.C. Stokes, M. Leopold, G. Multhaup, L.E. Goldstein, R.C. Scarpa, A.J. Saunders, J. Lim, R.D. Moir, C. Glabe, E.F. Bowden, C.L. Masters, D.P. Fairlie, R.E. Tanzi, A.I. Bush, J. Biol. Chem. 274 (1999) 37111. [22] D. Jiang, L. Men, J. Wang, Y. Zhang, S. Chickenyen, Y. Wang, F. Zhou, Biochemistry 46 (2007) 9270. [23] A. Loksztejn, W. Dzwolak, P. Krysinski, Bioelectrochemistry 72 (2008) 34. [24] M. Masarik, A. Stobiecka, R. Kizek, F. Jelen, Z. Pechan, W. Hoyer, T.M. Jovin, V. Subramaniam, E. Palecek, Electroanalysis 16 (2004) 1172. [25] I. Szymanska, H. Radecka, J. Radecki, R. Kaliszan, Biosens. Bioelectron. 22 (2007) 1955. [26] M. Vestergaard, K. Kerman, M. Saito, N. Nagatani, Y. Takamura, E. Tamiya, J. Am. Chem. Soc. 127 (2005) 11892. [27] X. Huang, C.S. Atwood, R.D. Moir, M.A. Hartshorn, R.E. Tanzi, A.I. Bush, J. Biol. Inorg. Chem. 9 (2004) 954. [28] H. Naiki, F. Gejyo, K. Nakakuki, Biochemistry 36 (1997) 6243. [29] J. Zou, K. Kajita, N. Sugimoto, Angew. Chem. Int. Ed. 40 (2001) 2274. [30] Y. Yoshiike, K. Tanemura, O. Murayama, T. Akagi, M. Murayama, S. Sato, X. Sun, N. Tanaka, A. Takashima, J. Biol. Chem. 276 (2001) 32293.
Contents lists available at ScienceDirect
Electrochemistry Communications journal homepage: www.elsevier.com/locate/elecom
Rapid label-free detection of metal-induced Alzheimer’s amyloid b peptide aggregation by electrochemical method Jie Geng, Haijia Yu, Jinsong Ren, Xiaogang Qu * Division of Biological Inorganic Chemistry, State Key Laboratory of Rare Earth Resource Utilization, Graduate School of the Chinese Academy of Sciences, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun, Jilin 130022, China
a r t i c l e
i n f o
Article history: Received 15 August 2008 Received in revised form 11 September 2008 Accepted 12 September 2008 Available online 20 September 2008 Keywords: Ab aggregation Metal Electrochemistry Tyrosine
a b s t r a c t Amyloid b peptide plays a critical role in the pathogenesis of Alzheimer’s disease (AD). Metal ions are highly enriched in cerebral amyloid deposits in AD and are proposed to be able to mediate Ab conformation. Therefore, a rapid, low-cost, and sensitive detection of metal-induced Ab aggregation and their relation to AD is clearly needed for the clinical diagnosis and treatment. In this report, we study metal-induced Ab aggregation by a rapid, label-free electrochemical method and monitor both the aggregation kinetics and the morphology in the absence or presence of Zn (II) and Cu (II). Different electrochemical behaviors of Ab in the absence or presence of metal ions have been observed both in the aggregation kinetics and final aggregates. Our results provide new insights into the application of the rapid, low-cost and sensitive electrochemical device for clinical diagnosis of metal effects on AD or other neurodegenerative diseases which are related to protein misfolding. Ó 2008 Elsevier B.V. All rights reserved.
1. Introduction Alzheimer’s disease (AD) is the most common form of neurodegenerative disorders among the elderly, which is characterized by numerous senile plaques and neurofibrillary tangles in specific regions of brain [1]. The major component of these plaques is the b-amyloid peptide (Ab). The mechanisms underlying the pathogenesis of AD remain poorly understood. Recent studies have suggested that amyloid b-aggregation is an essential event in the pathogenesis of AD [2–5]. Factors influencing Ab aggregation have attracted much attention. Transition metals such as copper and zinc are present at high concentrations in Ab plaques in AD patients [6]. Ab peptides have high affinity for metal ions and exposure to certain metal ions has been proposed as a risk factor for developing AD. Effects of metal ions, in particularly copper and zinc, on the aggregation of Ab have been widely studied [7–13]. We have reported that Cu (II) and Zn (II) can inhibit Ab induced DNA condensation [14] and Cu (II) or Zn (II) binding to Ab [15] can release water molecules. Further studies indicate that Zn (II) binding causes more water molecules released [15] demonstrating that hydration changes can be different upon different metal binding. Since metal ions are highly enriched in cerebral amyloid deposits in AD and are proposed to be able to mediate Ab conformation, a rapid, low-cost, and sensitive detection of metal-induced
* Corresponding author. Tel./fax: +86 431 85262656. E-mail address: [email protected] (X. Qu). 1388-2481/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.elecom.2008.09.020
Ab aggregation and their relation to AD is clearly needed for the clinical diagnosis and treatment. Electrochemical methods have been widely used to study the electron transfer process of protein and DNA [16–25]. Recently, Vestergaard and co-workers reported a rapid label-free electrochemical detection of Ab aggregation [26]. The electrochemical oxidation of tyrosine residue can be used as a probe to detect Ab aggregation. In this report, we study metal-induced Ab aggregation by electrochemical method and monitor both the aggregation kinetics and the morphology in the absence or presence of Zn (II) and Cu (II). 2. Experimental procedures 2.1. Reagents Ab1-40 (lot no. U10012) was purchased from American peptide cooperation. The synthetic Ab peptide was characterized and prepared as previously described [14,15]. All metal chloride salts were obtained from Sigma. HFIP was obtained from Acros organics. Solutions were all prepared in ultrapure water purified through a MilliQ system (Millipore, USA). 2.2. Electrochemical measurement Square wave voltammetry (SWV) was carried out with a CHI660B electrochemistry workstation (CHI, USA). All electrochemical experiments employed a conventional three-electrode
J. Geng et al. / Electrochemistry Communications 10 (2008) 1797–1800
2.3. Electron microscopy Ab1-40 (50 lM) was incubated at 37 °C in the absence or presence of two equivalents of Cu (II) or Zn (II) for 6 days. Samples (10 ll) of aggregated peptide were spotted onto carbon-coated copper grids for 30 min. The grids were blotted with filter paper to remove excess buffer and the sample was stained with 1.5% (w/v) phosphotungstic acid (pH 7.0). Grids were blotted again and air-dried before analysis on a transmission electron microscope (JEOL JEM-1011), operating with a voltage of 100 kV.
3. Results and discussion A well-defined oxidation peak of tyrosine residue in the square wave voltammogram (SWV) was observed at 0.71 V (Fig. 1A) on GCE in detection of Ab and its peak potential is in agreement with the previously reported value [26]. The current intensity decreased dramatically in the presence of Zn (II) or Cu (II) (Fig. 1) indicating that metal binding can cause Ab conformational change [11–15] and influence the microenvironment of tyrosine residue. On the contrary to Zn (II) or Cu (II), Mg (II) did not change the current intensity within experimental errors (Fig. 1B). This is consistent with the fact that Mg (II) hardly alters the peptide conformation. We also found that Ab aggregation induced by Zn (II) or Cu (II) was reversible upon EDTA chelation (Fig. 1A) in accordance with the previous results [8]. The influence of Zn (II)/Cu (II) on Ab aggregation is dose-dependent (Fig. 1B). The peak current decreased gradually as the ratio of [metal]/[Ab] increased. The decrease reached a plateau at 4:1 ratio, where the current intensity remained 45% for Zn (II) and 65% for Cu (II) to their individual initial values. Therefore, the ratio 4:1 might be the highest binding ratio when Zn (II) or Cu (II) binding to Ab [8,27]. The peak current of Ab-Zn (II) was more decreased than that of Ab-Cu (II) at the same binding ratio indicating that Zn (II) was more efficient to induce Ab aggregation than Cu (II) under physiological conditions [9]. We further studied time dependence of Ab aggregations in the absence or presence of metal ions by the electrochemical method. Ab or Ab-metal complex was incubated at 37 °C for indicated period of time before measurements. In the absence of metal ions, the aggregation profile was sigmoidal (Fig. 1C), which indicated the aggregation was a nucleation-dependent process [28]. The current signal of Ab nearly disappeared when incubated for 6 days due to formation of ordered fibrils (Fig. 1C), while in the presence of Zn (II) or Cu (II) (Fig. 1C), the current signal decreased dramatically
A
600
Current(nA)
500 400 300 200 100 0 0.5
0.6
0.7
0.8
0.9
Potential(V)
B Normalized Current
cell (1 ml, single electrolyte compartment) with a glassy carbon electrode (CHI, USA) used as the working electrode, a platinum wire (CHI, USA) as the auxiliary electrode, and an Ag/AgCl electrode (CHI, USA) as the reference electrode. The glassy carbon electrode (GCE) (Ø = 3.0 mm) was pretreated as previously reported [16]. To prepare the analytes, 50 lM Ab was incubated at 37 °C in the absence or presence of different concentration of metal ions for a defined period of time. For chelation experiment, EDTA was added to the Ab-metal complexes to a final concentration of 1 mM and incubated for 1 h. Unless otherwise stated, all buffers used were 20 mM Tris buffer, pH 7.0. For preparation of peptide-modified electrodes, aliquots of incubated Ab or Ab-metal complex were removed and diluted to the required concentrations (10 lM for Ab). A droplet of 10 ll of sample was dropped onto an inverted GCE and allowed to adsorb for 10 min. Analyses were carried out using the same buffer as the supporting electrolyte. SWV was carried out with initial potential 0.4 V, end potential l.0 V, step potential 4 mV, amplitude 25 mV, and frequency 15 Hz.
1.1 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0
2
4
6
8
10
[Metal Ions]/ A β
C Normalized Current
1798
1.0 0.8 0.6 0.4 0.2 0.0 0
20
40 60 80 100 120 140 Incubation Time (h)
Fig. 1. (A) Square wave voltammograms of Ab1-40 in the absence (black) or presence of two equivalents of Cu (II) (blue) or Zn (II) (red) and after EDTA chelation with Cu (II) (cyan) or Zn (II) (green); (B) dependence of current intensity on the molar ratio of metal ions to Ab:Cu (II), black squares; Zn (II), red circles; Mg (II), green triangles. Ab was incubated in the absence or presence of various concentrations of metals at 37 °C for 1 h; (C) kinetics of Ab1-40 aggregations in Tris buffer (20 mM Tris, pH7.0). Ab1-40 was incubated in the absence (black squares) or presence of two equivalents of Cu (II) (red circles) or Zn (II) (green triangles) at 37 °C for a defined period of time and measured by SWV.
in the first few days. Unlike Ab alone, the Ab-metal complexes did not show significant lag phase in the process of aggregation. We inferred that these two metal ions accelerated Ab aggregation, consistent with the earlier reports [8,9]. In the presence of Zn (II)/ Cu (II), the current decrease can reach a plateau in a short time where the current was about 35% for Zn (II) and 60% for Cu (II) of their individual initial values (Fig. 1C). However, for Ab alone, the current signals decreased almost to zero after a lag phase of 2 days. Previous studies have shown that Ab forms non-fibrillar aggregates in the presence of Zn (II) or Cu (II) [29–30] and metal binding can inhibit the transition to the more compact fibrillar conformation. Therefore, tyrosine residues in metal-induced non-fibrillar aggregates are more exposed than those in the ordered fibrils, and the
1799
J. Geng et al. / Electrochemistry Communications 10 (2008) 1797–1800
ET n
Monomer
l eta m o
Zn2+
ET
Cu 2+
ET
Scheme 1. Illustration of Ab aggregation in the absence or presence of Zn2+ or Cu2+.
dration [15] by osmotic stress method. Furthermore, zinc binding even causes water molecules released more than twice as much as copper binding [15]. Based on kinetic studies on Ab aggregation (Fig. 1C), it can be seen that the profile of Ab aggregation in the absence of metal ions was sigmoidal, in accordance with a nucleation process of Ab self-aggregation examined by Th T fluorescence [28]; while in the presence of metal ions, the lag phase almost disappeared due to a rapid metal-induced aggregation. However, the final current signals of Ab-metal complexes were significantly higher than that of Ab incubated alone. All these results indicate that metal ions can affect both the kinetics and the morphology of Ab aggregation. Different metal ions can have different effects and the difference can be detected by a rapid label-free electrochemical method (Scheme 1). Metal binding can induce Ab conformational transition and release a large amount of water molecules by forming Ab-metal intermediate [15], which can be followed by rapid Ab amorphous aggregations, thus preventing fibrillogenesis. 4. Conclusions In summary, different electrochemical behaviors of Ab in the absence or presence of metal ions and the influences of Zn (II) and Cu (II) can be used to detect metal-induced Ab aggregation and electrochemical method can monitor the aggregation kinetics and the morphology. This will be helpful for the potential application of the rapid, low-cost and sensitive electrochemical device for the clinical diagnosis of metal effects on AD or other neurodegenerative diseases which are related to protein misfolding. Acknowledgements Fig. 2. Transmission electron microscopy images of aggregated Ab1-40 in the absence (A) or presence of two equivalents of Cu (II) (B) or Zn (II) (C).
redox current can be detected in non-fibrillar aggregates. The aggregates induced by Zn (II) are more compact than Cu (II) because the current decrease is more significantly. This is also supported by our transmission electron microscopy (TEM) results (Fig. 2). In the absence of metal ions, Ab forms ordered fibrils (Fig. 2A). In the presence of Cu (II), TEM analysis shows that Ab forms amorphous aggregates which were approximately 10– 20 nm in size (Fig. 2B). Moreover, in the presence of Zn (II), Ab forms even larger amorphous aggregates (Fig. 2C) which are different from those induced by Cu (II), further supporting our electrochemical results. Metal ions play important roles in the pathogenesis of AD. We have reported that both copper and zinc binding to Ab cause dehy-
This project was supported by NSFC and Funds from the Chinese Academy of Sciences and Jilin Province. References [1] [2] [3] [4] [5] [6]
K.S. Kosik, Science 256 (1992) 780. J. Hardy, D.J. Selkoe, Science 297 (2002) 353. W.L. Klein, W.B. Stine Jr., D.B. Teplow, Neurobiol. Aging 25 (2004) 569. A. Lorenzo, B.A. Yankner, Proc. Natl. Acad. Sci. USA 91 (1994) 12243. Y.H. Suh, F. Checler, Pharmacol. Rev. 54 (2002) 469. M.A. Lovell, J.D. Robertson, W.J. Teesdale, J.L. Campbell, W.R. Markesbery, J. Neurol. Sci. 158 (1998) 47. [7] A.I. Bush, Trends Neurosci. 26 (2003) 207. [8] C.S. Atwood, R.D. Moir, X. Huang, R.C. Scarpa, N.M. Bacarra, D.M. Romano, M.A. Hartshorn, R.E. Anzi, A.I. Bush, J. Biol. Chem. 273 (1998) 12817. [9] A.I. Bush, W.H. Pettingell, G. Multhaup, M.d. Paradis, J.P. Vonsattel, J.F. Gusella, K. Beyreuther, C.L. Masters, R.E. Tanzi, Science 265 (1994) 1464.
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