Morphology and magnetic structure of the ferritin core during iron

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Morphology and magnetic structure of the ferritin core during iron loading and releasing by magnetooptical and NMR methods Marceli Koralewski, Lucia Balej#íková, Zuzana Mitroova, Miko#aj Pochylski, Miko#aj Baranowski, and Peter Kopcansky ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b18304 • Publication Date (Web): 08 Feb 2018 Downloaded from http://pubs.acs.org on February 9, 2018

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ACS Applied Materials & Interfaces

Morphology and magnetic structure of the ferritin core during iron loading and releasing by magnetooptical and NMR methods Koralewski Marceli1*, Balejčíková Lucia2,3, Mitróová Zuzana2, Pochylski Mikołaj1, Baranowski Mikołaj1, Kopčanský Peter2 1

Faculty of Physics, Adam Mickiewicz University, Umultowska 85, 61-614 Poznań, Poland Institute of Experimental Physics, SAS, Watsonova 47, 040 01 Kosice, Slovakia 3 Institute of Measurement Science, SAS, Dubravska cesta 9, 841 04 Bratislava 4, Slovakia * Corresponding author: E-mail address: [email protected] 2

ABSTRACT The ferritin is a protein, which serves as a storage and transportation capsule for iron inside living organisms. Continuous charging protein with iron and releasing it from the ferritin is necessary to assure proper management of this important ions within organism. From other side synthetic ferritin have great potential for biomedical and technological applications. In this work the behavior of ferritin during the processes of iron loading and releasing was examined using multiplicity of experimental technique. The quality of protein’s shell was monitored using circular dichroism, whereas the average size and its distribution was estimated from dynamic light scattering and TEM images, respectively. Because of the magnetic behavior of the iron mineral, number of magnetooptical methods were used to gain information on the iron core of the ferritin. Faraday rotation and magnetic linear birefringence studies provides evidence that the iron loading and the iron release processes are not symmetrical. The spatial organization of the mineral within the protein’s core changes depending on whether the iron was incorporated to, or removed from the ferritins shell. Magnetic optical rotatory dispersion spectra exclude contribution of Fe(II)-composed mineral, whereas joined magnetooptical and NMR results indicate that no mineral with high magnetization appear at any stage of the loading/release process. These findings suggest that the iron core of loaded/released ferritin consists of single-phase i.e. ferrihydrite. Presented results demonstrate usefulness of emerging magnetooptical methods in biomedical research and applications.

Keywords: Ferritin, Magnetic birefringence, Cotton–Mouton effect, Faraday rotation, MORD, NMR, TEM, CD, DLS

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1. INTRODUCTION Ferritins are a family of iron mineralization and storage proteins occurring in human, animals, plants, and microbial kingdoms1. Iron is stored in the protein hollow shell of ferritin (i.e. apoferritin) as a ferric complex with significant amounts of phosphate. The molecular weight of apoferritin sphere with an outer diameter of 12 nm, and an inner diameter up to 8 nm is about 450 kDa and it is composed of 24 protein subunits. Such protein structure can bound up to 4500 iron atoms within cavity according to the specific requirements of the organism. This flexible and dynamic apoferritin structure, is stable at temperatures up to 80 °C, capable by autocatalytic activity to control not only the transport of ferrous ions, but also ions of other metals (e.g. Co2+, Mn2+), small molecules and chelate ligands. Variety of ferritins exists in nature and there is no single identical process for incorporation of iron into each of them. However, basic mechanism involved in iron ion exchange is related with autocatalytic function regulated through ferroxidase centers. In 24-meric ferritin, the H- chain carries a ferroxidase center, which appears to be essential for iron incorporation, whereas the L-chain facilitates iron mineralization within the cavity1–5. By a suitable chemical process, it is possible to use the apoferritin as a confined environment in which magnetic iron oxide nanoparticles i.e. magnetite or/and maghemite can be synthesized, forming a biocompatible ferrofluid, called magnetoferritin6. It was found that in vitro apoferritin cage, aside from Fe, can incorporate variety of metals ions (Ag, Au, Ni, Cr, Cd, Co, Cu, Zn, Pd, Rh, Pt, Ti, Tb, Gd) as well as alloys, metals oxides and semiconductors (CdS, CdSe, ZnSe, PbS, FeS) or even organics compounds and their complexes (e. g. doxorubicin, ferrocene, cisplatin) 5–12. The interest in synthetic ferritins has been stimulated by possible applications of this unique superparamagnetic material in electronics13 and in biomedicine14,15. Discovery of biological magnetite in the human brain16 and recent studies17 indicated that neurodegenerative diseases are associated with iron metabolism18 and in some cases disruption of normal storage function of apoferritin19. It is well known that ferrous iron (Fe2+) can promote free radical damage of various biomacromolecules function via Fenton reaction 20,21. Such iron overloading related to disorder in functioning and structure of ferritin (for instance mutation of ferritin protein22) could be responsible for transformation of natural ferritin core to magnetite and/or maghemite leading to the emergence of biogenic magnetoferritin. In particular, Quintana and co-workers23 have demonstrated that significant proportion of brain ferritin cores extracted from tissues damaged by Alzheimer’s disease contain magnetite-like compound. The structure, quality and quantity of magnetic structures in the brain ferritin have not been fully recognized yet and it has not been established whether they are related to the origin of the neurodegenerative diseases or to their consequences. Although there is a basic consensus that ferritin iron core composition significantly differs between physiological and pathological ferritins23–25, the chemical composition of the magnetic core inside pathological ferritin has not been determined yet, and the precise mechanism of magnetite formation and its relation to many diseases is still poorly understood3,19–25. Possible role of ferritin in formation of biogenic magnetic nanoparticle (BMN) was recently reviewed3. Explanation of the management of iron in ferritin, especially its release and incorporation processes followed by phase transformation of the core, is of great importance for understanding of many diseases development, especially neurodegenerative or cancer disorders. Native or natural iron core of ferritin is formed through Fe2+ binding and subsequent migration to the enzyme site, called ferroxidase center, where oxidation to Fe3+ occurs. Next, iron migrates to the protein interior where it forms the specific iron mineral4,26.Various physical and chemical methods were used to identify chemical composition of the mineral core3,4,26,27. Now it is generally accepted that the native ferritin iron core forms the (FeOOH)8(FeO:OPO3H2) complex. X-ray diffraction studies confirmed that the structure is similar to a mineral of a 2-line ferrihydrite (Fh) or a 6-line ferrihydrite. Moreover, the type of


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the Fh mineral was found dependent on the number of iron atoms present within the proteins core4,26,27. Different result was obtained in a recent study where the structure of horse spleen ferritin (HSF) core was investigated by transmission electron microscopy (TEM), X-ray absorption Near Edge Spectroscopy (XANES), Electron Energy-Loss Spectroscopy (EELS), Small-Angle X-ray Scattering (SAXS), and superconducting quantum interference (SQUID) methods 28. In this work the ferritin core was gradually removed using thioglycolic acid, and the mineral properties and composition were observed to change as the total iron content in the core decreased. The relative amount of magnetite in ferritin containing 2200 to 200 iron atoms rose steadily from approximately 20% to around 70%, whereas the ferrihydrite mineral fell from 60% to approximately 20%. It was suggested that ferritin showed polycrystalline structure with three different mineral phases: ferrihydrite, magnetite, and hematite, which further occur at different locations in relation to the protein shell depending on the core size28. This result is contestable as another group29,30 showed that the mineral ferrihydrite is electron beam sensitive and will undergo internal rearrangements when exposed to the electron beam at relatively high flux. The possibility of ferritin core formed from two different phases was also indicated by nuclear magnetic resonance (NMR) relaxometry results, however the amount of brain ferritin with magnetite rather than ferrihydrite was found below 1% 31. Modeling the magnetic behavior of HSF with a two-phase core structure was also proposed by Brem et al.32, where two components with high and low coercivity were detected and ascribed to ferrihydrite and probably magnetite/maghemite respectively. Because iron ions embedded in different mineral form possess different magnetic properties, thus various magnetic methods provide valuable information on the ferritin core structure through investigations of its magnetic behavior. Such measurements indicated superparamagnetic behavior of mineral core of native ferritin33, whereas superantiferromagnetic behavior of HSF nanoparticles have been shown by a combination of low-field susceptibility and magnetization measurements (up to 55 × 104 Oe) in the superparamagnetic regime (30–250 K)34,35. As was shown recently, magnetooptical investigations are suitable methods to distinguish various magnetic core structures of aqueous suspension of magnetoferritin and native ferritin3,36,37,38,39,40. The purpose of present work is to gain new information on the structure of the mineral phase of ferritin core using different magnetooptical methods: magnetic linear birefringence (MLB), Faraday rotation (FR) and magnetic optical rotatory dispersion (MORD); and NMR technique. These measurements are supported by additional circular dichroism (CD), dynamic light scattering (DLS) and conventional TEM studies. The main interest concerns the determination of the magnetic properties and the structure of ferritin core and its relation to the number of iron atoms. Particular attention is put on the processes of iron loading and releasing, especially whether the core structure changes when a given number of iron atoms was reached after they were added or drawn off from the ferritin core. The results obtained are compared with those of similar measurements performed for magnetoferritin, as well as for ferritin mimetics, and discussed in the light of earlier investigations of ferritins and similar nanoscale systems. All together, these results allow us to propose the model of a single-phase core structure of the ferritin. The quantity and quality of the information derived from the magnetooptical studies make us to believe that these procedures are very promising tool for possible analytical application in biomedicine. 2. EXPERIMENTAL SECTION 2.1. Chemicals A native horse spleen ferritin and equine spleen apoferritin was obtained from Sigma – Aldrich and used for further treatment using the synthesis scheme described below. FeCl3.6H2O,


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FeSO4.7H2O and NH4OH were obtained from Sigma-Aldrich. All other chemicals 2,2´bipyridyl ,C10H8N2); acetic acid, CH3COOH; ascorbic acid, C6H8O6; Coomassie Brilliant blue G 250, hydrogen peroxide, H2O2; ethanol, C2H6O; ferrozine; HEPES; Mohr salt (Ammonium iron(II) sulfate hexahydrate, (NH4)2Fe(SO4)2.6H2O; phosphoric acid, H3PO4; potassium thiocyanate, KSCN; sodium acetate, C2H3O2Na; sodium dihydrogen phosphate dodecahydrate, Na2HPO4×12H2O; sodium hydroxide, NaOH; sodium phosphate monobasic dehydrate, NaH2PO4×2H2O; trimethylamine N-oxide, (CH3)3NO, thioglycolic acid (C2H4O2S)) were obtained from Sigma or Fluka and were of analytical reagent grade. 2.2. Management of iron content in Ferritin Loading iron ions into ferritin core (Preparation of reconstructed ferritins) Reconstructed ferritins were prepared by gradual additions of Fe2+ ions into apoferritin solution at 25°C and constant stirring. First, apoferritin was added to 0.02 M HEPES buffer treated to pH 6.5 with 2 M NaOH. Solution was deaerated using N2 for 1 hour and the reaction bottle was hermetically enclosed. Then, 0.1 M solution of ammonium iron(II) sulfate hexahydrate, as Fe2+ source (previously deoxygenated) was added to the reaction bottle using syringes. Each addition of Fe2+ was followed by additions of 0.07 M solution of trimethylamine N-oxide, (CH3)3NO as oxidant in stoichiometric ratio Fe2+: oxidant = 2:3 dropwise in 10 steps. After reaction, final solution was dialyzed against 0.02 M HEPES buffer with pH 6.5 at 4°C for 4 days to remove intermediates. Releasing iron ions from ferritin core (Preparation of reduced ferritins) Removing of iron ions from the ferritin’s interior was realized in two ways: 1. Reduced ferritins were prepared by gradual removing of iron in acidic conditions in the presence of chelate. First, ferritin (HSF) solution was added to 0.1 M acetate buffer with pH = 5.2 followed by addition of Fe2+chelate solution of 0.3 M 2,2´- bipyridyl. This mixture was dialyzed against 0.01 M thioglycolic acid buffer pH 4.25 for 30 – 120 minutes at 25°C. The pink color of the ferritin solution indicates the binding of Fe2+ with the chelate. After the reaction was finished, colored product was removed by dialysis against 0.15 M NaCl at 4°C for 4 days. 2. Horse spleen ferritin dispersion was prepared in 0.1 M phosphate buffer pH 7.4. Then, ferritin was reduced by adding of ascorbic acid and released Fe2+ ions were binding by ferrozine in the reaction bottle with observed color change. The reduction was performed in incubator of 1 hour at constant temperature 25°C. The number of released iron was regulated by the stoichiometric ratio of 0.02 M ascorbic acid and 0.02 M ferrozine solutions. After that, the complex of Fe-ferrozine was separated by dialysis against 0.1 M phosphate buffer pH 7.4 at 4°C for 4 days. Determination of loading factor – LF Loading factor (the average number of Fe atoms per one protein macromolecule) was determined using UV-VIS spectrophotometer SPECORD 40 (Analytik Jena) at 25 °C with precision about 1%. The mass concentration of iron atoms was determined after oxidation 2+ 3+ of Fe to Fe ions with 3% H2O2 in an acid environment of concentrated 35% HCl. After addition of 1M KSCN, the red thiocyanate complex of Fe[Fe(SCN)6] was formed and its


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absorbance was measured at the light wavelength 450 nm. The measured absorbance value was recalculated to corresponding mass concentration of iron atoms using earlier registered calibration curve. Standard Bradford method was used for calculation of the mass concentration ( ) of native apoferritin (NA). The absorbance of blue colored complex of Bradford agent with protein was detected at the light wavelength 595 nm after 5 minutes incubation at 25°C. From the calculated ratio of a in a given volume of sample using known molecular weights of NA and iron, respectively, loading factor of sample was calculated according to equation:

=

. .

(1)

2.3 Dynamic Light Scattering (DLS) The hydrodynamic diameter of prepared colloidal solutions was measured by Zetasizer Nano ZS 3600, (Malvern Instruments) using dynamic light scattering (DLS). The measurements were performed at 25°C, in triplicate, using disposable polystyrene cuvettes. The size distribution was displayed in the Zetasizer software, set to protein data analysis mode, as a dependence of the relative intensity of light scattered from particles as a function of their size. In this form of presentation, the average hydrodynamic diameter was represented by the maximum of the curve. 2.4 Circular Dichroism (CD) Ferritin folding and refolding was studied by circular dichroism (CD) using a JASCO spectropolarimeter model J-815 (JASCO, Japan) with a quartz cell of 0.02 cm path length. CD data were collected from 190 nm to 260 nm at room temperature with 50 nm/min scanning speed and with 1 nm bandwidth. All the spectra were the average of three executive scans for each sample. Spectral contribution from buffer was subtracted using software provided by JASCO. The molar ellipticity [deg cm2 dmol-1] was calculated on the basis of a mean residue molecular weight of 113 Da. Obtained spectra were deconvoluted and analyzed using CDSSTR module with Dichroweb - an online server for protein secondary structure analyses from CD spectroscopic data41. 2.5 Transmission Electron Microscopy (TEM) Transmission electron microscope (TEM) images were taken with JEM – 1200 EX II (JEOL, Japan) operated at 80 kV. The samples were diluted with water and placed on carbon-coated Cu grid. Afterwards the grids were dried in the air at room temperature. To gain information about core dimensions, the particles were manually identified and their size was determined from the number of pixels occupied in the image. At least 100 particles, in different areas of the image, have been subjected to the above analysis. The histogram of obtained sizes was created and described by log-normal size distribution function defined as42:

P(D) =

 ln 2 ( D / D0 )  exp  −  2s 2 2π sD  , 1


(2)

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where: D0 and s are parameters related to the average diameter and the standard deviation σ by the formulas43: = D0 exp (s2/2) and σ = D0 exp (s2/2)(exp s2 – 1)1/2. 2.6 Nuclear Magnetic Resonance (NMR) T1 relaxation time measurements were made using a saturation recovery method. The experiment was performed using TEL-PS15 pulsed NMR spectrometer (TEL - Atomic Inc.) operated at 15.251 MHz characterized with relative high homogeneity of magnetic field (Bo = 356.6 mT) generated by iron-core electromagnet with field stabilization. For NMR FID (Free Induction Decay) signals detection quadrature technique was used on two orthogonal channels. The other major experimental parameters were set: Gain = 35 dB, Phase = 106o, Time constant = 5 µs, Dwell time = 2.0 µs and NOP equal 1024. For each sample two accumulations was made. T1 profiles were recorded at 37 oC. The temperature of the sample was controlled by Peltier module installed inside the NMR probe. The uncertainty in the T1 relaxation time was < 5%. To obtain T1 relaxivity in [s-1/mM (protein)], reciprocal of T1 were calculated, the respective buffer values were subtracted and obtained results were divided by the molar concentration of ferritin (protein molecular weight = 450 kDa was used). 2.7 Magnetic Linear Birefringence (MLB) Magnetically induced linear optical birefringence, shortly magnetic linear birefringence (MLB) was measured using the laboratory made set-up described in details in our earlier paper37. The instrument allows precise measurements of the angle of rotation of the polarization plane, θ, for light passing through the sample placed in a magnetic field in the Voigt configuration. The light beam from a He-Ne laser (λ = 632.8 nm) was used. The angle θ is proportional to the birefringence ∆n = n║- n┴ of the sample by the well-known relation:

θ = πL ∆n , λ

(3)

where: L denotes the length of the cell and λ the wavelength of the light used, whereas n║ and n┴ are refractive index of light polarized parallel and perpendicular to direction of magnetic field respectively. The accuracy of the angle estimation was better than 0.001º. In order to conduct an automated experiment the current supply (Electro-Automatik GmbH) for iron-core electromagnet (H ~ 20 kOe), the gaussmeter, the refrigerant circulator, the analyzer rotary stage and the photomultiplier were all interfaced to a PC computer. A program written in LabVIEW® was used for synchronizing the measurement sequence and for data collection. When the sample is placed in a weak external magnetic field H, the induced birefringence is proportional to the square of magnetic field intensity. According to the relation (4) the proportionality constant is known as the Cotton-Mouton constant, CCM44: ∆n = CCM λ Η2 ,

(4)

The CCM constant provides information on the optical and magnetic behavior of the studied sample39: CM

C

ρ N ∆α  ∆χ  µ m  +  =  30nε o λ  µ o kT  kT 

2

 , 


(5)

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where: ∆α is anisotropy of volume optical polarizability, ρN is the volume concentration (particle number density), n is the refractive index of the solution, εo denotes the permittivity of free space, λ the wavelength of the light used, µo is the permeability of free space, k is the Boltzmann constant and T stands for the absolute temperature, µm is the permanent magnetic dipole moment and ∆χ is anisotropy of magnetic susceptibility.

2.8 Faraday Rotation (FR) and Magnetic Optical Rotatory Dispersion (MORD) The angle of rotation of the polarization plane of light was measured using the modified polarimeter model P2000 (JASCO, Japan)38. A static magnetic field was produced in a small water cooled homemade solenoid which fits into the polarimeter. The magnetic field, H, could be sweep from –2.6 to +2.6 kOe using a stabilized high-current power supply (ElektroAutomatik GmbH). The magnetic field was measured by Hall effect AC/DC current sensor calibrated by gaussmeter (TEL - Atomic Inc.). The set-up allowed for determination of the Faraday rotation angle (FR) with accuracy of 0.001o for the substances with absorbance below 2. To reach this condition respective dilution and the optical pathlength were adjust. Cylindrical cuvettes with quartz windows with an optical pathlength, L, of 2 or 8 mm were used. In constant field, magnetic optical rotatory dispersion (MORD) i.e. light wavelength dependence of Faraday rotation angle can be measured from 270 nm to 660 nm using external light source and double prism monochromator. In order to conduct an automated experiment the current supply, the monochromator, and the polarimeter output were all interfaced to a PC computer. A program written in LabVIEW® was used for synchronizing the measurement sequence and for data collection. The second instrument which allow FR measurements in higher magnetic fields, up to 12 kOe, operated with He-Ne or argon laser and in principle working similarly to MLB set-up (see above) was also used. In so called low magnetic field regime, that is when thermal energy of the system exceed the magnetic energy, the behavior of the measured FR angle on the applied magnetic field changes linearly, and can be described by the equation: FR = VLH,

(6)

where V is the Verdet constant characterizing the material. 3. RESULTS Composition, size and structure TEM Figure 1 shows typical TEM images taken on representative ferritin samples with low and relatively high LF obtained after reduction and reconstruction processes, as well as for native ferritin (see also Fig. 1S in Supporting Information). The images look similar to those reported in previous studies of native HSF45. Histograms presenting the particle size distribution were described by log-normal distribution function (as shown in the inset of Fig. 1) and average diameters obtained for selected ferritins are listed in Table 1. As follows from Fig. 1, the core diameter, , increases with the iron loading factor and the distribution in diameters is small, as indicated by relatively narrow histograms. Interestingly, there is no obvious difference between the behavior of (LF) obtained for loaded and partially reduced ferritin. It should be mention that conventional TEM image provides information on the size of the “shadow” casted by the object that does not have to be solid and whose shape may not be necessarily spherical. Also, the hollow sphere (or other perforated object) in the TEM image


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would be hardly distinguishable from the solid grain. Knowing parameters of particular mineral unit cell (ferrihydrite in our case46) it is easy to calculate the dimeter of the solid spherical grain containing given number of iron atoms. The dashed blue line in Fig.1 represents the dependence of solid ferrihydrite sphere versus LF. It is clear that this curve lies quite below the experimental points obtained from TEM images, especially for low values of LF. This means, the extracted from TEM images values cannot be attributed to the diameters of the spherical solid core. As a result, the cores within similar range of values may in fact have very different morphology and thicknesses, which in turn may specifically influence other measured parameters of the system. DLS Figure 2 presents the average hydrodynamic diameter, , of native apoferritin and native ferritin, as well as reconstructed and reduced ferritins in aqueous solutions, as obtained by dynamic light scattering (DLS). Unlike TEM method, which provide images of the shadow of the core and is not sensitive to the proteins shell without special staining, the DLS method provide information about the effective size of whole hydrated/solvated particles. The measured dimension depends on both mass and shape (conformation) of the protein outer shell and therefore is generally larger then Similarly to TEM results, also values are slightly depended on the loading factor. Unlike TEM diameters, (LF) behaviors presented on Fig. 2 are different whether the iron was loaded to ferritin core, or it was released. The observed deviation may be justified by various concentrations of proteins in solution and difference in chemical composition of the solvent, including pH, molality or polarity that can contribute to conformation change of protein shell. Direct observation of this effect can be seen in Fig. 2 for ferritin studied in three different solvents: 0.1 M phosphate buffer (pH ~ 7), 0.02 M HEPES buffer (pH ~ 6) and 0.15 M NaCl solution (pH ~ 7). All samples studied shows DH value in the range 11-25 nm i.e. δ = 14 nm. The spread of the measured values look rather wide, but if we consider single solution only, the change in the ferritin hydrodynamic radius is much smaller i.e. HEPES give δ = 8 nm and phosphate solution give δ = 3 nm, highest δ = 10 nm give NaCl solution. CD Detailed information about the condition of protein shell after chemical processes performed during preparation of modified ferritin samples can be gain by CD spectroscopy. The CD spectra of the reconstructed and partially reduced ferritins with different amount of iron atoms in the core are presented in Fig. 3. Two minima at around 208 nm and 222 nm, which are characteristic of α - helical structure of proteins, are clearly visible. The relation between the amplitudes of these minima change slightly with LF. Deconvolution of the CD spectra delivers an estimate of the secondary structure content in the protein shell of ferritin. The dependencies of helicity and β-sheet content on the iron loading factor obtained during iron reconstruction and reduction processes are presented in the insets on Fig. 3a and Fig. 3b respectively. Coherent changes between helicity and β-structuring can be noticed, i.e. when helicity is decreasing the β-sheet content is increasing. The information derived from CD spectra indicate that the protein shell in the prepared synthetic ferritins is most likely intact after the mineralization/remineralization processes. The iron core does not affect strongly the secondary structure of loaded or released ferritin. However, some small and broad maximum in the degree of helicity, accompanied by the minimum in β-sheet content dependence, may be noticed at around LF = 800 (see insets of Fig. 3). Earlier described DLS results indicated some effect of solvent on the ferritin hydrodynamic radius. Even if the question whether the extrema observed ACS Paragon Plus Environment

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for helicity and β-sheet content may be also related to specific protein-solvent interaction needs further and more dedicated studies, the CD results confirm earlier reports on the loose contacts between iron core and apoferritin cavity47. Preliminary measurements of temperature dependence (range 8-80 oC) of CD and MLB for aqueous suspension of HSF were also made (data not shown). It was found, that in this range of temperatures, the degree of helicity decreased over 30%, whereas the CCM constant decreased nearly 4 times. It was noticed that good condition of the ferritin suspension and repeatability of results could preserved when temperature was not higher than 70 oC. NMR In Fig. 4 T1 relaxivity, measured for loaded and released ferritins at temperature T = 37 °C and magnetic induction B = 0.36 T, were plotted versus LF and compared with respective results for magnetoferritins synthetizes in our laboratory 48. In this figure T1 relaxivity are presented in units of [sek-1mM-1 (protein)] for direct comparison with earlier extensive NMR studies of ferritin (loaded) by Brooks et al.49. At room temperature T1 relaxivity (not shown) are slightly higher than at 37 °C in in agreement with results reported by Brooks et al.49. These authors propose empirical relation describing LF dependence of T1 relaxivity: 1/T1 = A + D (LF)b .

(7)

The parameters of the above equation turned out to be dependent on both, the temperature and the magnetic field induction. For T = 37 °C and B = 1T, the values of parameters were found to be A = 11.3, D = 0.231 and b = 0.67 49. The solid line on Fig. 4 presents the plot of eq. 7, which qualitatively reproduce our experimental data. Small disagreement can be explained by smaller magnetic field induction used in our experiment (B = 0.36 T), difference in the solvent and in the sample preparation procedure. As follows from Fig. 4, all the results for loaded and released ferritins are much smaller than T1 relaxivity values obtained for a few samples of magnetoferritin, whose mineral core is composed mainly from maghemite48. T1 relaxivity was measured also for magnetite and maghemite nanoparticles covered with different surfactants. In order to make a directly comparison between different iron-composed samples, it is more useful to present the T1 relaxivity values related to concentration of iron ions instead of concentration of proteins. This is done by divided respective T1 relaxation time by molar concentration of iron. In these units T1 relaxivity of all studied ferritins cover the range of 0.035 - 0.165 [sek-1mM-1 (Fe)], whereas for magnetite T1 relaxivity span the range of 4 – 46 [sek-1mM-1 (Fe)]50,51. In other words, T1 relaxivity for the mineral core of studied ferritins is 2 - 3 orders of magnitude smaller than T1 relaxivity obtained for nanoparticles build from magnetite or maghemite mineral. Magnetooptical phenomena Magnetically induced linear optical birefringence (MLB), ∆n(H), was measured at room temperature for native horse spleen ferritin aqueous suspensions with various concentration of proteins (data not shown). The overall iron concentration, cFe, in the solution studied was in the range of 0.05 – 0.5 g/L. We found that the value of measured induced birefringence is increasing with the iron concentration. However, the values of specific birefringence (i.e. birefringence ∆n divided by the iron concentration cFe) were found similar for all the samples examined. This is in agreement with detailed study of native ferritins presented in recent papers36,39,52. Such a result suggest that, within such range of iron concentration, there is no aggregation of


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examined ferritins in the solution and any magnetic moment related with these nanoparticles is relatively small. Measurements of MLB for all reduced and loaded ferritins were made for one or two different concentrations within this range. None of the ferritin samples tested showed birefringence saturation in magnetic field range, up 20 kOe and a linear relation between ∆n and H2 is fulfilled. Such a dependence is demonstrative for a Cotton-Mouton effect which takes place at so called low field regime, where magnetic energy of the particle is lower than its thermal energy. In this case the Cotton – Mouton constant, CCM, can be determined with the help of eq. (4). The specific C - M constant values (i.e. CCM/cFe) are listed in Table 1 and their dependence on the iron loading factor are presented on Fig. 5. A good agreement with previously published results for relatively high loaded native HSF36,39,52 was obtained. The effect of carrier medium (solvent) on Cotton-Mouton constant is observed, particularly pronounced for a loading process. Similarly to magnetically induced birefringence, also the Faraday rotation (FR) measured at room temperature, does not show any sign of saturation at the magnetic field strengths applied in the experiment (up to 12 kOe, data not shown). By making a fit of eq. (6) to experimental data, the Verdet constant V can be obtained. The values of specific Verdet constant (i.e. Vsp = V/cFe) for wavelength equal 546 nm are collected in Table 1 and presented on the inset of Fig. 6. The value of Vsp shows a maximum for relatively low values of loading factor and stabilizes at constant value for the synthetic ferritins studied. The results of MORD measurements are presented on Fig. 6. The spectra were collected at room temperature in the spectral range 270 to 660 nm for a constant magnetic field H ≈ 2.6 kOe. Several aqueous suspensions of loaded and reduced ferritins of defined LF were studied. Above the negative edge at around 300 nm all the MORD spectra for loaded and reduced ferritins are relatively similar to each other and do not show any characteristic features. In the visible region of the spectrum FR effect is also quite small (see Fig. 6) introducing uncertainty and some scattering of the experimental points. Below the 300 nm edge the difference between loaded and released ferritins seems to emerge. This is, however, related to a very week FR signal and the contribution of different concentration of proteins and iron in studied samples. Because of experimental limitation we were not able go below 270 nm to study this effect in detail. 4. DISCUSION In the presented study, several methods were used to follow changes in magnetic properties of ferritin in aqueous solution during the iron loading and reducing processes. We used magnetooptical and NMR effects as, in contrary to many other methods, they allow examination of proteins dispersed in liquid solvent without any harmful influence on a mineral core inside their cavity. Number of supporting experimental methods were also used to examine the influence of the iron loading and releasing processes on the size, composition and internal structure of the ferritin mineral core. The DLS results (see Fig. 2) indicated that protein shell was not destroyed during a chemical procedures leading to reduced and reconstructed proteins, however slight modification of dimension was noticed. With the CD method we were able to confirm, that also the secondary structure of protein shell of synthetic ferritins was likely intact after mineralization/remineralization processes (see Fig. 3). Slight changes in the folding of the apoferritin was suggested to results from the difference in the solvent used in these procedures. Experiments performed on HSF at elevated temperatures indicated that this protein have unusually high thermal and denaturant stability i.e. denaturation temperature, Tm, is as high as ~93 oC47. It was recently12 found that ferritin from hyperthermophilic bacterium was virtually


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unaltered after 15 min of exposure to 80 and 90 oC. Our temperature measurements (range 8-80 o C) of CD and MLB for aqueous suspension of HSF demonstrate that the core magnetism is loosely correlated with protein’s shell integrity. The mineral core is not rigidly bined to the carboxyl group of the inside surface cavity amino acids. As long as primary structure composition of protein does not change, some alternations in the secondary structure composition (i.e. % helix or β-sheet etc.) have rather small influence on morphology of the mineral core especially at high loading factor values. Although this may somewhat modify the geometrical shape of mineralized layer, the magnetic phase of the core remains untouched, as will be discussed later. The size of the ferritin iron core was estimated by analyzing the TEM images and correlated with this of the solid-grain nanoparticles. As follows from Fig.1, the same number of iron ions within ferritin core seems distributed over larger area in comparison to ions forming massive grains (which fills the space entirely). This result proves that the shape of the ferritin core, as observed in conventional TEM images, does not correspond to the 2D projection of a massive grain. This result is in agreement with the relationship between the iron loading and iron core morphology established very recently by Jian et al.53. Authors of this work used highly elaborated methodology of HAADF-STEM technique and among other conclusions they noticed substantial core dimension for ferritin with relatively low loading factor. These microscopic results corroborate with our speculation that higher diameter observed using conventional TEM for low and moderately loaded ferritin (below ~1100 Fe atoms) corresponds not to a massive grain but rather to some specific geometrical object formed during mineralization/remineralization processes53. The morphology of such an object should in turn modify the optical polarizability and magnetic susceptibility, as well as the anisotropy of these properties, that is the characteristics which can be studied by magnetooptical methods. The shape of the ∆n(H) dependence can be the source of important information on the type of magnetic material forming the core of studied ferritins. The saturation of magnetic birefringence (MLB) observed for relatively weak magnetic field (as this used in this study) would be the manifestation of the existence of highly magnetic material36. Our experimental setup is able to distinguish this effect for iron concentration as low as 10-5 g/L54. However, the MLB measured for all examined ferritins shows only linear dependence on H2 without any sign of saturation. This observation is a direct evidence that for both, loaded and reduced ferritins, we observe the response of the magnetic material with very low magnetization, i.e. having small or even zero permanent magnetic moment. We can then state, that from the macroscopic point of view, the core of the examined ferritins is not made of magnetite or/and maghemite, or the concentration of these minerals is extremely low. More detailed information comes from the discussion of the Cotton-Mouton constant (CCM) describing the linear MLB characteristic. As follows from eq. 5, the value of CCM depends on two contributions. The first one is related to the magnetic susceptibility, ∆C∆χ ∝ ∆α ∆χ and so to the induced magnetic moment. The second part is associated to the permanent magnetic moment of the nanoparticle, ∆Cµm∝ ∆αµm. In both cases, the crucial role is played by the value of anisotropy of volume optical polarizability, ∆α, connected to morphology of mineralized/remineralized core in the cavity of the ferritin. Although the morphology of the core may also influence it’s magnetic dipolar properties, the details on the origin of magnetic disorder is still a fundamental question55. Even if the exact impact of the above factors on measured specific CCM is not unanimously established, differentiation between different possible mineral core structure can be still realized on the comparative basis. As follows from the data collected in Table 1, the values of CspCM (i.e. CCM/cFe ) for examined ferritins are much lower than those found for magnetite nanoparticle36, magnetoferritin36 or antiferromagnetic


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akaganeite massive grains of few nm in size 39. Substantial values of CspCM, observed for magnetoferritin and magnetite particles, can be easily justified by the high permanent magnetic dipole of the grain-forming mineral. This rationalization is, however, not sufficient to explain differences in CspCM values observed between akaganeite particles and currently studied loaded and released ferritins. Here, the effect of the core shape should be considered. It is well established that ferritin quaternary structure has eight hydrophilic channels that seem to mediate iron transit in and out of the protein cage 27. Autocatalytic processes are slightly different depending on ferritin source2,4,5 however mineralization process starts from the outer part of the cavity and proceeds toward the center as was shown for HSF56,57. Starting from the empty apoferritin, the core formation begins close to the eight channels of the protein shell. Therefore, at low LF it will not constitute a massive grain, but rather create lumps of iron mineral whose positions will be firstly related to the channel locations. As LF is increasing, also the size and the shape of the core progress. The initial iron nuggets turn into irregular surface with a flake-like shape, which further grows on the inner surface of the protein to eventually take the shape of the empty sphere. The gradual change of the core morphology affects anisotropy of both optical polarizability and magnetic susceptibility. This process is evidenced on Fig. 5 where the value CspCM constant is increasing during the iron loading and stabilize when LF reach ~1500 Fe atoms. At this point, the ferritin core resemble the empty ball, since about 1500 – 1600 iron atoms are enough to compose a 1-nm thick ideal hollow sphere inside 8 nm cavity of apoferritin. Further increasing of the number of antiferromagnetically ordered iron ions, inside a hollow ferrihydrite sphere, do not contribute significantly to its magnetic moment and in practice do not change much anisotropies of magnetic susceptibility and optical polarizability of the grain. As a result, both above described contributions to CspCM are more or less stable with increasing a LF over 1500 atoms inside a ferritin cavity. At limiting value of LF (above 4000 Fe atoms) massive grain have eventually a chance to form inside the cavity. The process of releasing iron from ferritin core affects the CspCM values differently than the charging procedure. This means that also the shape of the core undergoes a different change when the iron leaves the interior of the protein. Changes of CspCM (LF) dependence, measured during iron reduction, have the same tendency and initial curve shape like this observed for massive grain of mimetic antiferromagnetic akaganeite nanoparticle (see Fig.12 in ref.39). We can then speculate that during releasing of iron, the core behave more like a massive grain. This conclusion is in accordance with early TEM and SAXS measurements and modeling of ferritin core proposed by Ciasca et al.56,57 as well as with recently published “atlas” of ferritin morphology53. It should be noticed that the obtained results are to some extent dependent on the solvent used, particularly for the iron loading procedure. The explanation of this solvent effect deserve further dedicated studies, nevertheless, also here the lack of the symmetry in CspCM , observed during loading and releasing of iron, is still preserved. Similarly to the MLB results, in Faraday rotation (FR) experiment only the linear relation on H was observed in examined magnetic field range (up to 12 kOe). Absence of the FR saturation38 confirms our earlier suggestion about deficiency of material with high magnetization in studied ferritin samples. From the linear FR(H) dependence, the Verdet constant, V, was determined. For paramagnetic and diamagnetic liquids this parameter was found proportional to the magnetic susceptibility of their molecules i.e. V ∝ χ 58,59,60. For several diamagnetic liquids linear relation between V and optical polarizability α was also experimentally proven60. Because the values of these parameters correlate with the shape and size of the magnetic material, the Verdet constant, similarly to CCM, provides information on the morphology of the ferritin core. Likewise for CCM(LF) dependence, also FR(LF) shows lack of the symmetry in iron loading and releasing processes. This means, that both magnetooptic methods provide coherent picture of the influence of these procedures on the core morphology. The measured


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specific Verdet constant for ferritin examined in this work is about 2 orders of magnitude smaller than this established for magnetite nanoparticle or magnetoferritin (see Table 1 and Fig. 6b)38. Similar conculsion can be made from the comparison of NMR T1 relaxation time (see Fig. 4) measured for examined ferritins and for differently loaded magnetoferritins. These results allows to evidence the ferrihydrite (Fh) as a main component of the core with the irrelevant, if any, contribution from high magnetic minerals like magnetite or maghemite. This statement is also confirmed by MORD spectra (see Fig. 6a) taken for loaded and released ferritins. Firstly, the measured FR values are small in the visible region of spectrum. More importantly, the spectra do not reveal characteristic feature above negative edge at about 300 nm. Such a feature, especially bands around 450 nm, are related with Fe2+ (more spectroscopic information about Fe spectra can be find in38 and references therein) and are often observed for magnetite nanoparticles38,40,61,62. Lack of such feature in MORD spectra proofs that there is no magnetite in the core of examined ferritins. 5. CONCLUSION We have discussed for the first time both the structural and magnetic properties of synthetic ferritin during procedures leading to incorporation and reduction of iron into and out from its magnetic core. Different magnetooptical methods and NMR technique were used with additional characterization by conventional TEM, CD and DLS measurements. The results obtained were compared with literature data for magnetoferritin and ferritin mimetics. The magnetic field dependencies of the Faraday rotation and induced linear magnetic birefringence have been measured for so called low field region only. The dependencies of specific Verdet constant and specific Cotton-Mouton constant on the number of iron ions within the core, determined for loading and releasing processes, were found not symmetrical. These results were rationalized assuming that at the beginning of loading of iron into the empty apoferritin cage, the mineral core forms a hollow shell which eventually (at maximum number of ions that can be fitted in the ferritin cavity) transform into a solid grain. During releasing of iron, only the granule volume decreases without changing the massive-grain morphology of the core which is schematically presented on Fig. 7. The iron loading and release processes are to some extent affected by the solvent, whose role still requires more detailed and dedicated study. Comparison of the magnetooptical and NMR results obtained for studied ferritins with samples containing minerals with high magnetization allow discrimination between a ferrihydrite or magnetite/maghemite core of ferritin studied. No magnetite or maghemite was found in the suspension of studied ferritins at any stage of iron loading/release procedure. This result was confirmed by analysis of the MORD results, where the specific shape of the recorded spectra exclude contribution of Fe2+ composed mineral as building subdomains of the core of reconstructed or partially reduced ferritins. The results presented in this work shed new light on biomineralization and biomagnetism of synthetic ferritin. They allowed us to discuss the mineral phase of the ferritin core and the way its morphology changes during iron loading and its releasing. This information is crucial for better understanding of etiology of neurodegenerative diseases and it can be also very useful in emerging clinical application of magnetooptical methods63,64,65,66. Aside from the purely biomedical applications, the knowledge on the morphology of the core in various phases of its constitution should benefits in better control of procedures utilizing ferritins as nanoreactors for synthesis of different novel materials. Supporting Information


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TEM images of differently loaded ferritin, with particle size distributions obtained from analysis of TEM images. ACKNOWLEDGEMENTS We are grateful to Dr Wojciech Wieczorek for TEM measurements which were performed at Laboratory of Faculty of Biology AMU. We are very grateful to prof. Maciej Kozak (Faculty of Physics AMU) for granting access to the JASCO spectrometer and Witold Gospodarczyk MSc for the help in CD measurements. The work was supported by the project in the frame of SF EU No. 26110230097. REFERENCES (1) Bou-Abdallah, F. Special Issue on Ferritin. Biochimica et Biophysica Acta - General Subjects. 2010, pp 687–688. (2) Sun, S.; Chasteen, N. D. Ferroxidase Kinetics of Horse Spleen Apoferritin. J. Biol. Chem. 1992, 267 (35), 25160–25166. (3) Gorobets, O.; Gorobets, S.; Koralewski, M. Physiological Origin of Biogenic Magnetic Nanoparticles in Health and Disease: From Bacteria to Humans. Int. J. Nanomedicine 2017, Volume 12, 4371–4395. (4) Harrison, P. M.; Arosio, P. The Ferritins: Molecular Properties, Iron Storage Function and Cellular Regulation. Biochim. Biophys. Acta - Bioenerg. 1996, 1275 (3), 161–203. (5) Theil, E. C.; Behera, R. K.; Tosha, T. Ferritins for Chemistry and for Life. Coordination Chemistry Reviews. 2013, pp 579–586. (6) Meldrum, F. C.; Heywood, B. R.; Mann, S. Magnetoferritin: In Vitro Synthesis of a Novel Magnetic Protein. Science (80-. ). 1992, 257 (5069), 522–523. (7) Douglas, T.; Stark, V. T. Nanophase Cobalt Oxyhydroxide Mineral Synthesized within the Protein Cage of Ferritin. Inorg. Chem. 2000, 39 (8), 1828–1830. (8) Meldrum, F. C.; Douglas, T.; Levi, S.; Arosio, P.; Mann, S. Reconstitution of Manganese Oxide Cores in Horse Spleen and Recombinant Ferritins. J. Inorg. Biochem. 1995, 58 (1), 59–68. (9) Tosha, T.; Ng, H. L.; Bhattasali, O.; Alber, T.; Theil, E. C. Moving Metal Ions through Ferritin-Protein Nanocages from Three-Fold Pores to Catalytic Sites. J. Am. Chem. Soc. 2010, 132 (41), 14562–14569. (10) Uchida, M.; Kang, S.; Reichhardt, C.; Harlen, K.; Douglas, T. The Ferritin Superfamily: Supramolecular Templates for Materials Synthesis. Biochimica et Biophysica Acta General Subjects. 2010, pp 834–845. (11) Iwahori, K.; Yamashita, I. Size-Controlled One-Pot Synthesis of Fluorescent Cadmium Sulfide Semiconductor Nanoparticles in an Apoferritin Cavity. Nanotechnology 2008, 19 (49). (12) Kasyutich, O.; Ilari, A.; Fiorillo, A.; Tatchev, D.; Hoell, A.; Ceci, P. Silver Ion Incorporation and Nanoparticle Formation inside the Cavity of Pyrococcus Furiosus Ferritin: Structural and Size-Distribution Analyses. J. Am. Chem. Soc. 2010, 132 (10), 3621–3627. (13) Yamashita, I.; Iwahori, K.; Kumagai, S. Ferritin in the Field of Nanodevices. Biochimica et Biophysica Acta - General Subjects. 2010, pp 846–857. (14) Clavijo Jordan, V.; Caplan, M. R.; Bennett, K. M. Simplified Synthesis and Relaxometry of Magnetoferritin for Magnetic Resonance Imaging. Magn. Reson. Med. 2010, 64 (5), 1260–1266. (15) Figuerola, A.; Di Corato, R.; Manna, L.; Pellegrino, T. From Iron Oxide Nanoparticles towards Advanced Iron-Based Inorganic Materials Designed for Biomedical Applications. Pharmacological Research. 2010, pp 126–143.


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Jian, N.; Dowle, M.; Horniblow, R. D.; Tselepis, C.; Palmer, R. E. Morphology of the Ferritin Iron Core by Aberration Corrected Scanning Transmission Electron Microscopy. Nanotechnology 2016, 27 (46). Koralewski, M. Unpublished Data; Poznan, 2017. Michel, F. M.; Hosein, H.-A.; Hausner, D. B.; Debnath, S.; Parise, J. B.; Strongin, D. R. Reactivity of Ferritin and the Structure of Ferritin-Derived Ferrihydrite. Biochim. Biophys. Acta - Gen. Subj. 2010, 1800 (8). Ciasca, G.; Papi, M.; Chiarpotto, M.; Rodio, M.; Campi, G.; Rossi, C.; De Sole, P.; Bianconi, A. Transient State Kinetic Investigation of Ferritin Iron Release. Appl. Phys. Lett. 2012, 100 (7). Ciasca, G.; Chiarpotto, M.; Campi, G.; Bocca, B.; Rodio, M.; Pino, A.; Ricci, A.; Poccia, N.; Rossi, C.; Alimonti, A.; Amenitsch, H.; De Sole, P.; Bianconi, A. Reconstitution of Aluminium and Iron Core in Horse Spleen Apoferritin. J. Nanoparticle Res. 2011, 13 (11). Van Vleck, J. H.; Hebb, M. H. On the Paramagnetic Rotation of Tysonite. Phys. Rev. 1934, 46 (1), 17–32. Egami, S.; Watarai, H. Microscopic Faraday Rotation Measurement System Using Pulsed Magnetic Fields. Rev. Sci. Instrum. 2009, 80 (9), 93705. Vandendriessche, S.; Valev, V. K.; Verbiest, T. Faraday Rotation and Its Dispersion in the Visible Region for Saturated Organic Liquids. Phys. Chem. Chem. Phys. 2012, 14 (6). Brullot, W.; Reddy, N. K.; Wouters, J.; Valev, V. K.; Goderis, B.; Vermant, J.; Verbiest, T. Versatile Ferrofluids Based on Polyethylene Glycol Coated Iron Oxide Nanoparticles. J. Magn. Magn. Mater. 2012, 324 (11). Lin, C.-R.; Tseng, Y.-T.; Ovchinnikov, S. G.; Ivantsov, R. D.; Edelman, I. S.; Fedorov, a S.; Kuzubov, a a; Fedorov, D. a; Starchikov, S. S.; Lyubutin, I. S. Fe3S4 and Fe3O4 Magnetic Nanocrystals: Magneto-Optical and Mössbauer Spectroscopy Study. Mater. Res. Express 2014, 1, 25033. Newman, D. M.; Heptinstall, J.; Matelon, R. J.; Savage, L.; Wears, M. L.; Beddow, J.; Cox, M.; Schallig, H. D. F. H.; Mens, P. F. A Magneto-Optic Route toward the in Vivo Diagnosis of Malaria: Preliminary Results and Preclinical Trial Data. Biophys. J. 2008, 95 (2), 994–1000. Nour, B. Y. M.; Newman, D. M.; Schallig, H. Laboratory Evaluation on the Sensitivity and Specificity of a Novel and Rapid Detection Method for Malaria Diagnosis Based on Magneto-Optical Technology (MOT). Malar. J. 2010, 9 (1), 207. Ku, B. Y.; Chan, M. L.; Ma, Z.; Horsley, D. A. Frequency-Domain Birefringence Measurement of Biological Binding to Magnetic Nanoparticles. J. Magn. Magn. Mater. 2008, 320 (18), 2279–2283. Murdock, R. J.; Putnam, S. A.; Das, S.; Gupta, A.; Chase, E. D. Z.; Seal, S. HighThroughput, Protein-Targeted Biomolecular Detection Using Frequency-Domain Faraday Rotation Spectroscopy. Small 2017, 13 (12).

FIGURE CAPTIONS Fig. 1 The diameter of the core of native, reconstructed and reduced ferritins obtained from analysis of TEM images versus the number of Fe atoms per grain. Inset shows representative TEM images of native, reconstructed and reduced ferritins, and particle size distributions obtained from analysis of TEM images - solid lines represents log-normal distribution function. The blue line show LF dependence of diameter expected for massive grain.


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Fig. 2 Hydrodynamic diameter of reconstructed and reduced ferritins as obtained from DLS measurements. Fig. 3 CD spectra for a) reconstructed and b) reduced ferritin. Inset - LF dependencies of: in a) the degree of helicity, in b) the degree of β − sheet; dashed line are guide for eyes. Fig. 4 Comparison of T1 relaxivity obtained for reconstructed and reduced ferritins with several differently loaded magnetoferritin. Solid curve eq. (7) – see text. Fig. 5 Specific Cotton-Mouton constant of reconstructed and reduced ferritins versus LF. Stars, circles and triangles describe different methods of synthesis. The solid and dashed line are guide for eyes, full and empty points show loaded and released ferritin samples respectively. Fig. 6 Wavelength dependence of the specific Faraday rotation a), and specific Verdet constant b) for reconstructed and reduced ferritins (at several LF) in aqueous suspensions. Fig. 7 The model of geometrical structure of ferritin core during iron loading and release. The core’s morphology is different depending in the direction of iron incorporation. At any stage of loading/releasing procedure, the iron is stored in the form of ferrihydrite – Fh. Table 1 The specific Cotton-Mouton constant, and specific Verdet constant (λ = 546 nm, T = 295 K), dimension of the core and hydrodynamic diameter of native, released and loaded ferritins with different LF; magnetoferritin, magnetite and akaganeite nanoparticles.

Nanoparticle Native HSF

LF

(No of Fe)

C

(10-14mA-2Lg-1(Fe))

Vsp

µm

(oT-1m-1Lg-1(Fe))

(µB)

D

DH

6,78

18-34

(nm) a

(nm)

1235-2200

4-7

-50

(128-556)

Releasd HSF

138 228 351 421

-0,99 2,81 4,52 3,75

56,5 37,0 -9,4 46,4

-

5,88 6,07 5,50

18,5 19,3 22,0 24,5

Loaded HSF

893 1027 1295 2782

-0,86 0,28 -0,48 -1,20

-9,2 -5,5 -11,2 -13,5

-

(Hepes)

5,72 6,53 6,71 7,09

14,5 17,2 16,3 19,3

Magnetoferritinb

3280

6833

-299

6146

6,8

-

Magnetitec

6420

3,62x105

-477

5984

6,9

-

Akaganeited

377

203

-49

239

3,0

-

(0.15 NaCl)

ab-

estimated from low temperature measurement from ref. 32. ref.38 and 54; c – ref.36 and 38; d – ref.39 and 54.


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Fig.1

Fig.2


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Fig. 3


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Fig. 4

Fig. 5


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Fig.6

Fig.7


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Graphical Abstract


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Morphology and magnetic structure of the ferritin core during iron

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