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Electrospray ionization mass spectrometry as a tool to study transferrin interactions with metals of biological interest

Dmitry R Gumerov, University of Massachusetts Amherst

Abstract

Transferrins constitute a class of metalloproteins that sequester and transport iron in vertebrates. Understanding the metal-binding properties of transferrin is essential for studies of iron metabolism and metabolic disorders. However, dynamics of these systems is poorly understood, since they are not amendable to analysis by NMR due to the high spin of the metal ion and the large molecular weight of the protein (35–80 kDa). In this work we successfully developed and utilized an ESI MS based approach to study metal-release properties of the human serum transferrin N-lobe (hTF/2N), as well as the intact transferrin from human (hTF). Both composition and conformation of a protein complex was monitored under a variety of conditions that are designed to mimic extracellular and endosomal environments. It was determined that iron dissociation occurs in vitro at typical endosomal pH 5.5 when physiological chelators (e.g. citrate) are present at endogenous levels. In the absence of such chelating agents, detectable iron release occurs at much lower pH (<4.5) and is accompanied by unfolding of the protein. ESI MS data also indicated the transient nature of the anion in the holo-protein. Furthermore, studies of hTF revealed that the conformation of the iron-free N-lobe is stabilized when the C-lobe contains iron, confirming the existence of an interlobe interaction within the protein. Another surprising discovery was that the apo-N-lobe is significantly less flexible compared to the apo-C-lobe. These results provide strong support for the earlier suggestions that hTF interacts with its receptor (TfR) primarily through the C-lobe both at the cell surface and inside the endosome. We also successfully extended the above ESI MS approach to characterize interaction of intact transferrin (80kDa) with metals of medicinal relevance, such as In3+ and Bi3+. The results of our studies have revealed that indium-hTF complex exhibits the same pattern of conformational and metal release properties as iron-hTF. Because of conformational similarities between indium-hTF and iron-hTF, it is possible that indium-hTF is recognized by transferrin receptor protein (TfR) at the cell surface and is transported into the cells by the same mechanism of receptor mediated endocytosis as iron. On the other hand the results of our bismuth studies suggest that hTF adopts a semi-open conformation upon bismuth uptake. This conformation is different from that of iron-hTF, therefore it is likely that bismuth transport does not occur via the similar mechanism.

Subject Area

Analytical chemistry|Biochemistry

Recommended Citation

Gumerov, Dmitry R, "Electrospray ionization mass spectrometry as a tool to study transferrin interactions with metals of biological interest" (2003). Doctoral Dissertations Available from Proquest. AAI3110495.
https://scholarworks.umass.edu/dissertations/AAI3110495

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