Supplementary Information - Ferrous iron formation following the coaggregation of ferric iron and the Alzheimer’s disease peptide β-amyloid
(1-42)
J. Everett1, E. Céspedes1, L.R. Shelford2, C. Exley3, J.F. Collingwood4, J. Dobson5, G. van
der Laan6, C.A. Jenkins7, E. Arenholz7 and N.D. Telling1
1
Institute for Science and Technology in Medicine, Keele University, Stoke-on-Trent, Staffordshire
ST4 7QB, UK
2
College of Engineering, Mathematics and Physical Sciences, University of Exeter, Exeter EX4 4QL,
UK
3
The Birchall Centre, Lennard-Jones Laboratories, Keele University, Staffordshire ST5 5BG, UK
4
University of Warwick, School of Engineering, Coventry CV4 7AL, UK
5
J.Crayton Pruitt Family Department of Biomedical Engineering & Department of Materials Science
and Engineering, University of Florida, Gainesville, Florida 32611, USA
6
Magnetic Spectroscopy Group, Diamond Light Source, Didcot, Oxfordshire, OX11 ODE, UK
7
Advanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, California, 94720, USA
Corresponding Author: Neil David Telling1
E mail: n.d.telling@keele.ac.uk Tel: +44 (0)1782 674968
Anoxic Sampling
To prevent alterations to the oxidation state of the iron material used, anoxic conditions were
created and maintained throughout the sampling and examination process. Sample
manipulation was conducted under anoxic conditions within a nitrogen filled glove chamber.
Nitrogen filled, O-ring sealed, plastic containers were used to transfer sample probes to the
XAS/XMCD beamline for examination. Beamline chambers were backfilled with nitrogen
preventing iron oxidation once probes had been inserted. Sample materials were exposed to
oxygen for a maximum of one second when being lowered into the beamline chamber. Such
methodology has previously been utilized by Coker et al. (2009)(1).
Two dimensional x-ray absorbance iron mapping
In order to identify areas of iron accumulation under XAS/XMCD examination, two
dimensional maps depicting the iron content of the sample area to a spatial scale of 100 µm
were created. These maps were achieved by scanning the sample area slightly below (706
eV) and at (710 eV) the iron L3 peak energy. Off peak absorbance values were then subtracted
from peak values resulting in the removal of any background absorbance signals to reveal
areas of iron accumulation (Figure S1). These areas of high iron content were then examined
in greater detail by performing XAS/XMCD scans across the entire iron L2,3-edge
(700-740 eV).
Figure S1. Two dimensional x-ray absorbance maps of a sample containing Aβ and ferric
iron. (a) Off-peak (706 eV) image and (b) peak iron L3 (710 eV) image. (c) Difference map
revealing the iron distribution of the sample area. Iron can be seen in the bottom left quadrant
(yellow/white).
X-ray beam reduction
Generally crystalline iron minerals are not susceptible to x-ray beam induced reduction
effects at the soft x-ray energies used in this study. However as more amorphous iron forms
were used here it was necessary to explore the effect of the x-ray beam on these phases. To
do this we performed repeated x-ray absorbance scans across the iron L2,3 edge, and
monitored any changes in the XAS profile as a function of x-ray beam exposure. An example
of this process is shown in Figure S2, where four successive XAS scans were performed on a
ferric iron (ferrihydrite) aggregate known to be unstable in the x-ray beam. Despite an x-ray
beam mediated reduction effect being apparent over the first three XAS scans, this reduction
effect plateaus on the fourth scan with iron being present as an iron(II)/(III) intermediate, not
a pure iron(II) phase. More importantly, in all cases of amyloid induced reduction, iron was
found to be heavily reduced upon first XAS examination. Therefore even when using iron
forms unstable in the x-ray beam, it was easily possible to separate reduction effects resulting
from amyloid interaction, to those induced by x-ray beam exposure.
Figure S2. X-ray absorption spectra of an unstable ferric iron aggregate following four
successive XAS measurements. Fe2+ cation peak is indicated by a grey dotted line at 708eV.
1. Coker VS, Telling ND, van der Laan G, Pattrick RAD, Pearce CI, Arenholz E, Tuna F,
Winpenny REP, Lloyd JR. 2009 Harnessing the Extracellular Bacterial Production of
Nanoscale Cobalt Ferrite with Exploitable Magnetic Properties. ACS Nano. 3, 1922-1928.