Quantification of the Binding Constant of Copper ... - ACS Publications

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J. Phys. Chem. B 2008, 112, 8160–8164

Quantification of the Binding Constant of Copper(II) to the Amyloid-Beta Peptide Lanying Q. Hatcher, Lian Hong, William D. Bush, Tessa Carducci, and John D. Simon* Department of Chemistry, Duke UniVersity, Durham, North Carolina 27708 ReceiVed: NoVember 12, 2007; ReVised Manuscript ReceiVed: April 9, 2008

The amyloid beta (Aβ) peptide of Alzheimer’s disease binds copper(II), and the peptide-bound metal may be a source of reactive oxygen species and neurotoxicity. To circumvent peptide aggregation and reduce redox activity, there is growing interest in using metal chelates as drug therapeutics for AD, whose design requires accurate data on the affinity of Aβ peptides for copper(II). Reports on Cu2+ binding to Aβ range from ∼105 to ∼109; these values’ being obtained for different peptide lengths (1-16, 1-28, 1-40, 1-42) at varying pH. Herein, we report that Cu2+’s binding to Aβ(1-40) at 37 °C occurs in a 1:1 stoichiometry with a pHdependent binding constant: 1.1 ((0.2) × 109 M-1 and 2.4 ((0.2) × 109 M-1 at pH 7.2 and 7.4, respectively. Under identical conditions, Aβ(1-16) reveals a comparable binding constant, confirming that this portion of the peptide is the binding region. Several previously reported values can be reconciled with the current measurement by careful consideration of thermodynamics associated with the presence of competing ligands used to solubilize copper. TABLE 1: Literature Binding Constants and Stoichiometry of Cu2+:Aβ Complexes

Introduction Postmortem brain biopsies of patients with dementia of Alzheimer’s type show extracellular plaque deposits rich in aggregated amyloid beta peptides (Aβ)1 that contain high concentrations of the metals Fe, Cu, and Zn.2,3 Although amyloid beta peptides undergo spontaneous self-aggregation, the binding of Cu2+ accelerates aggregation,4 and Cu2+-mediated aggregation is distinct from self-assembled aggregates.5,6 There is also increasing evidence that Cu2+ binding to Aβ induces redox chemistry and subsequent neurotoxicity.7–9 In vitro studies have showed that Cu2+ in the presence of Aβ forms reactive oxygen species such as H2O2 and possibly · OH via the Fenton reaction.10,11 As a result of these studies, metal chelation therapy is one strategy being developed for the treatment of Alzheimer’s disease.12,13 For example, clioquinol (a Cu2+ chelator) showed diminished Aβ deposition in transgenic mice and favorable preliminary results in a pilot phase 2 clinical trial.14,15 A lipophilic metal chelator, DP-109, also reduced amyloid plaque deposition in transgenic mice.16 Novel multifunctional chelators are now being designed that combine metal chelation with antioxidant ability and blood-brain barrier permeability.17,18 Agents that specifically target amyloid beta19 and nanoparticle delivery systems20 are promising directions in developing metal chelation therapy for Alzheimer’s disease. The potential role of Cu2+ in the pathogenesis of Alzheimer’s disease and the promise of metal chelation therapy has resulted in a concerted effort to quantify the metal binding to the Aβ peptide. Unfortunately, the literature has not achieved consensus as to the stoichiometry and binding affinity (see Table 1). For example, a 2:1 stoichiometry was inferred from Cu2+ EPR signal intensities,21 but a recent study using high resolution electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry reveals a 1:1 complex.22 One important point to note in the table is the use of competing ligands for Cu2+ in * Corresponding author address: Box 90034, Duke University, Durham, NC 27708. Phone: (919) 660-0330. Fax: (919) 684-4421. E-mail: [email protected].

Aβ fragment

K/M-1

Cu2+/Aβ

pH, temperature

competing ligand

ref

1-40a 1-42a 1-40b 1-42b 1-40b 1-42b 1-28 1-40a 1-16 1-16a,b 1-28a 1-28a 1-28a 1-16

6.3 × 105 5.0 × 105 7.9 × 108 1.9 × 108 1.0 × 107 3.2 × 107 ∼107 6.3 × 105 ∼107 5 × 104 2.5 × 106 8.3 × 105 4.0 × 105 1.5 × 109

1:1 1:1 2:1 2:1 2.5:1 2.5:1 2:1 1:1 2:1 2:1 1:1 1:1 1:1 1:1

7.4 7.4 7.4, 37 °C 7.4, 37 °C 6.6, 37 °C 6.6, 37 °C 7.4, 25 °C 7.3, 5 °C 7.8 7.4 7.2, 20 °C 6.5, 20 °C 7.2, 20 °C 7.2, 37 °C

Tris Tris various various various various Gly, His None Gly, His Gly none none none Gly

23 23 24 24 24 24 21 25 26 27 28 28 28 29

a

Calculated by K ) 1/Kd. b Low affinity site.

many of the reported studies. The competing equilibria with these ligands must be correctly modeled to obtain an accurate binding constant. Herein, we use the technique of isothermal titration calorimetry and determine the binding constant of Cu2+ to the Aβ peptide, specifically address the effect of peptide chain length, and reconciled several previously reported values. Experimental Section Materials and Methods. Aβ(1-40) and Aβ(1-16) were purchased from Biosynthesis, Inc. (Lewisville, TX) at >95% purity as custom peptides with no N-terminal or C-terminal modifications or conjugations. CuCl2 · 2H2O, glycine, N-(2hydroxyethyl)piperazine-N′(2-ethanesulfonic acid) (HEPES), and 1,4-piperazinediethanesulfonic acid (PIPES) were purchased from Sigma-Aldrich and were of reagent grade or higher. HEPES and PIPES buffers were prepared in ultrapure (>18 MΩ/cm resistivity) water to a concentration of 20 mM containing 160 mM NaCl buffered to pH 7.2 at 37 °C. CuCl2 solutions were dissolved in HEPES buffer together with 4 mol equiv of glycine to prevent the precipitation of Cu2+ hydroxide species.

10.1021/jp710806s CCC: $40.75  2008 American Chemical Society Published on Web 06/18/2008

Copper binding to Aβ by ITC.

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Full length Aβ peptides are tenaciously self-aggregating, and sample preparation can affect the aggregation state of the peptide.30 It is imperative to rule out experimental discrepancies in thermodynamic measurements that could arise from peptide aggregation. To address this issue, we compared two different preparation methods used by previous groups to generate solubilized monomers of the peptide. The first method involves dissolution of lyophilized peptide in buffer, followed by centrifugation for 10 min at >10 000g, which completely sediments all aggregated peptide.31 Roughly 10 mg of lyophilized Aβ(1-40) peptide was dissolved into 24 mL of HEPES buffer. The mixture was sonicated at 37 °C for 5 min and then centrifuged at 15 500g for 10 min to sediment any aggregated Aβ(1-40). The soluble peptide fraction was frozen at -20 °C in aliquots of 2.2 mL (enough for one ITC run) and thawed immediately before use. The peptide concentration was determined by the Bradford assay (Pierce Biotechnology, Rockford, IL). The second method involves alkaline pretreatment of the lyophilized peptide.32 Approximately 2 mg of lyophilized peptide was dissolved in 100 µL of 1 M NaOH and sonicated for 1 min. The peptide was diluted in 5 mL of HEPES buffer and centrifuged for 10 min at 10 000g. The pH of the resulting soluble Aβ(1-40) was readjusted to 7.2 or 7.4 at 37 °C before running the ITC experiment. The concentration of the final, pHadjusted peptide solution was determined by the Bradford assay. Isothermal Titration Calorimetry. Calorimetric titrations were carried out on a VP-ITC ultrasensitive microcalorimeter (Microcal, Northampton, MA). All solutions were degassed prior to use. The reaction cell contained a 60 µM Aβ(1-40) solution in HEPES buffer. The titrant, 700 µM CuCl2 with 2800 µM glycine also in HEPES buffer, was injected in 8 µL aliquots with 300 s spacing between injections. The cell was stirred at 307 rpm, and the titration was performed at 37 °C. Titrations using glycine alone were run to ensure that glycine was not interacting with the peptide (data not shown). The ITC data were analyzed taking into account the competition between Aβ and the glycine for Cu2+. The details are presented elsewhere;29 here, we provide an abbreviated summary of this quantitative model. When Aβ is titrated with Cu2+-Gly2 from the syringe, the following equilibria must be considered.

Cu2++ Gly h Cu2+-Gly

K1 ) [Cu2+-Gly]/ [Cu2+][Gly] (1)

Cu2+- Gly + Gly h Cu2+-Gly2

K2 ) [Cu2+-Gly2]/ [Cu2+-Gly][Gly] (2)

Aβ + Cu2+ h Cu2+-Aβ

K ) [Cu2+-Aβ]/[Cu2+][Aβ] (3)

The binding constants, K1 and K2 (eqs 1 and 2), and the associated enthalpies of formation for Cu2+-Gly and Cu2+-Gly2, H1 and H2, respectively, are known.33 The fraction of Aβ bound to Cu2+, Θ, is given by

Θ)

K[Cu2+] (1 + K[Cu2+])

(4)

The percentage of Cu2+ present in solution as Cu2+-Gly, Ω1, is given by

Ω1 )

K1[Gly] (1 + K1[Gly] + K1K2[Gly]2)

(5)

Likewise, the percentage of Cu2+ present in solution as Cu2+-Gly2, Ω2, is given by

Ω2 )

K1K2[Gly]2 (1 + K1[Gly] + K1K2[Gly]2)

(6)

All possible species in solution are related by Θ, Ω1, and Ω2 (which are in turn related by K, K1, and K2) and can be written as functions of the total glycine ([Gly]T) and total Cu2+ ([Cu2+]T) concentrations,29 giving rise to the relationship

Θ + [Aβ]TΘ + (Ω1 + Ω2)([Cu]T - [Aβ]TΘ) K(1 - Θ) [Cu]T ) 0 (7) Equation 7 is used to determine [Gly], which is then used to solve for [Cu2+], [Cu2+-Aβ], [Cu2+-Gly] and [Cu2+-Gly2] using eqs 4-6. Knowing the concentrations of all the species in the reaction cell, the change in heat associated with the equilibria in eqs 1-3 can be accurately described,

∆Q ) V0(H∆[Cu-Aβ]) + (H1 + H2)∆[Cu-Gly2] + H1(∆[Cu-Gly]) - injV(H1 + H2)[Cu-Gly2]S injV(H1)[Cu-Gly]S (8) where V0 is the volume of the reaction cell, injV is the titrant injection volume, and [Cu2+-Gly]S and [Cu2+-Gly2]S are the titrant concentrations in the syringe. The heat produced or absorbed per injection must also take into account a dilution factor, Fi ) exp(-injV(i)/V0).34 Therefore, the heat per injection, ∆Qi, is given by

∆Qi ) V0(H([Cu-Aβ]i - [Cu-Aβ]i-1Fi)) + (H1 + H2)([Cu-Gly2]i - [Cu-Gly2]i-1Fi) + H1([Cu-Gly]i - [Cu-Gly]i-1Fi) - (1 - Fi)(H1 + H2) × [Cu-Gly2]S - (1 - Fi)(H1)[Cu-Gly]S (9) The ITC data was fit to eq 9 using nonlinear least-squares implements in Igor Pro (Wavemetrics Inc., Lake Oswego, OR) to determine n (stoichiometry), K, and H for the binding of Cu2+ to the peptide. The reported binding constants are the average of three titrations. Results ITC measures any heat associated with the injection of a titrant into the thermally equilibrated reaction cell. A titration of Cu2+-Gly2 into the ITC reaction cell containing only HEPES buffer is shown in Figure 1. The top figure portrays the net change in heat after each injection, and the bottom displays the integrated heat. The endothermic heats observed early in the titration are representative of the dissociation of Cu2+-Gly2 upon dissolution into the cell. After several injections, the concentration of free glycine in the cell begins to increase, the equilibrium shifts, and dissociation becomes negligible. Thus, the titration reflects the dissociation/association of Cu2+-Gly2 upon injection into the reaction cell. Figure 2 shows the titration of Cu-Gly2 with Aβ(1-40) in HEPES buffer. For this experiment, the solubilized protein was prepared using method 1 described in the Experimental Section. Initial processing of the data with the Origin software provided

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Figure 1. Isothermal titration calorimetry data for Cu2+-Gly2 dissociation upon injection into the reaction cell containing only HEPES buffer, pH 7.2 at 37 °C.

Hatcher et al. ∆H ) -6.80 kcal/mol. When K1, K2, H1, and H2 of Cu2+-Gly2 are incorporated into the thermodynamic analysis,29 the affinity and heat associated with the binding of Cu2+ to Aβ(1-40) is K ) 1.1 ((0.2) × 109 M-1 and ∆H ) -8.32 ((0.04) kcal/mol (pH 7.2, I ) 0.16 M at 37 °C). At pH 7.4 and 37 °C in HEPES, the binding constant increases to 2.4 ((0.2) × 109 M-1. Recent data indicate that HEPES has an affinity for Cu2+,35 so to address the possible role of this buffer interaction on the determined binding constant, the same measurements were performed in PIPES. The corresponding data in PIPES buffer at pH 7.2 results in a binding constant of K ) 0.9 ((0.2) × 109 M-1, indicating that HEPES binding has a negligible effect. The binding of Cu2+ to Aβ(1-16) was also examined in HEPES and PIPES buffer. At pH 7.2 and 37 °C the binding constant in HEPES is 1.5 ((0.2) × 109 M-1. At pH 7.4 at 37 °C, the binding constant increases to 2.9 ((0.2) × 109 M-1, similar to that observed for Aβ(1-40). In PIPES at pH 7.4 at 37 °C, the binding constant is 3.0 ((0.2) × 109 M-1, again confirming that the measurements is independent of the buffer being used. The thermodynamic model used to quantify the binding constant from the ITC isotherm can be extended to include association of Cu2+ with HEPES. This derivation is presented in the Supporting Information and also demonstrates that the effect of buffer binding is negligible. To further address sample preparation, the same experiments were performed on solubilized protein prepared using method 2, described above. Peptide produced in this manner reveals a binding constant of 1.3 ((0.2) × 109 M-1 at pH 7.2 and 37 °C. Thus, the result is independent of the method use to prepare the monomeric peptide and is only weakly dependent on pH over the small range representative of the majority of the studies reported to date. Discussion

Figure 2. Titration of Cu-Gly2 and 60 µM Aβ(1-40) with both solutions in 20 mM HEPES buffer, pH 7.2 at 37 °C, I ) 0.16 M. The solid line in the bottom plot results from a nonlinear least-squares fit of eq 9 to the data. This fit reveals the affinity of Cu2+ for Aβ(1-40) is K ) 1.1((0.2) × 109 M-1 and ∆H ) -8.32 ((0.04) kcal/mol under these conditions.

by the manufacturer, which treats the equilibrium of Cu2+ with the peptide but ignores all competing equilibrium between the metal and glycine, gave a binding constant of 4.8 × 105 and

Table 1 presents a compilation of the values reported for the Cu2+-Aβ binding constant. A close look at this table indicates that the experimental results span a range peptide chain lengths. One must therefore consider whether the range of values reported is due to the dependence of binding constant on these factors. At pH 7.2, 37 °C, an identical analysis of the ITC data for Aβ(1-16) gives a binding constant of 1.5 × 109 M-1.29 This comparison establishes that the range in reported values (Table 1) is not a result of the different peptide chain lengths studied. The binding constant of Aβ(1-42) could not be determined from ITC because considerable aggregation occurred during the titration. The dissociation/association of Cu2+-Gly depicted in Figure 1 clearly shows that additional equilibria must be addressed to perform an accurate thermodynamic analysis of Cu2+’s binding to Aβ(1-40). One final concern that needs to be addressed is the possible formation of ternary complexes of Gly with the Cu2+-Aβ complex. The Supporting Information presents the results of a mass spectral analysis that demonstrates glycine does not form a ternary complex with Cu2+-Aβ(1-16) under the experimental conditions used in the ITC measurements. We now demonstrate that the major reason for the disparity of results in Table 1 results from neglecting the effects of competing ligands or buffers. First, consider the binding constant of ∼107 for Aβ(1-16), which was determined by measuring how added Gly and histidine (His) restored peptide fluorescence initially quenched through complexation with Cu2+.26 The analysis used neglected the fact that the binding of either Gly or His to Cu(II) occurs in

Copper binding to Aβ by ITC.

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a stoichiometric ratio of 2:1. The reported results indicate that when the glycine concentration is 40 times that of peptide, the tyrosine fluorescence signal is half of the maximum value or that half of the peptides present are bound to Cu2+. When half of the peptides present are bound to Cu2+, the binding constant is given by K ) 1/[Cu2+], where [Cu2+] is the concentration of free Cu2+ in solution, not ligated to either peptide or glycine. We have previously shown that by taking into account the Cu2+ glycine equilibria and adjusting for pH, this experiment reveals a binding constant of ∼2.5 × 109 M-1 at pH 7.2 and 37°,29 which is in excellent agreement with the measurements reported herein. Now consider the dissociation constant for Cu2+ to Aβ(1-40) of 1.6 µM (K ) 6.25 × 105 M-1) determined by fluorescence quenching.23 Those experiments were performed using Tris buffer, which in excess concentration competes for Cu2+ and is not considered in the data analysis. Therefore, the binding constant determined corresponded to the following chemical process:

Aβ + Cu(Tris)nf r Cu-Aβ

(11)

Tris

with an associated equilibrium constant of

K′ )

[Cu-Aβ]

(12)

[Aβ]([Cu] + [Cu(Tris)] + [Cu(Tris)2] + [Cu(Tris)3] + [Cu(Tris)4]

The desired binding constant, K ) [Cu2+-Aβ]/([Cu2+][Aβ]), is related to K′ by:

K ) K ′ × (1 + K1[Tris] + K1K2[Tris]2 + K1K2K3[Tris]3 + 4

K1K2K3K4[Tris] ) (13) where Ki values represent the sequential binding constants of Cu2+ to Tris: K1 ) 2.5 × 103 M-1, K2 ) 6.3 × 102 M-1, K3 ) 6.3 × 102 M-1, and K4 ) 2.0 × 102 M-1 at pH 7.4 (these values were calculated for pH 7.4 using data from NIST, as pKa ) 8.1 and log K1)4.1, log K2 ) 3.5, log K3 ) 3.5, and log K4 ) 3.0 at 25 °C with ionic strength at 0.1 M),33 for the addition of 1, 2, 3, and 4 Tris molecules, respectively. The 10 mM Tris buffer used in that work leads to a proportionality factor between K and K′ of 3.2 × 103, and so the Cu2+-Aβ binding constant obtained was actually 1.6 × 109 M-1, which is in excellent agreement with our current finding. Now consider binding studies by Guilloreau and co-workers, who reported two binding sites determined from ITC and fluorescence experiments.27 The distinctly different ITC isotherm and fitting result are likely an outcome of experimental conditions. In particular, the Cu2+ titrant added in a 10 mM HCl solution would produce a huge signal for buffer ionization due to the large pH difference between the syringe and the cell (pH 2 and 7.4, respectively). However, they also present Cu-Aβ fluorescence quenching and recovery by competition with glycine. Analyzing the data with a thermodynamic 1:1 stoichiometry binding model, K ) 1/[Cu] when half the Aβ is bound to Cu2+. Given the 600 µM glycine added to restore half the fluorescence intensity, accounting for the glycine bound Cu2+, the concentration of free Cu2+ is actually ∼4 nM; thus, K ∼ 2.5 × 108 M-1. The exact values cannot be calculated from the graph estimations, but the binding constant determined independently of glycine competition is much higher than previously reported and is more in line with the binding constant determined in this report.

Finally, consider the results of a competitive metal capture analyses used to quantify the binding affinity of Cu2+ to Aβ(1-40) and Aβ(1-42).24 This approach yielded a highaffinity binding site and a low-affinity binding site at pH 7.4 and 37 °C. The substoichiometric high-affinity binding site has not been observed by other laboratories, so we therefore focus on the low-affinity site in this discussion. The thermodynamic analysis considered only the K1 of each chelator when several chelators bound Cu2+ in a 2:1 ratio having two equilibrium constants, K1 and K2. Omitting the second Cu-Gly2 binding constant leads to a significantly lower calculated Cu-Aβ binding constant. Although we can not calculate the offset from the given experimental details, the effect of considering only K1 in the analysis of our ITC data results in a binding constant 2 orders of magnitude lower than when both K1 and K2 of the chelator are taken into account. The disparate binding constants obtained from experiments using unchelated Cu2+ present different issues to consider. Free Cu2+ has several complications, including limited solubility at neutral or basic pH; the solubility product constant (Ksp) of Cu(OH)2 is 2.2 × 10-20.34 In the presence of phosphate buffer, Cu2+ may also precipitate as the phosphate salt; Ksp of Cu3(PO4)2 is 1.4 × 10-37.35 Unchelated Cu2+ was also used in a study in which the binding constant was derived from NMR signal intensities.25 Although the key structural insight of anomalous Cu2+ binding to Aβ(1-40) is valid, the derivation of a dissociation constant from the decrease in the NMR signal intensity could be problematic: the signal decrease can also occur from peptide aggregation or a general loss in signal-to-noise ratio, as noted in the article. Conclusion Metal chelators that remove Cu2+ from Aβ can be an effective strategy to suppress formation of reactive oxygen species and neurotoxicity in AD pathology. This work establishes that the Cu2+-Aβ binding constant is 1.1 ((0.2) × 109 M-1 and 2.4 ((0.2) × 109 M-1 at pH 7.2 and 7.4, respectively. Under identical conditions, Aβ(1-16) reveals a similar binding constant, confirming that this portion of the peptide is the binding region. Previous Cu2+ binding studies did not quantitatively account for the competition of the buffer of experimental chelators, and inclusing these competing equilibria in the analysis of those data also yields binding constants on the order of 109 M-1. Thus, in designing molecule chelating agents for Alzheimer’s disease that target Cu2+, the chelator affinity must be higher than 1 × 109 M-1 but lower than that of essential Cu-metalloenzymes, such as Cu-Zn-SOD, which is on the order of 1015 M-1. Acknowledgment. We are grateful to the MFEL program administered by the AFSOR for support of this research. Supporting Information Available: The theoretical model used to analyze the ITC is extended to take into account known binding of Cu2+ to HEPES. The generated isotherms ignoring and accounting for HEPES are compared. The results demonstrate that under the experimental conditions used in this study, HEPES binding to Cu2+ does not affect the determined binding constant for Cu2+ to the Aβ peptide. ESI-MS data is presented that also rules out the presence of ternary complexes between the peptide, copper and glycine. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Morgan, C.; Colombres, M.; Nunez, M. T.; Inestrosa, N. C. Prog. Neurobiol. 2004, 74, 323.

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