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Study of the interaction of Zn2+ with Aβ(10-21) by fluorescamine assay
Chinese Chemical Letters 18 (2007) 97–98 www.elsevier.com/locate/cclet
Study of the interaction of Zn2+ with Ab(10-21) by fluorescamine assay Chao Feng Zhang, Li Fan, Pin Yang * Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Molecular Science, Shanxi University, Taiyuan 030006, China Received 12 May 2006
Abstract Zinc may play a role as a co-factor in the pathogenesis of Alzheimer’s disease (AD) through influencing the conformation and neurotoxicity of amyloid b-protein (Ab). Using the fluorescamine assay, we show for the first time that Zn2+ induced Ab(10-21) aggregate in a concentration-dependent manner. These results indicate that Ab(10-21) can be used as an in vitro model in Zn2+induced Ab aggregation and that the region 10-21 to be the minimal fragment of zinc-binding domain of full length Ab(1-42). # 2006 Pin Yang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. Keywords: Ab(10-21); Fluorescamine; Zn2+; Aggregation
Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and the main form of dementia. Senile plaques are one of the typical AD hallmarks and their major constituent is amyloid-b peptide (Ab). Aggregation of Ab into insoluble fibrils is a key pathological event in AD. One factor that influences the aggregation state of Ab is the interaction of Ab with certain transition metal ions. In vitro, Ab specifically interacts with Zn2+, and Zn2+ induces rapid aggregation of the synthetic or endogenous Ab in a pH-dependent manner [1]. The obligatory zinc-binding sequences has been mapped to the region 6-28 of Ab. While the histidine residues at positions 6, 13, and 14 of full length Ab(1-42) have been implicated in zinc binding, 13 and 14 appeared to be most critical. Molecular modeling has shown that Zn2+ binds to the Nt atom of the histidine imidazole ring and induces Ab(10-21) aggregation through intermolecular His 13 (Nt)-Zn2+-His 14 (Nt) bridge [2]. However, no experimental data support the hypothesis. In this paper, Zn2+-induced aggregation of Ab(10-21) was determined by fluorescamine assay to expand our previous work [2]. Samples were prepared from peptide stock solution by dilution with 20 mmol/L sodium phosphate buffer (pH 7.4) containing 150 mmol/L NaCl. The concentration of peptide was determined from the UV absorption intensity of tyrosine (e275 = 1410 L/mol cm) at pH 7.4 [3]. To determine the influence of Zn2+ on peptide aggregation, Zn2+ was added from the 10 mmol/L stock solution. The final Ab(10-21) concentration was 1 mmol/L, containing various concentrations of Zn2+. Samples were incubated for 0–24 h (5% CO2, 37 8C), after which insoluble aggregates were removed by centrifugation at 13,000 g for 10 min. The ratio of Ab(10-21) concentrations, as determined by fluorescamine assay, of the supernatant relative to the total peptide was used to calculate the levels of aggregation [4]. * Corresponding author. E-mail address: [email protected] (P. Yang). 1001-8417/$ – see front matter # 2006 Pin Yang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. doi:10.1016/j.cclet.2006.11.016
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C.F. Zhang et al. / Chinese Chemical Letters 18 (2007) 97–98
Fig. 1. Effects of Zn2+-induced concentration-dependent aggregation of Ab(10-21). Ab(10-21) at concentration of 1 mmol/L in pH 7.4 phosphate buffer was incubated separately with 0 mmol/L (a), 1 mmol/L (b), 2 mmol/L (c), 3 mmol/L (d), 5 mmol/L (e), 10 mmol/L (f) Zn2+ for 3 h. % of aggregation = 100 [(centrifuged value/noncentrifuged value) 100].
The fluorescence values were gathered using a F-2500 fluorometer with the excitation wavelength at 390 nm and emission at 480 nm. In the absence of Zn2+, Ab(10-21) showed no b-sheet structure in aqueous solution and did not aggregate significantly. This is consistent with previous results, where C-terminal amino acids have been shown to be crucial for Ab aggregation in aqueous solution. In the presence of Zn2+, Ab(10-21) aggregated rapidly and this action progressed incrementally in Zn2+ concentrations from 0 to 10 mmol/L (Fig. 1), which showed that Zn2+ induced Ab(10-21) aggregation in a concentration-dependent manner. Ab aggregation by Zn2+ may be a response to its neurotoxicity. The effects of aggregated Ab(10-21) on central nervous system are under way. From experimental view, we demonstrated that Zn2+ induced Ab(10-21) aggregation rapidly. Our findings suggested that the region 10-21 to be the minimal fragment of zinc-binding domain of Ab. Acknowledgment This work was supported by the National Natural Science Foundation of China (No. 30470408). References [1] [2] [3] [4]
A.I. Bush, W.H. Pettingell, G. Multhaup, et al. Science 265 (1994) 1464. D.X. Han, P. Yang, Sci. Chin. B 48 (2004) 126. T. Miura, K. Suzuki, N. Kohata, H. Takeuchi, Biochemistry 39 (2000) 7024. S.T. Liu, G. Howlett, C.J. Barrow, Biochemistry 38 (1999) 9373.
Study of the interaction of Zn2+ with Ab(10-21) by fluorescamine assay Chao Feng Zhang, Li Fan, Pin Yang * Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Molecular Science, Shanxi University, Taiyuan 030006, China Received 12 May 2006
Abstract Zinc may play a role as a co-factor in the pathogenesis of Alzheimer’s disease (AD) through influencing the conformation and neurotoxicity of amyloid b-protein (Ab). Using the fluorescamine assay, we show for the first time that Zn2+ induced Ab(10-21) aggregate in a concentration-dependent manner. These results indicate that Ab(10-21) can be used as an in vitro model in Zn2+induced Ab aggregation and that the region 10-21 to be the minimal fragment of zinc-binding domain of full length Ab(1-42). # 2006 Pin Yang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. Keywords: Ab(10-21); Fluorescamine; Zn2+; Aggregation
Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and the main form of dementia. Senile plaques are one of the typical AD hallmarks and their major constituent is amyloid-b peptide (Ab). Aggregation of Ab into insoluble fibrils is a key pathological event in AD. One factor that influences the aggregation state of Ab is the interaction of Ab with certain transition metal ions. In vitro, Ab specifically interacts with Zn2+, and Zn2+ induces rapid aggregation of the synthetic or endogenous Ab in a pH-dependent manner [1]. The obligatory zinc-binding sequences has been mapped to the region 6-28 of Ab. While the histidine residues at positions 6, 13, and 14 of full length Ab(1-42) have been implicated in zinc binding, 13 and 14 appeared to be most critical. Molecular modeling has shown that Zn2+ binds to the Nt atom of the histidine imidazole ring and induces Ab(10-21) aggregation through intermolecular His 13 (Nt)-Zn2+-His 14 (Nt) bridge [2]. However, no experimental data support the hypothesis. In this paper, Zn2+-induced aggregation of Ab(10-21) was determined by fluorescamine assay to expand our previous work [2]. Samples were prepared from peptide stock solution by dilution with 20 mmol/L sodium phosphate buffer (pH 7.4) containing 150 mmol/L NaCl. The concentration of peptide was determined from the UV absorption intensity of tyrosine (e275 = 1410 L/mol cm) at pH 7.4 [3]. To determine the influence of Zn2+ on peptide aggregation, Zn2+ was added from the 10 mmol/L stock solution. The final Ab(10-21) concentration was 1 mmol/L, containing various concentrations of Zn2+. Samples were incubated for 0–24 h (5% CO2, 37 8C), after which insoluble aggregates were removed by centrifugation at 13,000 g for 10 min. The ratio of Ab(10-21) concentrations, as determined by fluorescamine assay, of the supernatant relative to the total peptide was used to calculate the levels of aggregation [4]. * Corresponding author. E-mail address: [email protected] (P. Yang). 1001-8417/$ – see front matter # 2006 Pin Yang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. doi:10.1016/j.cclet.2006.11.016
98
C.F. Zhang et al. / Chinese Chemical Letters 18 (2007) 97–98
Fig. 1. Effects of Zn2+-induced concentration-dependent aggregation of Ab(10-21). Ab(10-21) at concentration of 1 mmol/L in pH 7.4 phosphate buffer was incubated separately with 0 mmol/L (a), 1 mmol/L (b), 2 mmol/L (c), 3 mmol/L (d), 5 mmol/L (e), 10 mmol/L (f) Zn2+ for 3 h. % of aggregation = 100 [(centrifuged value/noncentrifuged value) 100].
The fluorescence values were gathered using a F-2500 fluorometer with the excitation wavelength at 390 nm and emission at 480 nm. In the absence of Zn2+, Ab(10-21) showed no b-sheet structure in aqueous solution and did not aggregate significantly. This is consistent with previous results, where C-terminal amino acids have been shown to be crucial for Ab aggregation in aqueous solution. In the presence of Zn2+, Ab(10-21) aggregated rapidly and this action progressed incrementally in Zn2+ concentrations from 0 to 10 mmol/L (Fig. 1), which showed that Zn2+ induced Ab(10-21) aggregation in a concentration-dependent manner. Ab aggregation by Zn2+ may be a response to its neurotoxicity. The effects of aggregated Ab(10-21) on central nervous system are under way. From experimental view, we demonstrated that Zn2+ induced Ab(10-21) aggregation rapidly. Our findings suggested that the region 10-21 to be the minimal fragment of zinc-binding domain of Ab. Acknowledgment This work was supported by the National Natural Science Foundation of China (No. 30470408). References [1] [2] [3] [4]
A.I. Bush, W.H. Pettingell, G. Multhaup, et al. Science 265 (1994) 1464. D.X. Han, P. Yang, Sci. Chin. B 48 (2004) 126. T. Miura, K. Suzuki, N. Kohata, H. Takeuchi, Biochemistry 39 (2000) 7024. S.T. Liu, G. Howlett, C.J. Barrow, Biochemistry 38 (1999) 9373.