Comparative Structural and Chemical Studies of Ferritin Cores with Gradual Removal of their Iron Contents

Natividad Galvez, Belen Fernandez, Purificacion Sanchez, Rafael Cuesta, Marcelo Ceolın,*, Miguel Clemente-Leon, Susana Trasobares, Miguel Lopez-Haro, Jose J. Calvino, Odile Stephan, and Jose M. Domınguez-Vera*.

The ferritin iron core consists of a polyphasic structure with a ferrihydrite-magnetite core-shell structure. The proportion of magnetite increases with iron removal (from 20 to 70%). The magnetite does not spontaneously remove iron(II), as previously believed.

J. Am. Chem. Soc., (2008), 130 (25), 8062–8068.

Covalent deposition of ferritin nanoparticles onto gold surfaces.

José M. Domínguez-Vera, Lorena Welte, Natividad Gálvez, Belén Fernández, Julio Gómez-Herrero, Félix Zamora.

Ferritin nanoparticles have been inmobilized onto a properly modified gold surface by specific covalent bonding through lysine rests at the ferritin external surface. AFM images confirmed the existence of a single ferritin monolayer. This is an easy and flexible route to form stable ferritin networks, which are covalently fixed to a gold substrate.

Nanotechnology (2008) 19 025302.

Fluorescence Resonance Energy Transfer (FRET) in Ferritin Labeled with Multiple Fluorescent Dyes.

Belén Fernández, Natividad Gálvez, Purificación Sánchez, Rafael Cuesta, Ruperto Bermejo and José M. Domínguez-Vera.

We simultaneously labeled ferritin with two Alexa Fluor fluorophores (AF350 and AF430). When both fluorophores are labeled to the same ferritin subunit, fluorescence energy transfer (FRET) takes place from the excited AF350 to the acceptor AF430. By varying the number and ratio of labeled fluorophores, FRET can be modulated such that the ferritin particles can exhibit multiple colors under UV illumination. Labeling of the ferritin shell does not affect properties of the metallic core.

J Biol Inorg Chem (2008) 13:349–355

Size-controlled water-soluble Ag nanoparticles .

José M. Domínguez-Vera*a, Natividad Gálveza, Purificación Sáncheza, Antonio J. Motaa, Susana Trasobaresb, Juan C. Hernándezb and Jose J. Calvino*.

Water soluble and extremely stable Ag nanoparticles have been prepared in the apoferritin cavity. Size control is possible by changing the initial Ag+-apoferritin stoichiometry ratio.

Eur. J. Inorg. Chem. (2007) 4823–4826

Permanent magnetism in apoferritin-encapsulated Pd nanoparticles.

Miguel Clemente-León, Eugenio Coronado, Alejandra Soriano-Portillo, Natividad Gálvez and José M. Domínguez-Vera.

Pd nanoparticles have been prepared within the apoferritin cavity. X-Ray powder diffraction, transmission electronic microscopy and magnetization measurements have been used for characterizing the nanoparticles. The nanoparticles exhibit permanent magnetism at room temperature.

J. Mater. Chem., 2007, 17, 49 - 51, DOI: 10.1039/b614592b

Apoferritin-encapsulated Ni and Co Superparamagnetic Nanoparticles.

N. Gálvez, P. Sánchez, J. M. Domínguez-Vera, M. Clemente-León, E. Coronado.

Superparamagnetic Ni and Co nanoparticles have been prepared within the apoferritin cavity. The protein shell prevents bulk aggregation of the metal particles, rendering them water soluble.

JOURNAL OF MATERIALS CHEMISTRY 2006, 16, 2757-2761.

Magnetic Langmuir-Blodgett Films of Ferritin with Different Iron Contents.

José M. Dominguez-VeraMiguel Clemente-León, Eugenio Coronado, Alejandra Soriano-Portillo, Enrique Colacio, José M. Domínguez-Vera, Natividad Gálvez, Rafael Maduenño, and María T. Martín-Romero.

Magnetic Langmuir-Blodgett films of four ferritin derivatives with different iron contents containing 4220, 3062, 2200, and 1200 iron atoms, respectively, have been prepared by using the adsorption properties of a 6/1 mixed monolayer of methyl stearate (SME) and dioctadecyldimethylammonium bromide (DODA). The molecular organization of the mixed SME/DODA monolayer is strongly affected by the presence of the water-soluble protein in the subphase as shown by ð-A isotherms, BAM images, and imaging ellipsometry at the water-air interface. BAM images reveal the heterogeneity of this mixed monolayer at the air-water interface. We propose that the ferritin is located under the mixed matrix in those regions where the reflectivity is higher whereas the dark regions correspond to the matrix. Ellipsometric angle measurements performed in zones of different brightness of the mixed monolayer confirm such a heterogeneous distribution of the protein under the lipid matrix. Transfer of the monolayer onto different substrates allowed the preparation of multilayer LB films of ferritin. Both infrared and UV-vis spectroscopy indicate that ferritin molecules are incorporated within the LB films. AFM measurements show that the heterogeneous distribution of the ferritin at the water-air interface is maintained when it is transferred onto solid substrates. Magnetic measurements show that the superparamagnetic properties of these molecules are preserved. Thus, marked hysteresis loops of magnetization are obtained below 20 K with coercive fields that depend on the number of iron atoms of the ferritin derivative.

Langmuir 2006, 22, 6993-7000.

Rate of iron transfer through the horse spleen ferritin shell determined by the rate of formation of Prussian Blue and Fe-desferrioxamine within the ferritin cavity.

Bo Zhang, Richard K. Watt, Natividad Gálvez, José M. Domínguez-Vera and Gerald D. Watt

Iron (2+ and 3+) is believed to transfer through the three-fold channels in the ferritin shell during iron deposition and release in animal ferritins. However, the rate of iron transit in and out through these channels has not been reported. The recent synthesis of [Fe(CN)₆]³⁻, Prussian Blue (PB) and desferrioxamine (DES) all trapped within the horse spleen ferritin (HoSF) interior makes these measurements feasible. We report the rate of Fe²⁺ penetrating into the ferritin interior by adding external Fe²⁺ to [Fe(CN)₆]³⁻ encapsulated in the HoSF interior and measuring the rate of formation of the resulting encapsulated PB. The rate at which Fe²⁺ reacts with [Fe(CN)₆]³⁻ in the HoSF interior is much slower than the formation of free PB in solution and is proceeded by a lag period. We assume this lag period and the difference in rate represent the transfer of Fe²⁺ through the HoSF protein shell. The calculated diffusion coefficient, D 5.8 × 10^− 20 m²/s corresponds to the measured lag time of 10–20 s before PB forms within the HoSF interior. The activation energy for Fe²⁺ transfer from the outside solution through the protein shell was determined to be 52.9 kJ/mol by conducting the reactions at 1040 °C. The reaction of Fe³⁺ with encapsulated [Fe(CN)₆]⁴⁻ also readily forms PB in the HoSF interior, but the rate is faster than the corresponding Fe²⁺ reaction. The rate for Fe³⁺ transfer through the ferritin shell was confirmed by measuring the rate of the formation of Fe-DES inside HoSF and an activation energy of 58.4 kJ/mol was determined. An attempt was made to determine the rate of iron (2+ and 3+) transit out from the ferritin interior by adding excess bipyridine or DES to PB trapped within the HoSF interior. However, the reactions are slow and occur at almost identical rates for free and HoSF-encapsulated PB, indicating that the transfer of iron from the interior through the protein shell is faster than the rate-limiting step of PB dissociation. The method described in this work presents a novel way of determining the rate of transfer of iron and possibly other small molecules through the ferritin shell.

Biophys. Chem. 2006, 120, 96.

Iron(III) Complexation of Desferrioxamine B Encapsulated in Apoferritin.

José M. Dominguez-Vera

Desferrioxamine B, DFO, was encapsulated in apoferritin by the pH-induced dissociation-reformation of apoferritin in presence of DFO. DFO remains encapsulated because is too large to exit through the apoferritin channels. However, iron(III) can diffuse through these channels and react with the DFO-encapsulated molecules, giving rise to the [DFOFe] complex, which is detected by UV-Vis spectroscopy through the appearance of its typical maximal absorption at 425 nm. The [DFOFe] complex does not exit the apoferritin structure either, and it remains encapsulated.

Journal of Inorganic Biochemistry 2004, 98, 469-472.

Preparation of Cu and CuFe Prussian Blue Derivative Nanoparticles Using  the Apoferritin Cavity as Nanoreactor

Galvez, Natividad; Sanchez, Purificacion; Dominguez-Vera, Jose M.

Copper and CuFe Prussian blue nanoparticles have been prepared by using a copperII-loaded apoferritin as a chemically and spatially confined environment for their construction.

Dalton Transactions 2005, 2492-2494.

Catechol releases iron(III) from ferritin by direct chelation without iron(II) production.

Purificación Sánchez, Natividad Gálvez, Enrique Colacio, Elena Miñambres, José M. Domínguez-Vera

The mechanism of the release of iron from ferritin by catechol does not take place by iron(II) reduction (the usual assumption) but by direct iron(III) chelation and therefore without iron(II) production. A possible extension of these findings to other catechols is discussed on the basis of the stability with respect to the internal redox reaction of the iron(III)-catechol complexes.

DALTON TRANSACTIONS 2005, 811-815.

Release of Iron from Ferritin by Aceto- and Benzohydroxamic Acids.

Natividad Gálvez, Beatriz Ruiz, Rafael Cuesta, Enrique Colacio, José M. Domínguez-Vera.

The release of iron from ferritin by aceto- and benzohydroxamic acids was studied in function of the chelator concentration, pH, and presence or absence of urea. The results collectively demonstrate that both aceto- and benzohydroxamic acid remove iron from ferritin, despite the greater size of benzohydroxamic acid compared with the ferritin channels, indicating the flexibility of these channels. Furthermore, the results show that the iron release from ferritin by aceto- and benzohydroxamic acid is not as slow as traditionally considered for direct iron chelation.

INORGANIC CHEMISTRY 2005, 44, 2706-2709.

Nanoparticles of Prussian-blue Ferritin: A New Route for Obtaining Nanomaterials.

José M. Domínguez-Vera, Enrique Colacio.

Encapsulated nanoparticles of Prussian blue in apoferritin were made by reaction of iron(II) with hexacyanoferrate(III)-loaded apoferritin, which was previously prepared by the pH-induced dissociation-reformation of apoferritin in presence of hexacyanoferrate(III). TEM images of the blue solution obtained showed discrete spherical electron dense iron particles with an average size of about 5 nm. This represents a new route for preparing metallic nanoparticles that offers control over the size and protection against aggregation. Moreover, the fact that the particles are obtained by reaction of hexacyanoferrate(III) and iron(II) building blocks opens up the possibility of obtaining not only homo- but also heterobimetallic nanoparticles.

Inorganic Chemistry 2003, 42, 6983-6984.