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  • Chapter 3

    InorganicProtein Interactions in the Synthesis

    of a Ferrimagnetic Nanocomposite

    T. Douglas1,6, J. W. M. Bulte2, D. P. E. Dickson3, R. B. Frankel4, Q. A. Pankhurst3, . M. Moskowitz5, and S. Mann1

    1School of Chemistry, University of Bath, Bath BA2 7AY, United Kingdom

    2National Institutes of Health, Bethesda, MD 20892 3Department of Physics, University of Liverpool, Liverpool L69 3BX,

    United Kingdom 4Department of Physics, California Polytechnic State University,

    San Luis Obispo, CA 93407 5Department of Geology and Geophysics, University of Minnesota,

    Minneapolis, MN 55455

    The iron-storage protein ferritin provides a spatially constrained reaction environment within a catalytically active bio-polymer. These properties have facilitated the synthesis of a ferrimagnetic iron oxide-protein composite, formed by tailoring conditions for the synthesis of magnetite. The controlled partial oxidation of Fe(II), at high pH and elevated temperature, in the presence of apo-ferritin resulted in the formation of a colloidal composite with a narrow particle size distribution. This material combines characteristics of both the protein and the inorganic phase. Direct magnetic measurements indicated 13,100 Bohr magnetons per particle. Electron and x-ray diffraction data indicated the presence of a cubic iron oxide mineral phase but could not distinguish between magnetite and maghemite. Data from Mssbauer spectroscopy, measured both in the presence and absence of an applied field as well as at low temperature, suggested that the predominant mineral phase was maghemite rather than magnetite.

    The protein ferritin is ubiquitous in biological systems. Ferritin stabilizes and stores colloidal iron oxide in the form of the hydrated mineral ferrihydrite (1), (5Fe203.9H20 with variable amounts of phosphate). The protein comprises 24 subunits, of two types (H (heavy) and L (light), that self-assemble forming a central cavity, approximately 80 in diameter, in which the iron oxide is mineralized. This arrangement gives rise to a highly symmetrical spherical protein, 120 in diameter, intersected by channels through which the ions move. As its biological function suggests, the protein has high specificity for iron but this has not prevented the synthetic formation of other, non-ferrous minerals, such as a

    6Current address: Department of Chemistry, Ithaca College, Ithaca, N Y 14850

    0097-6156/95/0585-0019$12.00/0 1995 American Chemical Society

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    In Hybrid Organic-Inorganic Composites; Mark, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

  • 20 HYBRID ORGANIC-INORGANIC COMPOSITES

    manganese oxyhydroxide (2) and a uranium oxyhydroxide, within the protein cavity (3). The mechanism for iron biomineralization in ferritin is thought to involve two distinct clusters of amino acids (on each subunit) that act as ferroxidase centers (H subunit only) and crystal nucleation sites (both H and L subunits) respectively (4).

    As a bio-polymer matrix ferritin combines a number of properties that make it ideal for the synthesis of nanocomposite materials: a) a well defined, constrained reaction environment b) stable protein matrix (pH4 to 9, and up to 90C) c) catalytic oxidation site and crystal nucleation sites d) non-immunogenic protein (high sequence homology amongst ferritins from varying sources) e) biodegradable, non-toxic polymer. This work reports the characterization of a magnetic core synthesized within the cavity of the ferritin protein.

    Synthesis of Magnetoferritin

    Chemical reduction of the native ferrihydrite followed by chelation and removal of the ferrous ion leaves the apo-protein intact and the central cavity available for remineralization (5). The synthesis of magnetoferritin (6) was carried out anaerobically at elevated temperature (60-65C) and pH (8.6) by the stepwise addition of Fe(II) to a solution of apoferritin followed by partial oxidation until an average loading of 2500 Fe atoms per ferritin had been achieved. The reaction product was a homogeneous black-brown solution, unlike the blood red color of the native ferritin, which could be precipitated by the application of a high gradient magnetic field.

    Characterization of Magnetoferritin

    Transmission Electron Microscopy (TEM). The magnetically isolated magnetoferritin cores were imaged by T E M (Figure la.) and revealed an average particle size of 73 diameter with a homogeneous size distribution ( = 14) (Figure 2). Electron diffraction confirmed a face-centered cubic mineral phase (Figure lb.), different from the native ferrihydrite, but it was not possible to distinguish between magnetite, Fe304, and maghemite, y-Fe203 (Table I). Control experiments using 0.1 M NaCl in place of apoferritin produced a black, extensively aggregated material (figure 3), which exhibited the same electron diffraction pattern as for the magnetoferritin core. The lattice image (figure 4) of a magnetoferritin core approximately 80 in diameter, obtained by high resolution transmission electron microscopy (HRTEM), clearly shows two sets of lattice fringes (d =

    Table I. Electron diffraction of magnetite, maghemite and magnetoferritin

    Fe304/y-Fe203 ( n ^) Magnetoferritin Control reaction d spacing () measured d spacing measured d spacing

    () () 4.85 111 4.80 4.85 2.97 220 3.01 3.11 2.53 311 2.54 2.53

    2.099 400 2.11 2.11 1.715 422 1.73 1.73 1.616 511 1.65 1.60 1.485 440 1.51 1.50

    (magnetite: space group Fd3m, a=8.39; maghemite: a=8.34)

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    In Hybrid Organic-Inorganic Composites; Mark, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

  • DOUGLAS ET AL. InorganicProtein Interactions

    Figure 1. (a) T E M image of magnetoferritin cores. Scale bar is 50 nm. (b) Electron diffraction from magnetoferritin cores.

    40 50 60 70 80 90 100 110

    Core diameter ()

    Figure 2. Core size distribution of magnetoferritin. The cores average 73 14 in size.

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  • HYBRID ORGANIC-INORGANIC COMPOSITES

    Figure 3. T E M image of the precipitate formed in the absence of ferritin.

    Figure 4. High resolution T E M (lattice image) of the magnetic core showing the {111} and (220) lattice fringes.

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    In Hybrid Organic-Inorganic Composites; Mark, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

  • 3. DOUGLAS ET AL. InorganicProtein Interactions 23

    4.85) 70 apart, as well as a another lattice fringe (d = 2.25) at 35 to these. These are consistent with the {111} and (220) lattice planes respectively of a mineral having cubic symmetry and lattice parameters of 8.4.

    Mossbauer spectroscopy. In an attempt to identify the magnetic iron oxide phase present, the ^ F e Mossbauer spectrum of magnetoferritin was measured (7). Spectra (Figure 5a.) were recorded both in the presence and absence (8) of an applied magnetic field and compared with those of crystalline ferrihydrite, a poorly crystalline ferrihydrite, magnetite and maghemite samples (Figure 5b.). At 100K applied magnetic field and compared with those of crystalline ferrihydrite, a poorly crystalline ferrihydrite, magnetite and maghemite samples (Figure 5b.). At 100K the zero field Mossbauer spectrum is clearly different from native ferritin (9), although the Fe?+ component of the spectrum could indicate the presence of some ferrihydrite-like material. At 4.2K the zero field spectrum shows a sextet with broad asymmetric lines. The lineshapes are consistent with the spectrum of maghemite but the line broadening precludes any definitive distinction between magnetite and maghemite. In addition, the value of the hyperfine field (50 at 4.2K) is consistent with both magnetite and maghemite (10).

    0.00

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    Magnetoferritin 100

    JL -10.0 -6.0 -2.0 2.0

    Velocity (mm/s) 6.0 10.0

    Figure 5a. Mossbauer spectra of the magnetoferritin sample at 100 K, and at 4.2K in zero field and with a magnetic field of 9T field applied perpendicular to the -ray beam.

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