redox cycling of iron by aβ42

/freeradbiomed

Free Radical Biology & M

Original Contribution

Redox cycling of iron by Ah42

Ayesha Khan a, Jon P. Dobson b, Christopher Exley a,*

a Birchall Centre for Inorganic Chemistry and Materials Science, Lennard-Jones Laboratories, Keele University, Staffordshire ST5 5BG, UKb Institute for Science and Technology in Medicine, Keele University, Staffordshire ST5 5BG, UK

Received 27 June 2005; revised 31 August 2005; accepted 7 September 2005

Available online 14 October 2005

Abstract

The amyloid cascade hypothesis and oxidative damage have been inextricably linked in the neurodegeneration that is characteristic of

Alzheimer_s disease. We have investigated this link and sought to suggest a mechanism whereby the precipitation of Ah42 might contribute to the

redox cycling of iron and hence the generation of reactive oxygen species via Fenton-like chemistry. We have shown that the critical step in the

auto-oxidation of Fe(II) under the near-physiological conditions of our study involved the generation of H2O2 via O2S� and that Ah42 influenced

Fenton chemistry through aggregation state-specific binding of both Fe(II) and Fe(III). The net result of these interactions was the delayed

precipitation of kinetically redox-inactive Fe(OH)3(s) such that Fe(II)/Fe(III) were cycled in redox-active forms over a substantially longer time

period than if peptide had been absent from preparations. The addition of physiologically significant concentrations of either Cu(II) or Zn(II)

reduced the role played by Ah42 in the Fe(II)/Fe(III) redox cycle whereas a pathophysiologically significant concentration of Al(III) potentiated

the redox cycle in favour of Fe(II) whether or not Cu(II) or Zn(II) was additionally present. The results support the notion that oxidative damage in

the immediate vicinity of, for example, senile plaques, may be the result of Fenton chemistry catalysed by the codeposition of Ah42 with metals

such as Fe(II)/Fe(III) and Al(III).

D 2005 Elsevier Inc. All rights reserved.

Keywords: Amyloid; Alzheimer_s disease; h-Sheet; Fenton chemistry; Iron; Aluminum; Biological oxidation; Free radical

Introduction

Fibrillar deposits of h-amyloid (Ah) are neurotoxic in vivo

and may be involved in the pathogenesis of Alzheimer_sdisease (AD) [1]. Neurotoxicity may be mediated by fibrillar

Ah per se, prefibrillar Ah in equilibrium with precipitated

amyloid, or a combination of solution (colloidal) and ‘‘solid

phase’’ Ah. The mechanism of in vivo toxicity is unknown

though neuronal damage occurring in the immediate vicinity of

fibrillar amyloid deposits is often associated with markers for

oxidative stress [2]. In addition there is also evidence that Ahin senile plaques is itself oxidatively modified [3] and that

codeposits of iron and Ah are significant sources of reactive

oxygen species (ROS) [4]. Senile plaques are sinks for metals

which are both redox active, such as iron and copper, and redox

inactive, such as zinc and aluminum [5], and synergies between

0891-5849/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.freeradbiomed.2005.09.013

Abbreviations: Ah, h-amyloid; ROS, reactive oxygen species; KH, Krebs-

Henseleit; ThT, Thioflavin T.

* Corresponding author.

E-mail address: [email protected] (C. Exley).

different metals codeposited with different forms of Ah might

define the neurotoxic potential of amyloid deposits in vivo [6].

Ferrous iron, Fe(II), catalyses the formation of ROS, such as

the hydroxyl radical (OHS), by its reaction with hydrogen

peroxide (H2O2). However, extremely low concentrations of

Fe2+ and competitive substrates for H2O2, such as the enzyme

catalase, would normally ensure that this reaction was not

favoured in vivo. Therefore, it was surprising to read that Fe(II)

had been identified associated with senile plaques [4] and even

more surprising to learn that the origin of such could be the

reduction of Fe(III) in the presence of Ah42 [7]. If senile

plaques were catalysts for the formation of Fe(II) then this

might explain their association with the generation of ROS and

neuronal damage in their immediate vicinities [8].

Ah42 localised to senile plaques has been shown to adopt h-pleated sheet conformation [3] and it is this fibrillar Ah in

concert with iron which is now implicated in the formation of

ROS [9]. However, several other metals are found associated

with senile plaques [5] and either alone or in tandem with iron

may influence the conformation of deposited Ah42 [10] and its

potential to catalyse the formation of ROS [11–13]. We have

edicine 40 (2006) 557 – 569

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A. Khan et al. / Free Radical Biology & Medicine 40 (2006) 557–569558

investigated the role of the aggregation state of Ah42 in

promoting the redox cycling of Fe(II) and the concomitant

effects of companion metals, aluminum, copper, and zinc.

Materials and methods

Peptide preparations

Ah42 was purchased as the lyophilised salt (Bachem,

Saffron Walden, UK), dissolved in 0.010 mM NaOH in

ultrapure water (conductivity <0.067 AS/cm, Elga, UK) to

give a ca 0.200 mM stock solution. This stock was then

centrifuged (15,000 rpm for 300 s) and divided into an

appropriate number of aliquots which were immediately frozen

Fig. 1. Summary of the different preparations, their conditions of incubation, the sam

of Fe(II) following the addition of either Fe(II) or Fe(III).

and thereafter thawed and used within 3 days of storage at

�20-C. Peptide aliquots were diluted into an appropriate

volume of modified Krebs-Henseleit (KH) medium (NaCl,

123.5 mM; KCl, 4.8 mM; MgSO4, 1.2 mM; CaCl2, 1.4 mM;

glucose, 11.0 mM) which was buffered at pH 7.40 T 0.05 with

100 mM Pipes (1,4-piperazinediethanesulfonic acid) to give

nominal [Ah42] of 0.29, 1.83, and 3.52 AM. Al(III), Fe(III),

Cu(II), and Zn(II) were added to preparations as certified

stocks of their nitrate salt (Perkin-Elmer, Beaconsfield, UK)

whereas Fe(II) was added each time from freshly prepared

stocks of 1.50 mM FeCl2 in 2% v/v HCl (Riedel de Haan,

Poole, UK). A summary of the peptide preparations and their

respective peptide-free controls is shown schematically in Fig.

1. The peptide preparations are also listed in Tables 1–3.

pling to determine aggregation states, and the spectrophotometric measurement

Table 1

The influence of the concentration of Ah42 on Thioflavin T fluorescence (AU)

of preparations (a–c) incubated at 37-C for 1, 24, or 48 h (pre-Fe(II) addition)

and the Thioflavin T fluorescence (AU) and concentration (AM) of Fe(II)

([Fe2+]T) following the addition of 5.0 AM Fe(II) and incubation at 37-C for a

further 30 min (post-Fe(II) addition)

Preparation Incubation time

1 hour 24 hours 48 hours

(a) 0.29 lM Ab42 + Fe(II)

ThT fluorescence

Pre-Fe(II) 4 (0.3) 6 (1.0) 4 (0.4)

Post-Fe(II) 4 (0.4) 5 (0.7) 3 (0.4)

Fe(II) concentration

[Fe2+]T 0.84 (0.055) 1.57 (0.204) 1.35 (0.123)

(b) 1.83 lM Ab42 + Fe(II)

ThT fluorescence

Pre-Fe(II) 46 (20.7) 60 (24.8) 41 (22.5)

Post-Fe(II) 26 (14.2) 36 (11.8) 21 (15.0)


[Fe2+]T 1.14 (0.223) 2.09 (0.303) 1.92 (0.368)

(c) 3.52 lM Ab42 + Fe(II)

ThT fluorescence

Pre-Fe(II) 189 (31.5) 192 (18.3) 130 (23.4)

Post-Fe(II) 137 (68.6) 110 (23.6) 60 (12.0)


[Fe2+]T 0.98 (0.163) 3.10 (0.300) 3.14 (0.901)

Mean and SD are given, n = 15.

Fig. 2. The auto-oxidation for 150 min of 5.0 AM Fe(II) added in modified KH

medium buffered at pH 7.40 T 0.05 and 37-C compared to the same conditions

but including either 8 mM sodium azide or 10 AM ascorbic acid. Mean and SD

are plotted, n = 5.

A. Khan et al. / Free Radical Biology & Medicine 40 (2006) 557–569 559

Spectrophotometric determination of Fe(II)

The reaction of Fe(II) with 1,10-phenanthroline to form a

coloured complex with an absorption maximum at around 510

nm is a tried and tested method for the determination of Fe(II) in

aqueous solutions at near-neutral pH [14]. The application of

the Beer-Lambert law and the extinction coefficient for the

Fe(II)–phenanthroline complex (11,110 M�1 cm�1) allows for

the precise determination of Fe(II) in the submicromolar range

and, providing that 1,10-phenanthroline is present to excess, the

method is free of interferences from other metals (e.g., Fe(III),

Al(III), Cu(II), Zn(II)) bound by this ligand. We tested the

possible interference of each and combinations of these metals

on the determination of Fe(II) and confirmed the lack of any

interferences under the conditions of our study. We optimised

the application of this method to our assay conditions by

measuring the auto-oxidation of added Fe(II) over time and

included either 8 mM sodium azide (NaN3) as an accelerant or

10 AM ascorbic acid as an inhibitor of auto-oxidation (Fig. 2).

We used these data to choose 30 min as an appropriate time

period for auto-oxidation for all further auto-oxidation assays.

Thioflavin T fluorescence

Thioflavin T (ThT) is a benzothiazole dye which undergoes

a characteristic spectral alteration upon being bound by h-pleated sheet structures [15]. It is not bound by monomers or

oligomers of Ah42 nor is it bound by amorphous precipitates

of the peptide and in this study it was used to identify and

loosely quantify the formation of h-pleated fibrils of Ah42.

The assay has been described in detail elsewhere [16] but

briefly 100 AL of peptide (or control) preparation was sampled

as per Fig. 1 and mixed with 10 AL of ThT such that the

concentration of the latter was 10 AM and significantly in

excess of the concentration of peptide. The ThT fluorescence

of each sample was then determined at 37-C over a 300 s

period (Perkin-Elmer LS50B Luminescence Spectrometer; Ex,

450 nm, 5 nm bandpass; and Em, 482 nm, 10 nm bandpass),

the latter ensuring that a stable ThT fluorescence had been

reached before the reading was taken. Fluorescence readings

for peptide-free preparations, though usually very low, less

than 5 fluorescence units (<5 AU), were substracted from the

appropriate peptide preparation.

Transmission electron microscopy

Samples for TEM were taken according to the protocol in

Fig. 1, mounted on formvar-coated copper grids, negatively

stained with 2% uranyl acetate, and viewed using a JEOL JEM-

1230 electron microscope.

Statistical analyses

Statistical analyses were carried out using the Mann-

Whitney test (Minitab, version 14). This performs a hypothesis

test of the equality of two population medians and calculates

the confidence level and was used as a nonparametric

alternative to the two-sample t test due to data not being

normally distributed. Confidence levels > p = 0.05 were

rejected as not being statistically significant.

Results

To determine how solution chemistry in the absence of Ah42

influenced the auto-oxidation of Fe(II) all peptide preparations

were compared with peptide-free preparations. In this way it

was possible to determine how the presence of peptide

contributed to the auto-oxidation of Fe(II) under each set of


experimental conditions. In addition this allowed computation

of the peptide-attributable [Fe2+] which was defined as the

difference between the [Fe2+] measured in the presence of

peptide and the [Fe2+] measured in the corresponding peptide-

free preparation:

½Fe2þ�PA

¼ Fe2þ� �

T� Fe2þ� �

C:

How did [Ab42] and its time-dependent formation of b-pleatedsheets affect [Fe2+]

At the lowest [Ah42] of 0.29 AM and following only a

1-h incubation at 37-C the presence of h-pleated conformers of

peptide was indicated by ThT fluorescence. The measured

values of 3–6 AU were close to the limit of detection of this

application of the assay and did not change significantly ( p >

0.05) following incubation of this concentration of peptide for a

further 24 and 48h (Table 1). This [Ah42] increased [Fe2+]Trelative to peptide-free controls at 1 h ( p = 0.003), 24 h ( p <

0.001), and 48 h ( p < 0.001) though the increases were not time

dependent with the highest [Fe2+]T (1.57 T 0.204 AM ) which

represented ca 30% of added Fe(II), being measured in peptide

incubated for 24 h. A sixfold increase in [Ah42] to 1.83 AMproduced significantly higher ThT fluorescence (20–80 AU)

though the extent of formation of h-pleated conformers was not

significantly influenced ( p > 0.05) by ageing peptide solutions

for a further 24 or 48 h (Table 1). Once again, the presence of

peptide significantly increased [Fe2+]T relative to peptide-free

controls at 1, 24, and 48 h ( p < 0.001) and the highest [Fe2+]T(2.09 T 0.303 AM), measured at 24 h, represented ca 40% of the

added Fe(II). At 3.52 AM Ah42 the formation of h-pleatedconformers was extensive after only a 1-h incubation (189 T31.5 AU), remained unchanged over the next 24 h (193 T 18.3

AU), and fell significantly ( p < 0.001) by 48 h (130 T 23.4 AU)(Table 1). At 1 h incubation [Fe2+]T was not significantly

increased with respect to the peptide-free control ( p = 0.054)

whereas the increases were highly significant at both 24 and

48 h ( p < 0.001). Ageing of the peptide solutions resulted in

significant increases in [Fe2+]T from 0.98 T 0.163 AM at 1 h to

3.10 T 0.300 AM at 24 h ( p < 0.001) and a further

insignificant increase ( p = 1.000) from 24 to 48 h (3.14 T0.901 AM). The latter was equivalent to ca 65% of added

Fe(II). Ah42 was clearly influential in determining [Fe2+]Tthough there was not a clear relationship between the extent

of formation of h-pleated conformers of Ah42 and [Fe2+]T.

For example, at 3.52 AM Ah42 after 1 h incubation the ThT

fluorescence was 189 T 31.5 AU and the corresponding

[Fe2+]T was 0.98 T 0.163 AM whereas after 24 h incubation of

the peptide the ThT fluorescence was unchanged at 193 T18.3 AU while the [Fe2+]T was 3.10 T 0.300 AM. The

apparent lack of an association between ThT fluorescence and

[Fe2+]T was supported by an insignificant correlation coeffi-

cient (r2 = 0.113) when ThT fluorescence prior to Fe(II)

addition for all [Ah42] was plotted against their corresponding

[Fe2+]T. However, when the same analyses were carried out

using the control-subtracted data, [Fe2+]PA, a much more

positive correlation (r2 = 0.267) suggested, if only weakly,

that higher pre-Fe(II) addition ThT fluorescence resulted in

higher [Fe2+]PA (Fig. 3a). When this analysis was broken

down to show how the correlation between pre-Fe(II) addition

ThT fluorescence and [Fe2+]PA was influenced by the three

different [Ah42] it was found to be strongest for the

intermediate [Ah42]; the r2 were 0.078, 0.279, and 0.027

for 0.29, 1.83, and 3.52 AM Ah42, respectively (Figs. 3b–d).

This, along with the observation that a 12-fold increase in

[Ah42] (i.e., 0.29 to 3.52 AM) only resulted in a 2-fold

increase in [Fe2+]PA, may have suggested that some aspect of

the aggregation state of Ah42 was more important than its

absolute concentration in determining [Fe2+]T.

A consistent effect which was measured for each of the

[Ah42] (though not a statistically significant effect for 0.29 AMAh42) was that the addition of Fe(II) and the subsequent

incubation for 30 min always resulted in a significant fall in

ThT fluorescence over this short period. This remarkable effect

was loosely associated with [Fe2+]T in that proportional

reductions in ThT fluorescence were consistently greater in

those treatments in which [Fe2+]T were highest. For example,

for 3.52 AM Ah42 incubated for 1 h the 28% reduction in ThT

fluorescence which followed the addition of 5.0 AM Fe(II) was

associated with an [Fe2+]T of 0.98 T 0.163 AM whereas for the

same peptide incubated for 48 h the 54% reduction in ThT

fluorescence which followed the addition of 5.0 AM Fe(II) was

associated with an [Fe2+]T of 3.14 T 0.901 AM.

How was the influence of 3.52 lM Ab42 on [Fe2+] affected by

the additional presence of Al(III), Cu(II), and Zn(II)

Peptide-free preparations

The auto-oxidation at 37-C over 30 min of 5.0 AM Fe(II)

added to peptide-free preparations which had previously been

incubated at 37-C for 1, 24, and 48 h resulted in each case in ca

85% of the available Fe(II) being oxidised to Fe(III) (Table 2).

For example, for the 1 h preparation, [Fe2+]C fell from a

maximum possible concentration of 4.75 T 0.206 to 0.83 T0.105 AM. When the peptide-free preparations also included

8 mM NaN3, a known scavenger of OHS, the auto-oxidation

was accelerated significantly ( p < 0.001) such that more than

95% of the available Fe(II) was oxidised (Table 2). For

example, for the 1 h preparation, [Fe2+]C fell from a maximum

possible concentration of 4.75 T 0.206 to 0.24 T 0.075 AM. The

opposite effect was found for peptide-free preparations which

additionally included 5.0 AM Al(III), a known pro-oxidant,

such that [Fe2+]C in these preparations was only reduced by

50% (Table 2). For example, for the 1 h preparation, [Fe2+]Cfell from a maximum possible concentration of 4.13 T 0.374 to

2.15 T 0.155 AM. When 5.0 AM Fe(III) was added to peptide-

free preparations instead of Fe(II) the resultant [Fe2+]C were

very low, for example, 0.14 T 0.050 AM for the 1 h preparation,

and suggested that the peptide-free preparations were not

themselves capable of reducing Fe(III) to Fe(II). The additional

presence of 5.0 AM Al(III) in these Fe(III) preparations did not

influence the measured [Fe2+]C and this showed that Al(III) per

se could not reduce Fe(III) to Fe(II) (Table 2). The additional

presence of 10.0 AM Cu(II), a redox active metal, accelerated

Fig. 3. (a–d) The relationship between ThT fluorescence prior to the addition of 5.0 AM Fe(II) and [Fe2+]PA measured 30 min after its addition for all incubations (1,

24, and 48 h) at each [Ah42]. (a) All data; (b) 0.29 AM Ah42; (c) 1.83 AM Ah42; (d) 3.52 AM Ah42.


the auto-oxidation of Fe(II) significantly, such that almost 99%

of the available Fe(II) was oxidised. For example, for the 1

h preparation, [Fe2+]C fell from a maximum possible concen-

tration of 4.42 T 0.191 to 0.11 T 0.038 AM. Cu(II) was

significantly more effective than NaN3 in accelerating the

oxidation of Fe(II) to Fe(III). The additional presence of 5.0

AM Al(III) in these ‘‘copper’’ preparations increased [Fe2+]Csignificantly ( p < 0.05) though only to concentrations which

were similar to those measured for Fe(II) in the presence of

NaN3 (Table 2). When 5.0 AM Fe(II) was added to peptide-free

preparations which also included 10.0 AM Zn(II) its auto-

oxidation was delayed such that more than 25% of the added

Fe(II) remained in this form after 30 min incubation at 37-C.For example, in the preparation which had previously been

aged for 48 h, [Fe2+]C fell from 4.61 T 0.198 to 1.21 T0.241 AM. The additional inclusion of 5.0 AM Al(III) in

these ‘‘zinc’’ preparations potentiated the influence of Zn(II),

for example, in preparations aged for 1 h the [Fe2+]C were

1.08 T 0.123 AM in the absence of Al(III) and 1.23 T 0.158

AM in the presence of Al(III). Intriguingly the latter [Fe2+]Cwere significantly less than the equivalent ‘‘aluminium’’

preparations in the absence of added Zn(II) (2.15 T 0.155 AM)

(Table 2).

Peptide preparations

In the preparation which included only 3.52 AM Ah42 and

no added metals incubation for 1 h did not have a significant

influence on [Fe2+]T when compared with peptide-free controls

( p > 0.05) whereas the same concentration of peptide aged for

24 and 48 h resulted in significantly increased [Fe2+]T ( p <

0.001). The influence of peptide was greatest in solutions aged

for 24 and 48 h, in which up to 70% of the available Fe(II) was

measured, though [Fe2+]T in these aged treatments were not

significantly different from each other ( p > 0.05). In peptide

preparations which had been aged for either 24 or 48 h, but not

the 1 h preparation, the major proportion of [Fe2+]T was

[Fe2+]PA (Table 2).

In preparations which included NaN3 the presence of 3.52

AM Ah42 which had been incubated for 1, 24, and 48 h resulted

in significantly higher [Fe2+]T at each time point relative to the

respective peptide-free preparations ( p < 0.001). When [Fe2+]Tfor Ah42 T NaN3 were compared the presence of NaN3 had no

significant influence at t = 1 h (0.98 T 0.163 and 0.93 T 0.326 AMfor Fe(II) in the absence and presence of NaN3, respectively) and

significantly reduced [Fe2+]T at both 24 h ( p < 0.001) (3.10 T0.300 and 1.33 T 0.361 AMfor Fe(II) in the absence and presence

of NaN3 respectively) and 48 h (3.14 T 0.901 and 1.65 T 0.632

AM for Fe(II) in the absence and presence of NaN3, respectively)

( p = 0.002). Control-subtracted data showed that [Fe2+]PA for

Fe(II) T NaN3 was significantly higher at 1 h in the presence of

NaN3 ( p = 0.003), significantly lower at 24 h in the presence of

NaN3 ( p < 0.001) and insignificantly lower at 48 h in the

presence of NaN3 ( p < 0.05) (Table 2).

In preparations which included Al(III) the presence of

peptide and incubation for 1, 24, and 48 h resulted in

significantly higher [Fe2+]T relative to peptide-free controls

Table 2

The concentrations (AM) of Fe(II) (T, total; C, control; PA, total–control) in

each of the preparations (a– i) following their incubation at 37-C for 1, 24, or

48 h and the subsequent addition of either 5.0 AM Fe(II) (a,c,e,f,g,h,i) or 5.0

AM Fe(III) (b,d) and incubation for a further 30 min at 37-C



(a) Ab42 alone + Fe(II)

[Fe2+]T 0.98 (0.163) 3.10 (0.300) 3.14 (0.901)

[Fe2+]C 0.83 (0.105) 0.73 (0.117) 0.95 (0.080)

[Fe2+]PA 0.15 (0.163) 2.38 (0.300) 2.19 (0.901)

(b) Ab42 alone + Fe(III)

[Fe2+]T 0.60 (0.089) 1.48 (0.219) 2.29 (0.479)

[Fe2+]C 0.14 (0.050) 0.15 (0.068) 0.15 (0.070)

[Fe2+]PA 0.47 (0.089) 1.34 (0.219) 2.13 (0.479)

(c) Ab42 + Al(III) + Fe(II)

[Fe2+]T 2.79 (0.103) 3.90 (0.375) 4.27 (0.586)

[Fe2+]C 2.15 (0.155) 2.14 (0.224) 2.25 (0.779)

[Fe2+]PA 0.64 (0.104) 1.76 (0.375) 2.01 (0.586)

(d) Ab42 + Al(III) + Fe(III)

[Fe2+]T 0.77 (0.182) 1.98 (0.536) 2.34 (0.477)

[Fe2+]C 0.10 (0.045) 0.12 (0.048) 0.11 (0.047)

[Fe2+]PA 0.67 (0.182) 1.86 (0.536) 2.23 (0.477)

(e) Ab42 + NaN3 + Fe(II)

[Fe2+]T 0.93 (0.326) 1.33 (0.361) 1.65 (0.632)

[Fe2+]C 0.24 (0.075) 0.23 (0.092) 0.21 (0.087)

[Fe2+]PA 0.69 (0.326) 1.10 (0.361) 1.44 (0.632)

(f) Ab42 + Cu(II) + Fe(II)

[Fe2+]T 0.66 (0.143) 0.76 (0.219) 0.72 (0.330)

[Fe2+]C 0.11 (0.038) 0.06 (0.041) 0.07 (0.039)

[Fe2+]PA 0.55 (0.143) 0.70 (0.219) 0.64 (0.330)

(g) Ab42 + Cu(II) + Al(III) + Fe(II)

[Fe2+]T 0.84 (0.213) 2.12 (0.437) 1.76 (0.242)

[Fe2+]C 0.33 (0.083) 0.31 (0.102) 0.26 (0.139)

[Fe2+]PA 0.52 (0.161) 1.81 (0.437) 1.50 (0.242)

(h) Ab42 + Zn(II) + Fe(II)

[Fe2+]T 1.63 (0.102) 1.57 (0.155) 1.17 (0.083)

[Fe2+]C 1.08 (0.123) 1.19 (0.117) 1.21 (0.241)

[Fe2+]PA 0.62 (0.102) 0.41 (0.155) 0.10 (0.080)

(i) Ab42 + Zn(II) + Al(III) + Fe(II)

[Fe2+]T 2.12 (0.349) 2.52 (0.233) 1.86 (0.094)

[Fe2+]C 1.23 (0.158) 1.44 (0.140) 1.71 (0.443)

[Fe2+]PA 1.07 (0.349) 1.29 (0.233) 0.36 (0.094)



( p < 0.001). In the presence of peptide [Fe2+]T increased

significantly ( p < 0.001) between preparations incubated for 1

(2.79 T 0.103 AM) and 24 h (3.90 T 0.375 AM) and

insignificantly ( p = 0.340) between 24 and 48 h (4.27 T0.586 AM). For Fe(II) T Al(III) the presence of Al(III)

increased [Fe2+]T significantly in preparations incubated for 1

h ( p < 0.001), 24 h ( p < 0.001), and 48 h ( p = 0.006) while for

[Fe2+]PA the increases were only significant at 1 h ( p < 0.001)

and 24 h ( p = 0.003) (Table 2).

When Fe(III) instead of Fe(II) was added to 3.52 AM Ah42

and incubated for 1, 24, and 48 h the [Fe2+]T were significantly

( p < 0.001) increased relative to peptide-free controls for each

time point. In addition, [Fe2+]T increased significantly ( p <

0.001) between peptide preparations incubated for 1 (0.60 T0.089 AM) and 24 h (1.48 T 0.219 AM) and 24 and 48 h (2.29 T0.479 AM). The [Fe2+]T measured for peptide + Fe(III) were

significantly lower than for peptide + Fe(II) at 1 and 24 h ( p <

0.001) and insignificantly lower ( p = 0.142) at 48 h. For

[Fe2+]PA the values for peptide + Fe(III) were significantly

higher at t = 1 h ( p < 0.001), significantly lower at t = 24 h ( p <

0.001) and not significantly different ( p = 1.000) at 48 h. The

additional presence of Al(III) in these Fe(III) preparations

resulted in [Fe2+]T which were significantly increased ( p <

0.001) relative to the respective peptide-free controls. In the

presence of added Al(III) there were also small, but significantly

increased, [Fe2+]T in Fe(III) preparations incubated for 1 h ( p =

0.031) and 24 h ( p = 0.010) but not at 48 h ( p = 0.724). The

same trends, significant increases at 1 and 24 h and no

significant difference at 48 h, were observed for [Fe2+]PA in

Fe(III) preparations which included added Al(III) (Table 2).

In preparations which included Cu(II) the presence of Ah42

resulted in [Fe2+]T which were significantly higher ( p < 0.001)

than the respective peptide-free controls. However, [Fe2+]Twere low (0.66 T 0.143 AM at t = 1 h), less than 20% of added

Fe(II), and significantly lower ( p < 0.001) than the respective

Cu(II)-free preparations and they were not influenced by

incubation of the peptide for 24 or 48 h (Table 2). They were

also not significantly different than their respective [Fe2+]PA,

which suggested a significant role for Ah42 in maintaining their

concentrations. The additional presence of 5.0 AM Al(III)

added in the Ah42 + Cu(II) preparations resulted in signifi-

cantly increased [Fe2+]T at each time point ( p < 0.001), for

example, at t = 24 h it increased from 0.76 T 0.219 to 2.12 T0.437 AM, which equated to approximately 50% of added

Fe(II). The majority of the influence of Al(III) appeared to be

mediated through the [Fe2+]PA fraction, for example, again at t =

24, this was increased from 0.70 T 0.219 AM in the absence of

Al(III) to 1.81 T 0.437 AM in its presence (Table 2).

When Fe(II) was added to Ah42 preparations which

included 10 AM added Zn(II) the resultant [Fe2+]T were, in

comparison with their peptide-free controls, significantly

higher at t = 1 h and t = 24 h ( p < 0.001) and not significantly

different at t = 48 h ( p = 0.442). The highest [Fe2+]T was

measured at t = 1 h (1.63 T 0.102 AM), approximately 35% of

added Fe(II), and remained unchanged following incubation of

the peptide for 24 h (1.57 T 0.155 AM) before falling

significantly ( p < 0.001) at t = 48 h (1.17 T 0.083 AM). The

[Fe2+]T at t = 1 h was significantly higher ( p < 0.001) than that

found in the absence of added Zn(II) whereas the values at 24

and 48 h were significantly lower ( p < 0.001) than the

corresponding Zn(II)-free preparations. At t = 24 and 48 h the

[Fe2+]PA were extremely low in the presence of Zn(II), which

suggested that Zn(II) reduced the contribution of Ah42 to

[Fe2+]T under these conditions. The additional presence of

Al(III) in these Zn(II) + peptide preparations significantly

increased [Fe2+]T at each time point ( p < 0.001). It also

significantly increased [Fe2+]PA at each time point, for

example, at t = 24 it was increased from 0.41 T 0.155 to


1.29 T 0.233 AM, though it did not prevent significant

reductions in both [Fe2+]T and [Fe2+]PA between 24 and

48 h (Table 2).

We have used ThT fluorescence to determine the extent to

which preparations which included 3.52 AM Ah42 incubated at

37-C for 1, 24, and 48 h formed h-pleated conformers and how

aggregation status subsequently changed following the addition

of 5.0 AM Fe(II) and incubation for a further 30 min. The data

were used to identify if the propensity for Ah42 to influence

[Fe2+]T was dependent upon its prior assembly into h-pleatedsheets of amyloid (Table 3). Significant ThT fluorescence

(>100 AU) was measured after 1, 24, and 48 h in preparations

which included Ah42 alone and Ah42 + Al(III). For the former,

ThT fluorescence was only weakly correlated with either

[Fe2+]T (r2 = 0.006) or [Fe2+]PA (r2 = 0.027) whereas for

Ah42 + Al(III) r2 values of 0.190 and 0.179 for [Fe2+]T and

[Fe2+]PA respectively indicated a closer relationship between

Table 3

Thioflavin T fluorescence (AU) in each preparation (a– i) following incubation

at 37-C for 1, 24, or 48 h (pre-Fe(II)/Fe(III) addition) and following the

addition of either 5.0 AM Fe(II) (a,c,e,f,g,h,i) or 5.0 AM Fe(III) (b,d) and

incubation for a further 30 min at 37-C (post-Fe(II)/Fe(III) addition)



(a) Ab42 alone + Fe(II)

Pre-Fe(II) 189 (31.5) 192 (18.3) 130 (23.4)

Post-Fe(II) 137 (68.6) 110 (23.6) 60 (12.0)

(b) Ab42 alone + Fe(III)

Pre-Fe(II) 162 (13.0) 160 (50.9) 189 (37.2)

Post-Fe(II) 158 (9.6) 143 (41.0) 173 (28.0)

(c) Ab42 + Al(III) + Fe(II)

Pre-Fe(II) 159 (44.9) 174 (25.4) 196 (7.3)

Post-Fe(II) 116 (21.6) 117 (20.4) 105 (28.0)

(d) Ab42 + Al(III) + Fe(III)

Pre-Fe(II) 151 (19.2) 145 (17.4) 149 (47.1)

Post-Fe(II) 138 (10.9) 115 (43.7) 112 (43.9)

(e) Ab42 + NaN3 + Fe(II)

Pre-Fe(II) 85 (45.3) 103 (36.6) 123 (31.4)

Post-Fe(II) 53 (31.5) 64 (20.1) 54 (14.1)

(f) Ab42 + Cu(II) + Fe(II)

Pre-Fe(II) 13 (2.8) 21 (4.5) 17 (5.8)

Post-Fe(II) 11 (2.1) 15 (3.7) 10 (3.3)

(g) Ab42 + Cu(II) + Al(III) + Fe(II)

Pre-Fe(II) 10 (1.5) 19 (8.0) 16 (5.2)

Post-Fe(II) 8 (1.0) 17 (7.9) 11 (4.7)

(h) Ab42 + Zn(II) + Fe(II)

Pre-Fe(II) 22 (5.0) 39 (4.5) 25 (8.9)

Post-Fe(II) 16 (2.5) 23 (1.6) 12 (2.1)

(i) Ab42 + Zn(II) + Al(III) + Fe(II)

Pre-Fe(II) 40 (30.7) 81 (22.2) 56 (11.4)

Post-Fe(II) 30 (25.0) 50 (10.0) 25 (4.3)


Fig. 4. (a and b) The relationship between ThT fluorescence prior to the

addition of 5.0 AM Fe(II) and [Fe2+]T measured 30 min after its addition for all

incubations (1, 24, and 48 h) for (a) Ah42 alone and (b) Ah42 + 5.0 AM Al(III).

these variables (Fig. 4). The presence of the OHSradical

scavenger, NaN3, resulted in ThT fluorescence which was

significantly lower ( p < 0.001) than those measured for Ah42

alone or Ah42 + Al(III) and which increased from 85 T 45.3 AUat t = 1 h to 103 T 36.6 AU at 24 h and to 123 T 31.4 AU at 48 h.

Though these increases were not statistically significant

between time periods ( p > 0.05) ThT fluorescence in these

preparations was positively correlated with both [Fe2+]T (r2 =

0.186) and [Fe2+]PA (r2 = 0.188). For Ah42 + Cu(II) the values

of ThT fluorescence at 1, 24, and 48 h were very low (<20 AU)

and corresponded to some of the lowest values for [Fe2+]T and

[Fe2+]PA. In these preparations there were no correlations

between ThT fluorescence and [Fe2+]T (r2 = 0.010) or [Fe2+]PA(r2 = 0.003). The additional presence of Al(III) in these Cu(II)

preparations neither increased nor decreased ThT fluorescence

at 1, 24, or 48 h ( p > 0.05) but it did result in strong positive

correlations between ThT fluorescence and both [Fe2+]T (r2 =

0.520) and [Fe2+]PA (r2 = 0.528) (Fig. 5). Similarly for Cu(II),

Ah42 + Zn(II) resulted in low values of ThT fluorescence for 1,

24, and 48 h (20–40 AU) and relatively weak correlations

between ThT fluorescence and both [Fe2+]T (r2 = 0.128) and

[Fe2+]PA (r2 = 0.026). However, the additional presence of

Al(III) in these Zn(II) preparations increased ThT fluorescence

significantly at each time point ( p < 0.05) and resulted in

Fig. 5. (a–b) The relationship between ThT fluorescence prior to the addition

of 5.0 AM Fe(II) and [Fe2+]T measured 30 min after its addition for all

incubations (1, 24, and 48 h) for (a) Ah42 + 10.0 AM Cu(II) and (b) Ah42 +

10.0 AM Cu(II) + 5.0 AM Al(III).


addition of 5.0 AM Fe(II) and [Fe2+]T measured 30 min after its addition for al

incubations (1, 24, and 48 h) for (a) Ah42 + 10.0 AM Zn(II) and (b) Ah42 + 10.0

AM Zn(II) + 5.0 AM Al(III).


stronger positive correlations between ThT fluorescence and

both [Fe2+]T (r2 = 0.388) and [Fe2+]PA (r2 = 0.179) (Fig. 6).

ThT fluorescence was also measured for each preparation at

1, 24, and 48 h following the 30 min incubation in the presence

of 5.0 AM added Fe(II). Data were used to determine if the

addition of Fe(II) influenced the aggregation status of Ah42 and

whether any change was related to [Fe2+]T or [Fe2+]PA for

similarly aged preparations (Table 3). For each preparation at

each incubation period the addition of 5.0 AM Fe(II) always

resulted in a fall in ThT fluorescence. The proportional

reductions in ThT fluorescence in preparations including

peptide incubated for 1 h were in the range 14–38% and

were statistically significant ( p < 0.05) for Ah42 + NaN3 and

Ah42 + Al(III). For preparations incubated for 24 h the

proportional reductions in ThT fluorescence were usually

greater than for 1 h, range 8–43%, and the falls were

statistically significant ( p < 0.05) for Ah42 alone, Ah42 +

NaN3, Ah42 + Al(III), Ah42 + Zn(II), and Ah42 + Zn(II) +

Al(III). The largest proportional reductions in ThT fluores-

cence were measured for preparations incubated for 48 h,

range 31–64%, and were statistically significant ( p < 0.05)

for all of the preparations. While the influence of Fe(II) on

the ThT fluorescence of different preparations was clearly

associated with their period of incubation prior to the addition

of Fe(II) no positive relationships were found when changes

in ThT fluorescence were correlated with either [Fe2+]T or

[Fe2+]PA (r2 values ranged from 0.002 to 0.075).

When 5.0 AM Fe(III), instead of Fe(II), was added to 3.52

AM Ah42 preparations incubated for 1, 24, and 48 h the

previously reported significant increases in both [Fe2+]T and

[Fe2+]PA were, as for Fe(II), only very weakly associated with

the initial ThT fluorescence (r2 = 0.043 and 0.042,

respectively). However, contrary to the results obtained with

Fe(II), the addition of Fe(III) to peptide preparations

incubated for 1, 24, or 48 h did not result in significant ( p >

0.05) reductions in ThT fluorescence. For example, at t = 48

h the addition of Fe(III) reduced ThT fluorescence from 189 T37.2 to 173 T 28.0 AU. When Fe(III) was added to preparations

of Ah42 which also included 5.0 AM Al(III) the relationship

between ThT fluorescence and [Fe2+]T or [Fe2+]PAwas stronger

(r2 = 0.227 and 0.131, respectively) than for the respective

Al(III)-free preparations (Fig. 7) and, in these preparations the

addition of Fe(III) resulted in reductions in ThT fluorescence

which at t = 48 h (149 T 47.1 to 112 T 43.9 AU) were

statistically significant ( p = 0.031). Despite these reductions in

l


addition of 5.0 AM Fe(III) and [Fe2+]T measured 30 min after its addition for all

incubations (1, 24, and 48 h) for (a) Ah42 alone and (b) Ah42 + 5.0 AM Al(III).

Fig. 8. Oligomers of Ah42 formed after only 1-h incubation, and prior to the

addition of 5.0 AM Fe(II), in the preparation which included 3.52 AM Ah42 +

10.0 AM Cu(II) + 5.0 AM Al(III). Magnification �50K.


ThT fluorescence there were no positive correlations between

the changes in ThT fluorescence and either [Fe2+]T or [Fe2+]PA.

Discussion

Auto-oxidation at 37 -C of 5.0 AM added Fe(II) in KH

medium buffered at pH 7.40 was >80% complete within 30

min and independent of preincubation for 1, 24, or 48 h. The

inclusion of 0.29, 1.83, or 3.52 AM Ah42 inhibited auto-

oxidation. For example, auto-oxidation was only 25%

complete in media which included 3.52 AM Ah42 which

had been preincubated for 48 h. [Ah42] were chosen to

encompass three saturation states of peptide, sub (0.29 AM),

near (1.83 AM), and super (3.52 AM) [17], distinguished by

their propensities to self-aggregate and form h-pleated sheet

conformers. The auto-oxidation of added Fe(II) was influ-

enced by synergies between peptide concentration and

incubation period though the formation of h-pleated sheets

was only part of any synergy. For example, the strongest

correlation between ThT fluorescence and [Fe2+]PA was found

for the near-saturation (1.83 AM) and not the supersaturation

(3.52 AM) concentration of Ah42 (Figs. 3a–d), indicating that

the form of peptide which influenced the auto-oxidation of

added Fe(II) was probably intermediate in structure between

the random turn/coil conformation of monomeric Ah42 and

the h-pleated sheets which bind ThT. To understand this

further the auto-oxidation of Fe(II) was determined in

preparations in which Ah42 was precipitated in the presence

of Cu(II), Zn(II), or Al(III). Cu(II) inhibited the formation of

h-pleated sheets and precipitated Ah42 as amorphous aggre-

gates which had no influence on the auto-oxidation of added

Fe(II). No correlation was found between ThT fluorescence

and [Fe2+]T (Fig. 5a). Zn(II) reduced the formation of h-pleated conformers of Ah42 and, concomitantly, the role of

Ah42 in influencing the auto-oxidation of added Fe(II). There

was a weak correlation between ThT fluorescence and [Fe2+]T(Fig. 6a). Al(III) reduced neither the formation of h-pleatedconformers nor the influence of Ah42 on the auto-oxidation of

added Fe(II). The latter was accentuated by Al(III) and

resulted in an improved correlation between ThT fluorescence

and [Fe2+]T (Fig. 4b). The additional presence of Al(III) also

resulted in a significantly improved positive correlation

between ThT fluorescence and [Fe2+]T in Ah42 + Cu(II)

preparations (Fig. 5b). Though not reflected in statistically

significant changes in ThT fluorescence this influence of

Al(III) was accompanied by the presence of oligomers of

Ah42 at t = 1 h (Fig. 8) and the disappearance of these

oligomers and the appearance of distinctly fibrillar structures

at t = 24 and 48 h. The additional presence of Al(III) in Ah42 +

Zn(II) preparations resulted in similar effects except that the

improved correlation between ThT fluorescence and [Fe2+]T(Fig. 6b) was also accompanied by significant increases in ThT

fluorescence. These observations suggested that Al(III) slowly

reversed the inhibitive influences of Cu(II) and Zn(II) on both

the formation of h-pleated conformers of Ah42 and the

propensity for Ah42 to influence the auto-oxidation of added

Fe(II). That the improved correlations between ThT fluores-

cence and [Fe2+]T occurred concomitantly with the transforma-

tion between non-ThT-reactive oligomeric forms of Ah42 and

ThT-reactive h-pleated conformers supported the previous

notion that either the conformational transformation per se or

an intermediate involved in the transformation between


conformers was active in influencing the auto-oxidation of

added Fe(II).

With the notable exception of preparations which included

Cu(II), Ah42 increased [Fe2+]T relative to preparations which

included neither peptide nor added metal. This may be due

to binding of Fe(II) by particular conformers or polymers of

Ah42 which, upon addition of 1,10-phenanthroline, released

the metal to form the characteristic 1,10-phenanthroline–

Fe(II) complex or it may indicate a role for Ah42 in the

redox cycling of iron. That the addition of Fe(III) to Ah42

resulted in the formation of Fe(II) supports the latter though

does not discount an additional role for peptide binding of

Fe(II).

To discriminate between these two mechanisms it is

necessary to identify the redox chemistry which in the absence

of Ah42 is occuring under the physiological-like conditions of

the assay. These redox reactions ((1)–(13)) and some of their

rate constants [18] are shown in Table 4. The oxidation of

Fe(II) by molecular oxygen (O2) is slow (1). However, it is

facilitated by the subsequent formation of the superoxide

radical anion (O2S�) which acts as an accelerant both directly

(3) and indirectly (2) via the formation of hydrogen peroxide

(H2O2) (4). Anything that influences this cascade of reactions

will impact significantly upon [Fe2+]C. These reactions involve

soluble forms of iron and their individual contributions to

redox cycling will be heavily influenced by factors affecting

iron solubility. Some reactions (e.g.,(6)) are not favoured due to

the extreme insolubility of iron (III) hydroxides (Ksp ca 10�37).

The OHS

scavenger NaN3 (7) accelerated auto-oxidation

(Table 2) which suggested that the reaction of Fe(II) with

H2O2 (4) was a significant rate-determining step in its auto-

oxidation. Cu(II) was more efficient than NaN3 in accelerating

the auto-oxidation of Fe(II) (Table 2) and this was almost

certainly due to its known influence upon the dismutation of

O2S� (8 and 9). There is controversy as to whether O2

S� acts as

a reductant or an oxidant ((10) and (11)) in the first instance

[19]. However, either way Cu(II) resulted in the lowest [Fe2+]C,

most probably through its acceleration of the formation of both

H2O2 and O2. The influence of Cu(II) pointed toward the

reaction of Fe(II) with H2O2 (4) as being of most importance in

Table 4

Redox reactions and, where appropriate, their rate constant

Reaction Rate constant k (M�1 s�1)

1. Fe(II) + O2 Y Fe(III) + O2S� 13

2. 2O2S� + 2H2O Y H2O2 + 2OH� 1.0 � 105

3. Fe(II) + O2S� + 2H+ Y Fe(III) + H2O2 1.0 � 107

4. Fe(II) + H2O2 Y Fe(III) + OHS+ OH� 3.1 � 104

5. Fe(II) + OHS Y Fe(III) + OH� 5.0 � 108

6. Fe(III) + O2S� Y Fe(II) + O2 1.5 � 108

7. N3� +SOH Y SN3 + OH�

8. Cu(II) + O2S� Y Cu(I) + O2 6.6 � 108

9. Cu(I) + O2S� + 2H+ Y Cu(II) + H2O2 2.0 � 109

10. Cu(II) + O2S� + 2H+ Y Cu(III) + H2O2

11. Cu(III) + O2S� Y Cu(II) + O2

12. H+/Fe2+/Al 3+ + O2S� Y AlO2

S2+ + Fe2+ + H+

13. Fe 3+ + AlO2S2+ Y Fe2+ + O2 + Al3+

All rate constants taken from [18].

the auto-oxidation of Fe(II). The highest [Fe2+]C were

measured in the presence of Al(III) and despite the fact that

these solutions were saturated with respect to aluminum

hydroxide and therefore during 48 h incubations would be

prone to considerable changes in their colloidal chemistry the

measured [Fe2+]C were independent of whether the prepara-

tions had been aged for 1, 24, or 48 h. It has been suggested

that previously noted effects of Al(III) on the auto-oxidation of

Fe(II) were due to Fe(II) being bound by particulate aluminum

such that it was protected from oxidation but still available for

binding by 1,10-phenanthroline [13]. The lack of influence of

the colloidal chemistry of Al(III) on the auto-oxidation of

Fe(II) combined with the observation that Al(III) per se did not

reduce Fe(III) to Fe(II) may suggest an alternative mechanism

involving the formation of a strong and fairly stable complex

between Al(III) and a reactant involved in the auto-oxidation of

Fe(II). Such a reaction and complex, the binding of Al3+ by

O2S� to form the semireduced radical cation AlO2

S2+, have

already been postulated [20] and would increase [Fe2+]C by

limiting the formation of H2O2 via reactions involving O2S�

(12) and accelerating the reduction of Fe(III) (13). Which of

these mechanisms was responsible for the remarkable influence

of Al(III) on the auto-oxidation of Fe(II) remains to be

determined. Zn(II) also increased [Fe2+]C and, like Al(III),

the explanation may be an interaction between O2S� and Zn2+.

Zn(II) as part of a Zn(II)/Cu(II) complex in superoxide

dismutase (SOD) is known to bind O2S� and binding is

thought to stabilise O2S� prior to it being ‘‘handed-on’’ to Cu(II)

for dismutation [21]. In this way Zn(II) would delay the

formation of H2O2 and thus, for this study, the critical step in

the oxidation of Fe(II). That Al(III) potentiated this activity of

Zn(II) or, looking at it in reverse, Zn(II) inhibited the

potentiation of [Fe2+]C due to Al(III), suggested a common

mechanism of effect and competition for either metal cation to

be bound by O2S�.

Control experiments indicated that under the conditions of

this study the reactions which involved the formation of

H2O2 via O2S� were critical in determining the net rate of

oxidation of Fe(II). The presence of Ah42 always resulted in

higher concentrations of Fe(II) than respective peptide-free

controls (i.e., [Fe2+]T > [Fe2+]C), which suggested a role in

the redox cycling of iron. To identify this role it is revealing

to consider how Fe(II) was generated in preparations in

which Fe(III) was substituted for Fe(II). In the absence of

peptide ca 0.1–0.2 AM Fe(II) was measured following the

addition of 5.0 AM Fe(III). The origin of this Fe(II) was

either as a contaminant of the Fe(III) stock solution or the

reduction of Fe(III) by a constituent of control preparations.

For the latter the rate of reduction of added Fe(III) would be

limited by its rapid precipitation as inert and extremely

insoluble Fe(OH)3(s). This might explain why, unlike Fe(II) +

Al(III), the additional presence of Al(III) in Fe(III) prepara-

tions did not result in higher [Fe2+]C. Contrary to control

preparations, in the presence of Ah42, Fe(III) was rapidly

reduced to the extent that in preparations incubated for 48 h over

50% of added Fe(III) was converted to Fe(II). The rate of

reduction of added Fe(III) increased with increased period of


incubation and though there were no statistically significant

differences in ThT fluorescence between these periods this

dependence was almost certainly associated with the aggrega-

tion state of Ah42. This might be understood in terms of

different affinities of age-dependent forms of Ah42 for binding

either Fe(II) or Fe(III). Binding of iron by organic ligands will

influence the redox potential of the Fe(II)/Fe(III) couple though

not necessarily in a predictable manner [18]. Binding of Fe(III)

will prevent its immediate precipitation as hydroxide and is

likely to increase its susceptibility to reduction whereas binding

of Fe(II) could accelerate its oxidation though it may also have

the opposite effect due to changes in the accessibility of O2 to

the iron centre.

A three-step mechanism is proposed to explain the role of

Ah42 in the auto-oxidation of Fe(II), the reduction of Fe(III),

and how these processes are influenced by Al(III), Zn(II), and

Cu(II) (Fig. 9).

Step 1

Fe(II) and Fe(III) are bound by monomeric Ab42 to increase

the likelihood of their oxidation and reduction, respectively.

The net result of these processes which we measured as [Fe2+]Twill depend upon (i) equilibria among complexed, free, and

precipitated iron, (ii) the redox potential of each of the soluble

forms of iron, and (iii) the dilution of each of these forms in the

preparation. There is no direct evidence in this study that

monomeric Ah42 bound either Fe(II) or Fe(III). There is

quantitative evidence in the literature for an Ah42–Fe(II)

complex though its calculated Kd of 36 AM [22] should mean

that it is unlikely to be stable in preparations herein. Evidence

is presented that preparations predicted to include predomi-

nantly monomeric Ah42 had no significant influence on [Fe2+]T

(Table 1). This would support the tendency for Ah42–Fe(II)

not to be formed in these preparations. There are no

quantitative data in the literature to support the formation of

an Ah42–Fe(III) complex. However, evidence is presented that

Ah42 incubated for only 1 h prevented the immediate

precipitation of Fe(III) and concomitantly accelerated its

reduction (Table 2). Whether these effects were mediated

Fig. 9. Schematic describing the role of the aggregation state of Ah42 in the redox c

Fe(II) and Fe(III), respectively.

through the formation of monomeric Ah42–Fe(III) remains to

be proven.

Step 2

Monomers of Ab42 form oligomers which may include

bound iron. Where oligomerisation brings iron atoms into

closer proximity thus countering any dilution effects redox

reaction rates are accelerated and the influence on [Fe2+]T is

accentuated relative to peptide in monomeric conformations.

There is not any direct evidence in the literature either that

oligomers of Ah42 include bound iron (or other metals) or that

oligomers act as catalysts for the oxidation/ reduction of iron.

Evidence is presented for significant increases in [Fe2+]T in

preparations of Ah42 in which oligomers were predicted to

have formed and in which further ageing of peptide did not

result in higher ThT fluorescence (Table 1). However,

increased [Fe2+]T were not recorded in preparations of Ah42

in which oligomers but not h-pleated fibrils were identified

(Table 2; Fig. 8). Because of the rapidity with which 3.52 AMAh42 aggregated under the conditions of this study it is not

possible to identify unequivocally if either monomeric or

oligomeric forms of peptide bound either form of iron.

Therefore it cannot be determined from the data if these forms

of bound iron were involved in the effects of Ah42 on the

Fe(II)/Fe(III) couple.

Step 3

Oligomers assemble into immature and mature b-pleatedfibrils. Iron may be retained by or titrated from newly formed

fibrils and free iron may be bound by newly formed fibrils. This

step is probably most representative of aggregation states in the

majority of peptide preparations investigated in this study. The

most significant effects upon [Fe2+]T were measured in

preparations incubated for 24 or 48 h and implicated either a

particular form of peptide or a specific change in peptide form

in the observed increases in [Fe2+]T. There is evidence in the

literature that fibrillar Ah42 binds Fe(II) much more tightly

(Kd = 0.2 AM) than monomeric Ah42 [22] though similar data

ycling of added Fe(II). Open and filled circles are qualitative representations of


are not available for Fe(III). Evidence is presented that some

form of Ah42 bound Fe(III) and accelerated its reduction.

Fe(II), either added or originating from the reduction of

Fe(III), could have been bound by fibrillar peptide, thereby

stabilising it against oxidation to the extent that it accumu-

lated in preparations in this form. Fe(II) would remain

available for binding by 1,10-phenanthroline and would,

therefore, be measured as [Fe2+]T. The slower oxidation and

subsequent titration of peptide-bound Fe(II) would result in its

precipitation as Fe(OH)3(s) and concomitantly the deposition

of Ah42 as mature double-stranded fibrils. The delayed

precipitation of Fe(III) in many of the preparations was

observed by TEM and may have reflected the kinetically

preferred formation of Fe(OH)3(s) relative to its complexation

by fibrillar forms of Ah42.

This three-step mechanism predicts that Ah42 influenced the

redox cycling of iron by delaying its precipitation as highly

insoluble and consequently redox-inactive Fe(OH)3(s). Ah42 is

not redox active per se, a conclusion which is supported by a

consensus in the literature that Ah42 does not spontaneously

form free radicals in the absence of either added or contaminant

metals [23,24]. [Fe2+]T was measured as both Fe(II) in solution

and Fe(II) bound to particular forms of Ah42 with the latter

being important in the mechanism by which high concentra-

tions of Fe(II) were generated from the addition of Fe(III). The

reduction of Fe(III) was fueled by the presence in all

preparations of contaminating concentrations of Fe(II) which

through its auto-oxidation and the formation of O2S� facilitated

the reduction of peptide-bound Fe(III) and the subsequent

titration of Fe(II) into solution. Auto-oxidation of Fe(II)

catalysed the further reduction of Fe(III) with higher [Fe2+]Tin Ah42 preparations aged for 24 and 48 h reflecting a role for

aggregated Ah42 in either binding of a higher proportion of

added Fe(III) or binding of Fe(II) such that its oxidation was

delayed. A combination of these competitive equilibria,

including the propensity for 1,10-phenanthroline to titrate

peptide-bound Fe(II), was responsible for [Fe2+]T in each

preparation and any such combination was further influenced

by the presence of Al(III), Cu(II), and Zn(II). For example, the

action of Al(III) in increasing [Fe2+]T in all preparations is

explained by both its unusual redox activity, the formation of

the putative AlO2S2+ inhibiting the formation of H2O2 while at

the same time accelerating the reduction of Fe(III), and its

acceleration of the formation of the fibrillar form of Ah42. The

latter was clearly evident in preparations which included both

Cu(II) and Al(III) in which in the absence of peptide Al(III)

had little influence upon [Fe2+]C due to the rapid dismutation of

O2S� by Cu(II). In the presence of peptide Al(III) reversed the

effects of Cu(II) on [Fe2+]T by promoting the oligomerisation

and fibrillisation of Ah42 and, since a proportion of the added

Cu(II) was bound by Ah42 and was no longer available for the

dismutation of O2S�, by competing more effectively to bind

O2S� thereby inhibiting the formation of H2O2. Cu(II) in the

absence of additional Al(III) effectively inhibited the redox

cycling of iron by both accelerating the formation of H2O2 [7]

and, importantly, precipitating Ah42 in a nonfibrillar and

critically non-Fe(II)-binding form [10]. The latter was con-

firmed by TEM though a limited number of oligomeric forms

of peptide were observed in these preparations. Zn(II)

influenced peptide effects on [Fe2+]T primarily through its

binding of O2S�. The formation of a putative Zn(II)–O2

S�complex acted to increase [Fe2+]T through the inhibition of the

formation of H2O2 [25] though this effect was not influenced

significantly by the presence of Ah42. Thus there was little

evidence that the oligomeric and fibrillar forms of Ah42 which

were present in Zn(II) preparations contributed to the redox

cycling of iron. Similarly there was no evidence that the

putative Zn(II)–O2S� complex was effective in reducing

Fe(III) only in competing with Al(III) for binding by O2S�.

Despite the observations by both ThT fluorescence and TEM

that preparations including Zn(II) T Al(III) included both

oligomeric and fibrillar Ah42 the highest [Fe2+]T for these

preparations were recorded for peptide preparations aged for

only 1 h and this tended to exclude a major role for the

aggregation state of the peptide in the redox cycling of iron in

the presence of Zn(II). The possible explanation for this is that

the free radical which was most closely associated with the

peptide_s role in these processes, O2S�, was effectively

mopped up by Zn(II). Support for this notion is that Al(III)

increased both [Fe2+]C and [Fe2+]T in the presence of Zn(II) and

this was most probably due to the competitive binding of

Al(III) and Zn(II) by O2S�.

One further major observation appeared to support the

notion that a fibrillar form of Ah42 bound and stabilised Fe(II).

In every preparation in which fibrillar material was recorded by

ThT fluorescence or TEM the addition of Fe(II) was followed

immediately by a statistically significant reduction in ThT

fluorescence (Table 3). These effects upon ThT fluorescence

were not observed in those preparations in which Fe(III) was

substituted for Fe(II) even though Fe(II) was subsequently

generated from the reduction of the added Fe(III). We contend

that the changes in ThT fluorescence which followed the

addition of Fe(II) were not brought about by redox events per

se, for example, the oxidation of amino acid residues, such as

methionine-35 [26], altering the propensity of peptide to form

fibrils, but by the Fe(II)-induced precipitation of fibrillar

peptide such that fewer sites were subsequently available for

binding of ThT [10,15]. The bound Fe(II) might still be

available to 1,10-phenathroline if, as is predicted, binding of

Fe(II) stabilised it against auto-oxidation.

In conclusion, the major role of Ah42 in the redox cycling of

iron was probably in binding Fe(III) and thereby delaying its

precipitation as redox-inactive Fe(OH)3(s). Further aggregation

state-specific binding of both Fe(II) and Fe(III) determined

critical equilibria involved in the formation of H2O2 via O2S� in

favour of maintaining Fe(II) in solution. The additional presence

of Al(III), Cu(II), and Zn(II) influenced both the aggregation

state of Ah42, and therefore its binding of Fe(II) and Fe(III), and

the redox chemistry, most specifically through direct interactions

with O2S�. It is the latter free radical anion which is now heavily

implicated in ROS-mediated neurotoxicity [27–30] and it is

demonstrated herein that in the presence of Ah42 any predicted

O2S�-induced toxicity would be exacerbated by a pathophysio-

logically significant concentration of Al(III) while both Cu(II)


and Zn(II), providing that Al(III) was absent, would mitigate

against any oxidative damage.

Acknowledgments

A.K. is funded by NIH Grant R01AG02030-01A1. Dr. O.

Exley is thanked for her help with the figures.

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