Native Horse Spleen Ferritin Research Articles

Fabrication of Gold Nanoparticles Inside Unmodified Horse Spleen Apoferritin

Size-constrained gold nanoparticles are successfully synthesized within the cavity of native horse spleen ferritin by using a two-step process relying on the initial formation of nanoclusters. The particles co-elute with the nanocage ferritin by chromatography, indicating that the particles are associated with intact protein cages. Further characterization reveals that the growth of the monodisperse particles is limited by the protein cage.

Radio frequency magnetic field effects on molecular dynamics and iron uptake in cage proteins

The protein ferritin has a natural ferrihydrite nanoparticle that is superparamagnetic at room temperature. For native horse spleen ferritin, we measure the low field magnetic susceptibility of the nanoparticle as 2.2 x 10(-6) m(3) kg(-1) and its Néel relaxation time at about 10(-10) s. Superparamagnetic nanoparticles increase their internal energy when exposed to radio frequency magnetic fields due to the lag between magnetization and applied field. The energy is dissipated to the surrounding peptidic cage, altering the molecular dynamics and functioning of the protein. This leads to an increased population of low energy vibrational states under a magnetic field of 30 microT at 1 MHz, as measured via Raman spectroscopy. After 2 h of exposure, the proteins have a reduced iron intake rate of about 20%. Our results open a new path for the study of non-thermal bioeffects of radio frequency magnetic fields at the molecular scale.

Very high field magnetization and AC susceptibility of native horse spleen ferritin

The magnetization of native horse spleen ferritin protein is measured in pulsed magnetic fields to 55 T at T=1.52 K. The magnetization rises smoothly with negative curvature due to uncompensated Fe 3+ spins and with a large high field slope due to the underlying antiferromagnetic ferritin core. Even at highest fields the magnetic moment is only ∼4% of the saturation moment of the full complement of Fe 3+ in the ferritin molecule. The AC magnetic susceptibility, χ AC( T, f), responding to the uncompensated spins, reaches a maximum near the superparamagnetic blocking temperature with the temperature of the maximum, T M, varying with excitation frequency, T M −1 α log f for 10⩽ f⩽10 4 Hz.

Apparent magnetic energy-barrier distribution in horse-spleen ferritin: Evidence for multiple interacting magnetic entities per ferrihydrite nanoparticle

Magnetic viscosity measurements were made on native horse-spleen ferritin in zero applied magnetic field at temperatures between 2 and 21 K. The data have been used to calculate the apparent magnetic-moment-weighted energy barrier distribution for the sample of ferritin. The distribution is composed of a log-normal distribution plus a second distribution that is well described by an exponential decay of barrier frequency with increasing barrier height. The two distributions contribute approximately equally to the overall distribution. The log-normal distribution has its peak at an energy barrier of approximately $3\ifmmode\times\else\texttimes\fi{}{10}^{\ensuremath{-}21}\mathrm{J},$ while the decay constant for the second distribution has a value of approximately $2\ifmmode\times\else\texttimes\fi{}{10}^{\ensuremath{-}21}\mathrm{J}.$ The existence of the low-energy barrier distribution with exponentially decaying shape in conjunction with the observation of shifted field-cooled magnetic hysteresis loops is interpreted as strong evidence for the existence of multiple interacting magnetic entities within each ferritin particle.

Fine particle iron oxide prepared by aerosol pyrolysis of the iron storage protein ferritin

Ferritin is a naturally occurring iron storage protein comprising spherical 6 mn diameter cores of hydrated iron oxyhydroxide surrounded by an organic shell. We present here early results on the aerosol pyrolysis of dilute dispersions of ferritin in water, a project undertaken with a view towards preparing monodisperse magnetic nanoparticles as model systems for studies of fine particle magnetism. Ferritin solution was prepared by rehydrating 4.2 mg of freeze dried native horse spleen ferritin in 50 ml of distilled, deionised and millipore filtered water. This solution was passed through a Bernoulli effect nebuliser and the resultant aerosol was carried through a pair of tube furnaces by compressed nitrogen gas, at a rate of approximately 3 1 s-l. The first oven temperature was held at 600 “C while the second oven temperature was held at 500, 600 and 700 “C in three separate runs. Particulate deposits were captured on nuclepore filters over 40 to 60 minute periods.

Magnetic interactions in native horse spleen ferritin below the superparamagnetic blocking temperature

The magnetic interactions in a freeze-dried native horse spleen ferritin were quantified by measuring 5 K IRM and DCD remanence curves using a SQUID magnetometer. Irreversible and reversible components of magnetic moment were determined for a range of fields to ± 30 kOe and attributed to blocked and unblocked SPM particles, respectively. This separation allowed the proportion and mean magnetic properties of each population to be determined. By fitting to a modified Langevin, the mean magnetic moment per particle of the unblocked population was calculated to be 15 μ B, whilst constituting 67% of the magnetic moment at 5 K. Inter-particle interactions in ferritin were quantified using Henkel and Δ I plots, showing that ferritin particles weakly interact, with Δ I < 3%. This finding is consistent with a weak dipole-dipole interaction between otherwise magnetically independent particles.

Immunomagnetic isolation of magnetoferritin-labeled cells in a modified ferrograph

Pan T, helper, and cytotoxic lymphocytes were isolated from the human peripheral blood mononuclear cell fraction by antibody staining, ferritin labeling, and deposition on glass slides. Two distinct forms of ferritin were used: one was native horse spleen ferritin, and the other was magnetoferritin. Magnetoferritin was obtained by reconstituting the horse spleen ferritin iron core with superparamagnetic magnetite instead of the usual paramagnetic ferrihydrite crystal. The cell deposition on microscopic glass slides in the magnetic field was obtained by an instrument that was adapted from an industrial magnetic deposition analyzer, the ferrograph. The identity of cells in the magnetic deposits was confirmed by comparing the cell fractions in the feed and in the eluate with the use of flow cytometry. The immunostaining protocol amplified the number of ferritin molecules per cell surface antigen 20-70 times. Magnetoferritin, but not native ferritin, imparted a sufficient magnetic moment to cells to deplete the labeled cell population between 67 and 88% of its initial concentration in a magnetic field of 1.67 Tesla (T), a field gradient of 2.57 T/mm, and a flow rate of 0.01 ml/min. This study showed that the magnetic moment of magnetoferritin was sufficient for immunomagnetic isolation of lymphocytes from mononuclear cell preparations in the modified ferrograph.

Immunomagnetic isolation of magnetoferritin‐labeled cells in a modified ferrograph

Pan T, helper, and cytotoxic lymphocytes were isolated from the human peripheral blood mononuclear cell fraction by antibody staining, ferritin labeling, and deposition on glass slides. Two distinct forms of ferritin were used: one was native horse spleen ferritin, and the other was magneto-ferritin. Magnetoferritin was obtained by reconstituting the horse spleen ferritin iron core with superparamagnetic magnetite instead of the usual paramagnetic ferrihydrite crystal. The cell deposition on microscopic glass slides in the magnetic field was obtained by an instrument that was adapted from an industrial magnetic deposition analyzer, the ferrograph. The identity of cells in the magnetic deposits was confirmed by comparing the cell fractions in the feed and in the eluate with the use of flow cytometry. The immunostaining protocol amplified the number of ferritin molecules per cell surface antigen 20–70 times. Magnetoferritin, but not native ferritin, imparted a sufficient magnetic moment to cells to deplete the labeled cell population between 67 and 88% of its initial concentration in a magnetic field of 1.67 Tesla (T), a field gradient of 2.57 T/mm, and a flow rate of 0.01 ml/min. This study showed that the magnetic moment of magnetoferritin was sufficient for immunomagnetic isolation of lymphocytes from mononuclear cell preparations in the modified ferrograph. © 1996 Wiley-Liss, Inc.

The effect of temperature on the radial distribution function for iron in native horse spleen ferritin

Iron K-edge X-ray absorption spectra were recorded for a sample of freeze-dried horse spleen ferritin over a range of temperatures from 40 to 300 K. Gaussian-type radial distribution functions were fitted to the data from all temperatures simultaneously and the obtained mean-square relative displacements σ 2 (EXAFS Debye-Waller factors) were fitted using an Einstein model. For iron atoms in the second and/or third coordination shell an Einstein temperature of 330 ± 20 K was obtained. If oxygen was assumed in the second or third shell, its Einstein temperature was 460 ± 20 K. This indicates that the core of ferritin may have a somewhat more rigid structure than those of previously studied ferritin analogues (polysaccharide iron complexes).

Reaction of hydrogen sulfide with native horse spleen ferritin

Ferritin is a protein that consists of 24 subunits that selfassemble to form a roughly spherical shell with external and internal diameters of approximately 12 and 8 nm, re~pectively.~ Hydrophilic and hydrophobic channels connect the internal cavity with the exterior space. These channels are thought to facilitate the passage of ions into and out from the central cavity of the protein. Different forms of ferritin are found in all five living kingdoms and function variously as iron-storage, -detoxification, -regulation, and -transport protein^.^ Iron is usually bound in the form of an inorganic solid phase of hydrated iron oxyhydroxide or phosphate within the central cavity of the m~lecu le .~ The composition, size, and structure of the inorganic phase vary depending on the source and history of the ferritin. Recently it was proposed that the supramolecular protein structure of ferritin could be used as a reaction cage to engineer novel inorganic nanospace structures with structural and optical properties that may find application in catalysis and optoelectronic d e ~ i c e s . ~ , ~ Ferritin has already been used to synthesize discrete inorganic nanospace structures containing iron sulfides, manganese oxides, uranium oxides! and ferrimagnetic iron oxides.1° It was shown using transmission electron microscopy (TEM) and energy-dispersive X-ray analysis (EDXA) that iron and sulfur were present within ferritin cages after reaction of native ferritin (containing ferrihydrite (5Fe203*9HzO)-like cores) with hydrogen sulfide gas.8 The aim of the present work is to use Mdssbauer spectroscopy (i) to determine the extent of the conversion of ferrihydrite to iron sulfide phases and (ii) to determine the nature of the iron sulfide phase(s) generated.

Reconstituted and native iron-cores of bacterioferritin and ferritin

The structural and magnetic properties of the iron-cores of reconstituted horse spleen ferritin and Azotobacter vinelandii bacterioferritin have been investigated by high-resolution transmission electron microscopy, electron diffraction and Mossbauer spectroscopy. The structural properties of native horse spleen ferritin, native Az. vinelandii, and native and reconstituted Pseudomonas aeruginosa bacterioferritins have also been determined. Reconstitution in the absence of inorganic phosphate at pH 7.0 showed sigmoidal behaviour in each protein but was approximately 30% faster in initial rate for the Az. vinelandii protein when compared with horse spleen apoferritin. The presence of Zn 2+ reduced the initial rate of Fe(II) oxidation in Az. vinelandii to 22% of the control rate. The iron-cores of the reconstituted bacterioferritins adopt defect ferrihydrite structures and are more highly ordered than their native counterparts, which are both amorphous. However, the blocking temperature for reconstituted Az. vinelandii (22.2 K) is almost identical to that for the native protein (20 K). Particle size measurements indicate that the reconstituted Az. vinelandii cores are smaller in median diameter than the native cores and this reduction in particle volume ( V) offsets the increased magnetocrystalline contribution to the magnetic anisotropy constant ( K) in such a way that the magnetic anisotropy barrier ( KV), and hence the blocking temperature, is similar for both proteins. Reconstituted horse spleen ferritin exhibits a similar blocking temperature (38 K) to that determined for the native protein, although it is structurally more disordered. The possibility of introducing structural and compositional modifications in both horse ferritin and bacterioferritins by in-vitro reconstitution suggests that these proteins do not function primarily as a crystallochemical-specific interface for core development in vivo.

Ultramicroscopic localization of cationized antigen in the glomerular basement membrane in the course of active, in situ immune complex glomerulonephritis.

The sequence of antigen localization and the interaction of immune deposits with the anionic sites of the glomerular basement membrane (GBM) were investigated in an active model of in situ immune complex glomerulonephritis using a cationized ferritin. Three weeks after immunization with native horse spleen ferritin, the left kidneys of rats were perfused with 500 micrograms of cationized ferritin through the left renal artery. One h after renal perfusion, most of ferritin particles localized subendothelially, corresponding to the anionic sites of the lamina rara interna. In the glomerular capillary loops, infiltrating polymorphonuclear leukocytes and monocytes were seen. Some of these monocytes were in direct contact with immune complexes containing ferritin aggregates associated with anionic sites of the lamina rara interna. At 24 h, numerous ferritin aggregates were present subepithelially, preferentially beneath the slit membrane. The subepithelial location of ferritin did not always correspond to the anionic sites of the lamina rara externa. From days 3 to 7, there was remarkable endocapillary cell proliferation in some loops and pronounced effacement of epithelial foot processes. Focal detachment of epithelium from the GBM was observed occasionally. From days 14 to 28, most of ferritin aggregates were located intramembranously and subepithelially. Membranous transformation has already begun around the subepithelial deposits. This morphological study provides insight into the fate of immune deposits and injury to the GBM in the glomerulonephritis.