Anal. Chem. 2001, 73, 2565-2570
Dynamics of Iron Release from Transferrin N-Lobe Studied by Electrospray Ionization Mass Spectrometry Dmitry R. Gumerov and Igor A. Kaltashov*
Department of Chemistry and Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, Massachusetts 01003
Transferrins constitute a class of metalloproteins that are involved in circulatory iron transport in a variety of species. The metal ion-binding properties of these proteins have been the focus of extensive research efforts in the past decade due to their extreme importance in a variety of biological and healthcare-related fields. The large size of these proteins, as well as the presence of high-spin metal ions (e.g., Fe3+), limits the use of NMR. In this work, we report on the use of electrospray ionization mass spectrometry (ESI MS) to study dynamics of the transferrin system in vitro under conditions that are designed to mimic the endosomal environment. ESI MS is shown to provide valuable insights into the mechanistic aspects of metal ion-binding/release by transferrins and is complementary to other spectroscopic techniques. Conformational stability of the complex is evaluated based on the appearance of the charge-state distribution of protein ions, while the composition of the protein-ligand complex is determined based on the mass of the protein ions. In the absence of iron chelators, a stepwise dissociation of the ternary complex (protein-metal ionsynergistic anion) is observed as the solution pH is gradually decreased. Although the release of synergistic anion from the complex is initiated at typical endosomal pH levels (i.e., 5.5), metal ion remains largely bound to the protein until the pH is lowered to a level of ∼4.5. Under these conditions, a significant fraction of the protein populates unfolded conformations. In stark contrast to this behavior, addition of an iron chelating agent (citrate) to the protein solution results in facile iron release at typical endosomal pH levels without any detectable unfolding of the protein. The mass spectral data lends further credibility to the notion that the holoprotein samples conformations that are specific to the apo form (e.g., “open conformation”), from which iron dissociation most likely occurs. The results of the present study demonstrate that ESI MS can be used to model metal ion release from transferrin under conditions that are designed to mimic the physiological environment.
* To whom correspondence should be addressed: (Tel) 413 545-1460; (fax) 413 545-4490; (e-mail)
[email protected]. 10.1021/ac0015164 CCC: $20.00 Published on Web 04/14/2001
© 2001 American Chemical Society
Transferrins are glycosylated multidomain proteins that sequester and transport insoluble Fe3+ in extracellular fluids in a variety of species.1 The uptake and transport of iron under physiological conditions require special mechanisms, since insoluble ferric ion Fe3+ (Ksp
