title: Incorporation of (57)Fe-isotopically enriched in apoferritin: formation and characterization of isotopically enriched Fe nanoparticles for metabolic studies.
abstract: The use of (57)Fe-isotopically enriched ferritin for the accurate measurement of Fe : ferritin ratios is proposed for metabolic studies. Thus, the synthesis of (57)Fe-isotopically enriched ferritin from horse apo-ferritin and isotopically enriched (NH4)2(57)Fe(II)(SO4)2 (Mohr's salt) is conducted. Size exclusion chromatography on-line with UV-VIS absorption (at 380 nm) is used in order to monitor the loading process of apo-ferritin. These studies revealed that the Fe-incorporation process involves also the formation of protein aggregates (oligomers) showing higher molecular mass than ferritin. A final optimized protocol involving incubation of the synthesized standard with guanidine hydrochloride (pH 3.5) has provided the best conditions for maintaining a stable protein structure without aggregates. Such (57)Fe-isotopically enriched ferritin was characterized and contained an average of 2200 atoms of Fe per mole of ferritin. The evaluation of the Fe-core after saturation with (57)Fe by Transmission Electron Microscopy (TEM) has revealed the formation of (57)Fe nanoparticles with a similar diameter to that of the commercial Fe-containing ferritin, confirming the process of Fe uptake, oxidation and mineralization within the protein cavity. The synthesized (57)Fe-ferritin shows great potential as a nanometabolic tracer to study the kinetics of Fe release in the cases of iron metabolic disorders.
authors.affiliation: Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, C/Julián Clavería 8, 33006 Oviedo, Spain. montesmaria@uniovi.es asm@uniovi.es.
authors.forename: T
authors.initials: T
authors.lastname: Konz
authors.forename: M
authors.initials: M
authors.lastname: Montes-Bayón
authors.forename: A
authors.initials: A
authors.lastname: Sanz-Medel
mesh_terms: D001052:Apoferritins / Q000737:chemistry*; D002850:Chromatography, Gel; D007503:Iron Isotopes / Q000737:chemistry*; D013058:Mass Spectrometry; D053768:Metal Nanoparticles; D046529:Microscopy, Electron, Transmission; D013056:Spectrophotometry, Ultraviolet
chemical_list: D007503:Iron Isotopes; D001052:Apoferritins