Rapid and efficient isolation of transferrin and ferritin from Manduca sexta

Joy J. Winzerling, Patricia Nez, Jerker Porath, John H. Law

Research output: Contribution to journalArticle

33 Scopus citations

Abstract

We report methods for the rapid purification of two iron-binding proteins from larval hemolymph of Manduca sexta. Ferritin was purified in two steps by density gradient ultracentrifugation. To accomplish this, we utilized the relatively high level of ferritin present in the hemolymph of this animal and augmented the density of the protein in vivo by injection of iron sulfate. Nitrocellulose blots analyzed by laser densitometry showed hemolymph from iron-injected insects contained about 0.4 mg of ferritin per ml (approximately 0.7% of total hemolymph protein); of this, 62% was found as pure ferritin in the pellet formed during ultracentrifugation. Following the density ultracentrifugation, we purified transferrin from the hemolymph subphase by immobilized metal ion affinity chromatography using a new gel, Novarose-SE1000/40 coupled to dipicolylamine (DPA) chelated with nickel. Higher capacity Ni2+DPA-gel permitted good resolution of transferrin in the first chromatography; a lower capacity of the same gel allowed purification of transferrin in a second step. Overall transferrin recovery was 52%. Larval hemolymph contained 0.770 mg transferrin/ml, representing about 1.3% of the total protein.

Original languageEnglish (US)
Pages (from-to)217-224
Number of pages8
JournalInsect Biochemistry and Molecular Biology
Volume25
Issue number2
DOIs
StatePublished - Feb 1995

Keywords

  • Ferritin
  • Immobilized metal ion affinity chromatography
  • Iron-binding proteins
  • Transferrin

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Insect Science

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