Ferritin is a ubiquitous intracellular iron storage protein of animals, plants, and bacteria. The cavity of this protein acts like a reaction chamber for natural formation and storage of nano-sized particles via biomineralization. Knowledge of the chemical composition and structure of the iron core is highly warranted in the fields of nano technologies as well as biomolecules and medicine. Here, we show that Raman microspectroscopy (RM) is a suitable nondestructive approach for an analysis of proteins containing such nano-sized iron oxides. Our approach addresses: (1) synthesis of suitable reference materials, i.e., ferrihydrite, maghemite and magnetite nanoparticles; (2) optimization of parameters for Raman spectroscopic analysis; (3) comparison of Raman spectra from ferritin with apoferritin and our reference minerals; and (4) validation of Raman analysis by X-ray diffraction and Mössbauer spectroscopy as two independent complementary approaches. Our results reveal that the iron core of natural ferritin is composed of the iron(III) hydroxide ferrihydrite (Fe2O3 ∙ 0.5 H2O).
TheilE.C. “Ferritin: The Protein Nanocage and Iron Biomineral in Health and in Disease”. Inorg. Chem. 2013. 52(21): 12223–12233.
2.
FanK.CaoC.PanY., et al.“Magnetoferritin Nanoparticles for Targeting and Visualizing Tumor Tissues”. Nat. Nanotech. 2012. 7(833): 459–464.
3.
MassnerC.SigmundF.PettingerS., et al.“Genetically Controlled Lysosomal Entrapment of Superparamagnetic Ferritin for Multimodal and Multiscale Imaging and Actuation with Low Tissue Attenuation”. Adv. Funct. Mater. 2018. 28(19): 1706793.
UenumaM.ZhengB.ImazawaT., et al.“Metal-Nanoparticle-Induced Crystallization of Amorphous Ge Film Using Ferritin”. Appl. Surf. Sci. 2012. 258(8): 3410–3414.
6.
GuptaA.K.GuptaM. “Synthesis and Surface Engineering of Iron Oxide Nanoparticles for Biomedical Applications”. Biomaterials. 2005. 26(18): 3995–4021.
7.
JordanV.C.CaplanM.R.BennettK.M. “Simplified Synthesis and Relaxometry of Magnetoferritin for Magnetic Resonance Imaging”. Magn. Reson. Med. 2010. 64(5): 1260–1266.
8.
MichelF.M.EhmL.AntaoS.M., et al.“The Structure of Ferrihydrite, a Nanocrystalline Material”. Science. 2007. 316(5832): 1726–1729.
9.
ItohH.SugimotoT. “Systematic Control of Size, Shape, Structure, and Magnetic Properties of Uniform Magnetite and Maghemite Particles”. J. Colloid Interface Sci. 2003. 265(2): 283–295.
10.
RümenappC.GleichB.HaaseA. “Magnetic Nanoparticles in Magnetic Resonance Imaging and Diagnostics”. Pharm. Res. 2012. 29(5): 1165–1179.
11.
NistlerA.HartmannC.RümenappC., et al.“Production and Characterization of Long-Term Stable Superparamagnetic Iron Oxide-Shell Silica-Core Nanocomposites”. J. Magn. Magn. Mater. 2017. 442: 497–503.
12.
BabayS.MhiriT.ToumiM. “Synthesis, Structural and Spectroscopic Characterization of Maghemite γ-Fe2O3 Prepared by One-Step Coprecipitation Route”. J. Mol. Struct. 2015. 1085: 286–293.
13.
de FariaD.L.A.VenâncioS.de OliveiraM.T. “Raman Microspectroscopy of Some Iron Oxides and Oxyhydroxides”. J. Raman Spectrosc. 1997. 28(11): 873–878.
14.
SchwamingerS.P.BauerD.Fraga-GraciaP., et al.“Oxidation of Magnetite Nanoparticles: Impact on Surface and Crystal Properties”. Cryst. Eng. Comm. 2017. 19(2): 246–255.
15.
RebodosR.L.VikeslandP.J. “Effects of Oxidation on the Magnetization of Nanoparticulate Magnetite”. Langmuir. 2010. 26(22): 16745–16753.
16.
NurdinI.YaacobI.I.JohanM.R., et al.“Characterization and Stability Monitoring of Maghemite Nanoparticle Suspensions”. Adv. Mater. Res. 2012. 576: 398–401.
17.
MuradE.SchwertmannU. “The Mössbauer Spectrum of Ferrihydrite and its Relations to those of other Iron Oxides”. Am. Mineral. 1980. 65(9–10): 1044–1049.
18.
KimW.SuhC.-Y.ChoS.-W., et al.“A New Method for the Identification and Quantification of Magnetite-Maghemite Mixture Using Conventional X-ray Diffraction Technique”. Talanta. 2012. 94: 348–352.
19.
SchwertmannU.StanjekH.BecherH.-H. “Long-Term In-Vitro Transformation of Two-Line Ferrihydrite to Goethite/Hematite at 4, 10, 15 and 25 ℃”. Clay Miner. 2004. 39(4): 433–438.
20.
KukkadapuR.K.ZacharaJ.M.FredricksonJ.K., et al.“Transformation of Two-Line Ferrihydrite to six-line Ferrihydrite under Oxic and Anoxic Conditions”. Am. Mineral. 2003. 88(11): 1903–1914.
21.
HaneschM. “Raman Spectroscopy of Iron Oxides and (Oxy)hydroxides at Low Laser Power and Possible Applications in Environmental Magnetic Studies”. Geophys. J. Int. 2009. 177(3): 941–948.
22.
IvlevaN.P.HuckeleS.WeinzierlB., et al.“Identification and Characterization of Individual Airborne Volcanic Ash Particles by Raman Microspectroscopy”. Anal. Bioanal. Chem. 2013. 405(28): 9071–9084.
23.
TumaR. “Raman Spectroscopy of Proteins: from Peptides to Large Assemblies”. J. Raman Spectrosc. 2005. 36(4): 307–319.
24.
PalonponA.F.SodeokaM.FujitaK. “Molecular Imaging of Live Cells by Raman Microscopy”. Curr. Opin. Chem. Biol. 2013. 17(4): 708–715.
25.
PeltonJ.T.McLeanL.R. “Spectroscopic Methods for Analysis of Protein Secondary Structure”. Anal. Biochem. 2000. 277(2): 167–176.
26.
CockellC.S.PybusD.Olsson-FrancisK., et al.“Molecular Characterization and Geological Microenvironment of a Microbial Community Inhabiting Weathered Receding Shale Cliffs”. Microb. Ecol. 2011. 61(1): 166–181.
27.
ReicheM.LuS.CiobotăV., et al.“Pelagic Boundary Conditions Affect the Biological Formation of Iron-Rich Particles (Iron Snow) and their Microbial Communities”. Limnol. Oceanogr. 2011. 56(4): 1386–1398.
28.
GomezM.A.HendryM.J.HossainA., et al.“Abiotic Reduction of Two-Line Ferrihydrite: Effects on Adsorbed Arsenate, Molybdate, and Nickel”. RSC Adv. 2013. 3(48): 25812–25822.
29.
BoucharM.DillmannP.NeffD. “New Insights in the Long-Term Atmospheric Corrosion Mechanisms of Low Alloy Steel Reinforcements of Cultural Heritage Buildings”. Materials. 2017. 10(6): 670–683.
30.
JanusB.BućkoM.S.ChrobakA., et al.“Magnetic Characterization of Human Blood in the Atherosclerotic Process in Coronary Arteries”. J. Magn. Magn. Mater. 2011. 323(5): 479–485.
31.
Abdus-SalamN.M’CiverF.A. “Synthesis, Characterization and Application of Two-Line and Six-Line Ferrihydrite to Pb(II) Removal from Aqueous Solution”. J. Appl. Sci. Environ. Manage. 2012. 16(4): 327–336.
32.
PetcharoenK.SirivatA. “Synthesis and Characterization of Magnetite Nanoparticles via the Chemical Co-Precipitation Method”. Mater. Sci. Eng. B. 2012. 177(5): 421–427.
33.
U. Schwertmann, R.M. Cornell. “Synthesis”. In: U. Schwertmann, R.M. Cornell, editors. The Iron Oxides: Structure, Properties, Reactions, Occurrence and Uses. Weilheim, Germany: Wiley-VCH Verlag GmbH and Co., 2003. Chap. 20, Pp. 535–539.
34.
AshtonL.BrewsterV.L.CorreaE., et al.“Detection of Glycosylation and Iron-Binding Protein Modifications Using Raman Spectroscopy”. Analyst. 2017. 142(5): 808–814.
35.
MuradE. “The Mössbauer Spectrum of ‘Well'-Crystallized Ferrihydrite”. J. Magn. Magn. Mater. 1988. 74: 153–157.
36.
PollardR.J.CardileC.M.LewisD.G., et al.“Characterization of FeOOH Polymorphs and Ferrihydrite Using Low-Temperature, Applied-Field, Mössbauer Spectroscopy”. Clay Miner. 1992. 27(1): 57–71.
37.
SchwertmannU.FriedlJ.StanjekH. “From Fe(III) Ions to Ferrihydrite and then to Hematite“. J. Colloid Interfaces Sci. 1999. 209(1): 215–223.
38.
MichelF.M.HoseinH.-A.HausnerD.B., et al.“Reactivity of Ferritin and the Structure of Ferritin-Derived Ferrihydrite”. Biochim. Biophys. Acta. 2010. 1800(8): 871–885.
39.
JubbA.M.AllenH.C. “Vibrational Spectroscopic Characterization of Hematite, Maghemite, and Magnetite Thin Films Produced by Vapor Deposition”. ACS Appl. Mater. Interfaces. 2010. 2(10): 2804–2812.
GriffeteN.LamouriA.HerbstF., et al.“Synthesis of Highly Soluble Polymer-Coated Magnetic Nanoparticles Using a Combination of Diazonium Salt Chemistry and the Iniferter Method”. RCS Adv. 2012. 2(3): 826–830.
42.
LeeN.SchuckP.J.NicoP.S., et al.“Surface Enhanced Raman Spectroscopy of Organic Molecules on Magnetite (Fe3O4) Nanoparticles”. J. Phys. Chem. Lett. 2015. 6(6): 970–974.
43.
ButlerH.J.AshtonL.BirdB., et al.“Using Raman Spectroscopy to Characterize Biological Materials”. Nat. Protoc. 2016. 11(4): 664–687.
44.
RygulaA.MajznerK.MarzecK.M., et al.“Raman Spectroscopy of Proteins: A Review”. J. Raman Spectrosc. 2013. 44(8): 1061–1076.
45.
ShebanovaO.N.LazorP. “Raman Study of Magnetite (Fe3O4): Laser-Induced Thermal Effects and Oxidation” J. Raman Spectrosc. 2003. 34(11): 845–852.
46.
StagiL.De ToroJ.A.ArduA., et al.“Surface Effects Under Visible Irradiation and Heat Treatment on the Phase Stability of γ-Fe2O3 Nanoparticles and γ-Fe2O3–SiO2 Core-Shell Nanostructures”. J. Phys. Chem. C. 2014. 118(5): 2857–2866.
47.
MendiliY.E.GrassetF.RandrianantoandroN., et al.“Improvement of Thermal Stability of Maghemite Nanoparticles Coated with Oleic Acid and Oleylamine Molecules: Investigations Under Laser Irradiation”. Phys. Chem. C. 2015. 119(19): 10662–10668.
48.
MatthäusC.BirdB.MiljkovićM., et al.“Infrared and Raman Microscopy in Cell Biology”. Methods Cell Biol. 2008. 89(0): 275–308.
49.
SolbrigR.M.DuffL.L.ShriverD.F., et al.“Raman and Infrared Spectroscopy of the Oxo-Bridged Iron(III) Complex, [Cl3Fe–O–FeCl3]-2 as a Spectroscopic Model for the Oxo Bridge in Hemerythrin and Ribonucleotide Reductase”. J. Inorg. Biochem. 1982. 17(1): 69–74.
50.
PlowmannJ.E.LoehrT.M.SchauerC.K., et al.“Crystal and Molecular Structure of the (μ-Oxo)bis[aquobis(phenanthroline)iron(III)] Complex, a Raman Spectroscopic Model for the Binuclear Iron Site in Hemerythrin and Ribonucleotide Reductase”. Inorg. Chem. 1984. 23(22): 3553–3559.
51.
WangJ.RousseauD.L.Abu-SoudH.M., et al.“Heme Coordination of NO in NO Synthase”. Proc. Natl. Acad. Sci. U.S.A. 1994. 91(22): 10512–10516.
52.
PapaefthymiouG.C. “The Mössbauer and Magnetic Properties of Ferritin Cores”. Biochim. Biophys. Acta. 2010. 1800(8): 886–897.
53.
DobsonJ. “Nanoscale Biogenic Iron Oxides and Neurodegenerative Disease”. FEBS Lett. 2001. 496(1): 1–5.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
