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.
Highlights
Iron is an essential element necessary for human life which is stored in the ubiquitous and highly-conserved protein ferritin
Commercial solution preparations (Sigma-Aldrich) of horse spleen apoferritin and ferritin were found to contain aggregates and low molar mass contaminants and both samples were purified to yield the monomeric species by size-exclusion chromatography prior to analysis
The apoferritin preparation had an average diameter of 12.9 nm with a sample polydispersity of 0.03; the ferritin preparation returned an average diameter of 12.1 nm with a sample polydispersity of 0.15 (Figure 1A)
Summary
Iron is an essential element necessary for human life which is stored in the ubiquitous and highly-conserved protein ferritin. Ferritin is an iron-storage protein distributed in high concentrations in the liver and spleen and found in the heart and kidney. Ferritins from all species have 24 protein subunits arranged in 4,3,2 symmetry to form a spherical and hollow complex with an approximately 8 nm diameter cavity capable of storing up to 4500 iron atoms [3]. The protein shell is highly conserved with a combined molar mass of around 500 kDa [4, 5], and both the apo- and iron-loaded form have been well-characterized by a wide range of spectroscopic, crystallographic, and biochemical assays, to determine their structure and function. Research efforts on ferritin were concentrated on its mechanism and regulation in diseased states [2, 6]
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