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.
Ying
L
L
Department of Biology, New York University, NY 10003.
He
J
J
Furmanski
P
P
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