Iron is an essential element necessary for human life which is stored in the ubiquitous and highly-conserved protein ferritin. The protein plays a key role in iron metabolism and its ability to sequester the element allows ferritin to be essential to iron detoxification and reserve. Regulation of iron is critical to many biological processes and deviation leads to many diseased states.
Ferritin is an iron-storage protein distributed in high concentrations in the liver and spleen but also 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. The protein shell is highly conserved with a combined molar mass of around 500 kDa, 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 also concentrated on its mechanism and regulation in diseased states.
Much of the recent research on ferritin transitioned from basic structure-function relationships to a nanoparticle model for metal load and colloid mixture analysis, as the protein cage of ferritin has been proven useful as a storage vessel for a multitude of minerals of other metals including gadolinium, lead, cadmium, nickel, cobalt, chromium, and gold. Ferritin has also recently been used to create magnesium-, cobalt-, and copper-based nanoparticles for electronics with conductive and magnetic properties. However, it has become increasingly apparent that control of the size and mineral load is critical to fabrication of nanomaterials such as memory devices and for fluorescent labeling of biomolecules.
Here, we describe a standardized method for determining the maximum load of several sources of ferritin by purification of the monomeric protein by size exclusion chromatography, sizing by dynamic light scattering, isolation of maximally-loaded species by preparative centrifugation, and finally, mass determination by sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation. We also present procedures for improving core size homogeneity utilizing sucrose-gradient preparative ultracentrifugation (Ghirlando, et. al., Nanotechnology, 2015, In Press)