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. 2016 Jan 29;27(4):045102.
doi: 10.1088/0957-4484/27/4/045102. Epub 2015 Dec 14.

Enrichment and characterization of ferritin for nanomaterial applications

Rodolfo Ghirlando  1 , Radina Mutskova, Chad Schwartz
Affiliations

Enrichment and characterization of ferritin for nanomaterial applications

Rodolfo Ghirlando et al. Nanotechnology. .

Abstract

Ferritin is a ubiquitous iron storage protein utilized as a nanomaterial for labeling biomolecules and nanoparticle construction. Commercially available preparations of horse spleen ferritin, widely used as a starting material, contain a distribution of ferritins with different iron loads. We describe a detailed approach to the enrichment of differentially loaded ferritin molecules by common biophysical techniques such as size exclusion chromatography and preparative ultracentrifugation, and characterize these preparations by dynamic light scattering, and analytical ultracentrifugation. We demonstrate a combination of methods to standardize an approach for determining the chemical load of nearly any particle, including nanoparticles and metal colloids. Purification and characterization of iron content in monodisperse ferritin species is particularly critical for several applications in nanomaterial science.

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Figures

Figure 1:
Figure 1:
Characterization of monomeric ferritin and apoferritin. (A) Ferritin and apoferritin appear homogenous by dynamic light scattering. Regularized CONTIN size-distributions based on dynamic light scattering intensity data collected for monomeric apoferritin (blue) and ferritin (red) purified by size exclusion chromatography. (B) Sedimentation velocity c(s) profiles for monomeric apoferritin (blue) and ferritin (red) following purification by size exclusion chromatography.
Figure 2:
Figure 2:
Fractionation and characterization of ferritin. (A) Monomeric Sigma-Aldrich ferritin was fractionated on a 5–30% (w/v) sucrose gradient. Absorbance profiles for apoferritin (blue) and ferritin (red) following centrifugation at 38,000 rpm for 2.5 hours on a SW40Ti rotor, and fractionation. Fraction 1 is the top of the gradient, whereas fraction 24 is the bottom. (B) Selected sedimentation velocity c(s) profiles for monomeric ferritin fractions resolved on a 5–30% sucrose gradient and purified by size exclusion chromatography. Profiles for fractions 5 (orange), 8 (dark yellow), 11(blue), 14 (red) and 17 (green) are shown, along with data for apoferritin (blue, scaled plot) and unfractionated monomeric ferritin (dashed black line). (C) Sedimentation velocity c(s) profiles for monomeric apoferritin (blue, scaled plot) and fractionated ferritin, fraction 17 corresponding to fully loaded ferritin (red). Fully loaded ferritin has a sedimentation coefficient of 60S.
Figure 3:
Figure 3:
Characterization of ferritin and apoferritin. (A) Approximately 5 mg of unpurified apoferritin (blue), 2.7 mg of unpurified Sigma-Aldrich ferritin (red) and 5 mg of Amersham ferritin (green) were resolved by size exclusion chromatography. The species eluting at 110 min. represent the monomeric fractions. (B) Size exclusion profiles for monomeric Amersham ferritin fractions 14 (red), 15 (blue), 16 (grey), and 17 (green) resolved on a preparative 5–30% sucrose gradient. Pooled fractions (total volume of 9 mL) contain ~15 – 30 μg/mL of protein. Peak fractions (0.5 mL) contain 20 μg/mL (fraction 17), 30 μg/mL (fraction 16), 63 μg/mL (fraction 15) and 67 μg/mL (fraction 14) of protein based on the modified Lowry assay.
Figure 4:
Figure 4:
Characterization of ferritin. (A) Sedimentation velocity c(s) profiles for Amersham ferritin (red) and monomeric Amersham ferritin purified by size exclusion chromatography (blue). (B) Sedimentation velocity c(s) profiles for monomeric Amersham ferritin fractions resolved on a 5–30% sucrose gradient and purified by size exclusion chromatography. Profiles for fractions 14 (red), 15 (blue), 16 (grey), and 17 (green) are shown.
Figure 5:
Figure 5:
Determination of the partial specific volume for the iron ferritin core. The product of the experimental sedimentation coefficient and calculated frictional coefficient, in the form of SNf is plotted against the estimated iron core molar mass. Data are shown for apoferritin, sucrose gradient fractions 8, 11, 14 and 17 of Sigma-Aldrich ferritin and fraction 17 of Amersham ferritin.
Figure 6:
Figure 6:
Buoyant molar mass of apoferritin. (A) Sedimentation equilibrium profiles at 280 nm of 0.3 A280 load apoferritin in PBS at 20°C and 3,000 (red), 5,000 (blue) and 8,000 (green) rpm. Data are modeled globally in terms of a single ideal species with the best-fit shown as solid lines. The corresponding residuals are displayed. For clarity only every third radial data point is shown. (B) Buoyant molar mass of apoferritin as a function of solvent density in sucrose and PBS. Errors in the buoyant molar mass are obtained from an analysis of the individual concentrations studied for each sample. The best-fit line provides values of the intercept and slope for the determination of the partial specific volume v¯2 and hydration parameter B1.
Figure 7:
Figure 7:
Buoyant molar mass of ferritin. (A) Sedimentation equilibrium profiles at 280 nm of 0.35 A280 load Amersham ferritin (fraction 17) in PBS at 20°C and 3,000 (red) and 5,000 (blue) rpm. Data are modeled globally in terms of a single ideal species with the best-fit shown as solid lines. The corresponding residuals are displayed. For clarity only every third radial data point is shown. (B) Buoyant molar mass of Amersham ferritin (fraction 16) as a function of solvent density in sucrose and PBS. The best-fit line provides values of the intercept and slope for the determination of the partial specific volume v¯2 and hydration parameter B1.
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