Iron-induced conformational change in human lactoferrin: demonstration by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analysis of effects of iron binding to the N and C lobes of the molecule.
Analysis of Fe-saturated- and apo-lactoferrin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) without heating the samples prior to application revealed a substantial difference in mobility. The mobility shift was fully reversible on repetitive removal and readdition of Fe. Binding of a single Fe to the N-lobe binding site was sufficient to cause the gel shift; binding of a second Fe to the C-lobe site did not further alter mobility. Removal of Fe from the N lobe of Fe2 lactoferrin did not restore mobility to the position of apolactoferrin. No change in mobility with Fe binding was detected in N and C lobes isolated from intact lactoferrin by controlled trypsin digestion. The data indicate that a conformational change induced by Fe binding to a single site on lactoferrin is detectable by SDS-PAGE and that this change requires an intact molecule, possibly due to the need for interactions between the two homologous lobes of the molecule.
Department of Biology, New York University, NY 10003.
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Ying
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Furmanski
D001059:Apoproteins / Q000737:chemistry; D001665:Binding Sites; D002244:Carbon; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007501:Iron / Q000737:chemistry*; D007781:Lactoferrin / Q000737:chemistry*; D009584:Nitrogen; D011485:Protein Binding; D011487:Protein Conformation
D001059:Apoproteins; C039223:apolactoferrin; D002244:Carbon; D007501:Iron; D007781:Lactoferrin; D009584:Nitrogen