European Biophysics Journal

, Volume 47, Issue 6, pp 641–653 | Cite as

Ferritin from the haemolymph of adult ants: an extraction method for characterization and a ferromagnetic study

  • Eliane Wajnberg
  • Odivaldo C. Alves
  • Jonas Perales
  • Surza Lucia G. da Rocha
  • André Teixeira Ferreira
  • Luiz Cláudio Cameron
  • Darci M. S. Esquivel
  • Maria de Lourdes Barriviera
Original Article


Ferritin has been studied in many animals, plants and bacteria. The main functions of ferritin in mammals are iron concentration and stabilization, protection against oxidants and iron storage for later developmental or iron-dependent activities. Although insect ferritin plays a key role in iron transport, only a few studies to date have examined its properties and function. Ferritin isolation from the haemolymph of adult Camponotus sericeiventris ants involved heating at 75 °C, followed by protein fractionation with 3.2 M KBr gradients and ferritin sedimentation with KBr. Protein identification was performed using high-resolution proteomics techniques. SDS-PAGE revealed three subunits with molecular weights (MW) of 26, 28 and 31 kDa. Native PAGE indicated a MW higher than 669 kDa. Proteomic analysis strongly suggested the 26 and 31 kDa bands as F2LCH and F1HCH subunits of ferritin, respectively. Ferromagnetic resonance (FMR) at 100 K showed, at low field, a characteristic broad component of the ferritin iron core, suggesting that its distribution was shifted to values greater than 3000, a higher content than in mammals. The protein yield and MW were comparable to those reported in other studies of insects. To the best of our knowledge, this is the first report on ferritin extracted from adult ants to date. These results are discussed on the basis of the protein structure–function relation of secreted insect and mammal ferritins. This purification method will allow the use of magnetic techniques, which are relevant for understanding the role of ferritin in the biomineralization of magnetic nanoparticles in insects.


Ferritin Eusocial insect Ants Ferromagnetic resonance (FMR) Iron transport 



We are grateful to Dr. Jacques H. C. Delabie, do Lab. de Mirmecologia do CEPEC, for his assistance with the taxonomic classification of Camponotus sericeiventris. M. L. Barriviera was financially supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico. We thank the anonymous reviewers for their constructive comments, which helped us to improve the manuscript and Dr. E. Ellis for linguistic revision.

Supplementary material

249_2018_1293_MOESM1_ESM.pdf (128 kb)
Supplementary material 1 (PDF 127 kb)
249_2018_1293_MOESM2_ESM.pdf (61 kb)
Supplementary material 2 (PDF 61 kb)
249_2018_1293_MOESM3_ESM.pdf (51 kb)
Supplementary material 3 (PDF 50 kb)


  1. Alenkina IV, Oshtrakh MI, Klencsár Z, Kuzmann E, Chukin AV, Semionkin VA (2014) {57Fe} Mössbauer spectroscopy and electron paramagnetic resonance studies of human liver ferritin, Ferrum Lek and Maltofer®. Spectrochim Acta A Mol Biomol Spectrosc 130C:24–36. CrossRefGoogle Scholar
  2. Arosio P, Ingrassia R, Cavadini P (2009) Ferritins: a family of molecules for iron storage, antioxidation and more. Biochim Biophys Acta Gen Subj 1790(7):589–599. CrossRefGoogle Scholar
  3. Arosio P, Carmona F, Gozzelino R, Maccarinelli F, Poli M (2015) The importance of eukaryotic ferritins in iron handling and cytoprotection. Biochem J 472(1):1–15. CrossRefPubMedGoogle Scholar
  4. Atkinson BG, Dean RL, Tomlinson J, Blaker TW (1989) Rapid purification of ferritin from lysates of red blood cells using proteinase-K. Biochem Cell Biol 67(1):52–57.
  5. Bradley JM, Le Brun NE, Moore GR (2016) Ferritins: furnishing proteins with iron Topical Issue in Honor of R.J.P. Williams. J Biol Inorg Chem 21(1):13–28. CrossRefPubMedPubMedCentralGoogle Scholar
  6. Capurro MDL, Iughetti P, Ribolla PE, de Bianchi AG (1996) Musca domestica hemolymph ferritin. Arch Insect Biochem Physiol 32(2):197–207CrossRefGoogle Scholar
  7. Charlesworth A, Georgieva T, Gospodov I et al (1997) Isolation and properties of Drosophila melanogaster ferritin—molecular cloning of a cDNA that encodes one subunit, and localization of the gene on the third chromosome. Eur J Biochem 247(2):470–475.
  8. Chasteen ND, Harrison PM (1999) Mineralization in ferritin: an efficient means of iron storage. J Struct Biol 126(3):182–194. CrossRefPubMedGoogle Scholar
  9. Chung MCM (1985) A specific iron stain for iron-binding proteins in polyacrylamide gels: application to transferrin and lactoferrin. Anal Biochem 148(2):498–502. CrossRefPubMedGoogle Scholar
  10. Cohen LA, Gutierrez L, Weiss A et al (2010) Serum ferritin is derived primarily from macrophages through a nonclassical secretory pathway. Blood 116(9):1574–1584. CrossRefPubMedGoogle Scholar
  11. Corpet F (1988) Multiple sequence alignment with hierarchical clustering. Nucl Acids Res 16(22):10881–10890CrossRefPubMedGoogle Scholar
  12. Cowley JM, Dawn EJ, Janney DE, Gerkin RC, Buseck PR (2000) The structure of ferritin cores determined by electron nanodiffraction. J Struct Chem 131:210–216. CrossRefGoogle Scholar
  13. de Oliveira JF, Wajnberg E, Esquivel DMDS, Weinkauf S, Winklhofer M, Hanzlik M (2010) Ant antennae: are they sites for magnetoreception? J R Soc Interface 7(42):143–152. CrossRefPubMedGoogle Scholar
  14. Dunkov BC, Georgieva T (2006) Insect iron binding proteins: insights from the genomes. Insect Biochem Mol Biol 36(4 SPEC. ISS.):300–309. CrossRefPubMedGoogle Scholar
  15. Gasdaska JR, Law JH, Bender CJ, Aisen P (1996) Cockroach transferrin closely resembles vertebrate transferrins in its metal ion-binding properties: a spectroscopic study. J Inorg Biochem 64(4):247–258. CrossRefPubMedGoogle Scholar
  16. Gálvez N, Fernández B, Sánchez P, Cuesta R, Ceolín M, Clemente-León M, Trasobares S, López-Haro M, Calvino JJ, Stéphan O, Domínguez-Vera JM (2008) Comparative structural and chemical studies of ferritin cores with gradual removal of their iron contents. J Ann Chem Soc 130(25):8062–8068CrossRefGoogle Scholar
  17. Gould JL, Kirschvink JL, Deffeyes KS (1978) Bees have magnetic remanence. Science 201(12):1026–1028. CrossRefPubMedGoogle Scholar
  18. Gutiérrez L, Zubow K, Nield J, Gambis A, Mollereau B, Lazaro FJ, Mirssilis F (2013) Biophysical and genetic analysis of iron partitioning and ferritin function in Drosophila melanogaster. Metallomics 5:997–1005CrossRefPubMedGoogle Scholar
  19. Hajdusek O, Sojka D, Kopacek P et al (2009) Knockdown of proteins involved in iron metabolism limits tick reproduction and development. Proc Natl Acad Sci 106(4):1033–1038. CrossRefPubMedGoogle Scholar
  20. Hamburger AE, West AP Jr, Hamburger ZA, Hamburger P, Bjorkman PJ (2005) Crystal structure of a secreted insect ferritin reveals a symmetrical arrangement of heavy and light chains. J Mol Biol 349:558–569. CrossRefPubMedGoogle Scholar
  21. Hsu CY, Chan YP (2011) Identification and localization of proteins associated with biomineralization in the iron deposition vesicles of honeybees (Apis mellifera). PLoS One 6(4).
  22. Hsu CY, Li CW (1994) Magnetoreception in honeybees. Science 265:95–97. CrossRefPubMedGoogle Scholar
  23. Hsu C-Y, Ko F-Y, Li C-W, Fann K, Lue J-T (2007) Magnetoreception system in honeybees (Apis mellifera). PLoS ONE 2(4):e395. CrossRefPubMedPubMedCentralGoogle Scholar
  24. Huebers HA, Huebers E, Finch CA et al (1988) Iron binding proteins and their roles in the tobacco hornworm, Manduca sexta (L.). J Comp Physiol B 158(3):291–300.
  25. Ibrahim MM, Edwards G, Seehra MS, Ganguly B, Huffman GP (1994) Magnetism and spin dynamics of nanoscale FeOOH particles. J Appl Phys 75(10):5873–5875. CrossRefGoogle Scholar
  26. Ikeya M (1993) New application of ESR, dating, dosimetry and microscopy. In: Zimmerman MR, Whitehead N (eds) Silica and silicates. Geotherm and vulcanism, Cap. 10. World Scientific Ed., SingaporeGoogle Scholar
  27. Jung JH, Eom TW, Lee YP, Rhee JY, Choi EH (2011) Magnetic model for a horse-spleen ferritin with a three-phase core structure. J Magn Magn Mater 323(23):3077–3080. CrossRefGoogle Scholar
  28. Kim BS, Lee CS, Yun CY, Yeo SM, Park WM, Kim HR (2001a) Characterization and immunological analysis of ferritin from the hemolymph of Galleria mellonella. Comp Biochem Physiol A Mol Integr Physiol. 129(2–3):501–509. CrossRefPubMedGoogle Scholar
  29. Kim BS, Yun CY, Yeo SM, Lee HJ, Kim HR (2001b) Cloning and expression of a ferritin subunit for Galleria mellonella. Arch Insect Biochem Physiol 47(1):8–17. CrossRefPubMedGoogle Scholar
  30. Kim EY, Lee CS, Kim HR (2004) Purification and properties of ferritin from the last instar larval hemolymph of Protaetia brevitarsis (Coleoptera). Entomol Res 34(2):91–99CrossRefGoogle Scholar
  31. Kuterbach DA, Walcott B. (1986) Iron-containing cells in the honey-bee (Apis mellifera). I. Adult morphology and physiology. J Exp Biol 126(1):375–387.
  32. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685. CrossRefPubMedPubMedCentralGoogle Scholar
  33. Lucano MJ, Cernicchiaro G, Wajnberg E, Esquivel DMS (2006) Stingless bee antennae: a magnetic sensory organ? Biometals 19(3):295–300. CrossRefPubMedGoogle Scholar
  34. Mandilaras K, Pathmanathan T, Missirlis F (2013) Iron absorption in Drosophila melanogaster. Nutrients 5(5):1622–1647. CrossRefPubMedPubMedCentralGoogle Scholar
  35. Nichol H, Locke M (1989) The characterization of ferritin in an insect. Insect Biochem 19(6):587–602. CrossRefGoogle Scholar
  36. Nichol H, Law J, Winzerling J (2002) Iron metabolism in insects. Annu Rev Entomol 47(1):535–559. CrossRefPubMedGoogle Scholar
  37. Ong ST, Shan Ho JZ, Ho B, Ding JL (2006) Iron-withholding strategy in innate immunity. Immunobiology 211(4):295–314. CrossRefPubMedGoogle Scholar
  38. Pham DQD, Winzerling JJ (2010) Insect ferritins: typical or atypical? Biochim Biophys Acta Gen Subj 1800(8):824–833. CrossRefGoogle Scholar
  39. Pierre TGS, Tran KC, Webb J et al (1991) Organ-specific crystalline structures of ferritin cores in P-thalassemia/hemoglobin E. 4. Biol Met 4:162–165. CrossRefGoogle Scholar
  40. Searle BC (2010) Scaffold: a bioinformatic tool for validating MS/MS-based proteomic studies. Proteomic 10(6):1265–1269. CrossRefGoogle Scholar
  41. Seo D-H, Nam K-P, Han J et al (2004) Rapid and efficient purification of haemolymph ferritin from cricket, Gryllus bimaculatus. Entomol Res 34(1):59–64. CrossRefGoogle Scholar
  42. Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal Chem 68(5):850–858. CrossRefPubMedGoogle Scholar
  43. Sibille JC, Kondo H, Aisen P (1988) Interactions between isolated hepatocytes and Kupffer cells in iron metabolism: a possible role for ferritin as an iron carrier protein. Hepatology 8:296–301 [PubMed: 3356411] CrossRefPubMedGoogle Scholar
  44. Tang X, Zhou B (2013) Iron homeostasis in insects: insights from Drosophila studies. IUBMB Life 65(10):863–872. CrossRefPubMedGoogle Scholar
  45. UniProt Consortium (2017) UniProt: the universal protein knowledgebase. Nucleic Acids Res 45:D158–D169. CrossRefGoogle Scholar
  46. Wagstaff M, Worwood M, Jacobs A (1978) Properties of human tissue isoferritins. Biochem J 173(3):969–977.
  47. Wajnberg E, El-Jaick LJ, Linhares MP, Esquivel DM (2001) Ferromagnetic resonance of horse spleen ferritin: core blocking and surface ordering temperatures. J Magn Reson 153(1):69–74. CrossRefPubMedGoogle Scholar
  48. Wajnberg E, Acosta-Avalos D, Alves OC, de Oliveira JF, Srygley RB, Esquivel DMS (2010) Magnetoreception in eusocial insects: an update. J R Soc Interface 7(Suppl 2):S207–S225. CrossRefPubMedPubMedCentralGoogle Scholar
  49. Wang W, Knovich M, Coffman L (2010) Serum ferritin: past, present and future. Biochim Biophys Acta 1800(8):760–769. CrossRefPubMedPubMedCentralGoogle Scholar
  50. Weir MP, Peters TJ, Gibson JF (1985) Electron spin resonance studies of splenic ferritin and haemosiderin. Biochim Biophys Acta (BBA) Protein Struct Mol 828(3):298–305. CrossRefGoogle Scholar
  51. Werner WE (1995) Ferguson plot analysis of high molecular weight glutenin subunits by capillary electrophoresis. Cereal Chem 72(3):248–251Google Scholar
  52. Winzerling JJ, Nez P, Porath J, Law JH (1995) Rapid and efficient isolation of transferrin and ferritin from Manduca sexta. Insect Biochem Mol Biol 25(2):217–224. CrossRefPubMedGoogle Scholar

Copyright information

© European Biophysical Societies' Association 2018

Authors and Affiliations

  • Eliane Wajnberg
    • 1
  • Odivaldo C. Alves
    • 2
  • Jonas Perales
    • 4
  • Surza Lucia G. da Rocha
    • 4
  • André Teixeira Ferreira
    • 4
  • Luiz Cláudio Cameron
    • 3
  • Darci M. S. Esquivel
    • 1
  • Maria de Lourdes Barriviera
    • 1
  1. 1.Coordenação de Materiais, Física Aplicada e NanociênciasCentro Brasileiro de Pesquisas FísicasRio de JaneiroBrazil
  2. 2.Departamento de Físico-QuímicaUniversidade Federal FluminenseNiteroi, RJBrazil
  3. 3.Laboratório de Bioquímica de ProteínasUniversidade Federal do Estado do Rio de JaneiroRio de Janeiro, RJBrazil
  4. 4.Laboratório de Toxinologia, Instituto Oswaldo CruzFundação Oswaldo CruzRio de Janeiro, RJBrazil

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