Urea Gel Electrophoresis in Studies of Conformational Changes of Transferrin on Binding and Transport of Non-Ferric Metal Ions.

Aviva Levina,Boer Wang,Peter A Lay

doi:10.3390/gels8010019

Abstract

Transferrin (Tf) is a crucial transporter protein for Fe(III), but its biological role in binding other metal ions and their delivery into cells remain highly controversial. The first systematic exploration of the effect of non-Fe(III) metal ion binding on Tf conformation has been performed by urea-polyacrylamide gel electrophoresis (urea-PAGE), which is commonly used for nucleic acids but rarely for proteins. Closed Tf conformation, similar to that caused by Fe(III)-Tf binding, was formed for In(III), V(III) or Cr(III) binding to Tf. In all these cases, metal distribution between Tf lobes and/or the rate of metal release under acidic conditions differed from that of Fe(III)-Tf. By contrast, Ga(III) and V(IV) did not form closed Tf conformation under urea-PAGE conditions. Apart from Fe(III), only In(III) was able to increase the proportion of closed Tf conformation in whole serum. These results suggest that Tf is unlikely to act as a natural carrier of any metal ion, except Fe(III), into cells but can reduce toxicity of exogenous metal ions by binding them in serum and preventing their entry into cells.

Highlights

Transport of Non-Ferric Metal Ions.The transferrin (Tf) family of proteins, which evolved for the purpose of safe and efficient transport of Fe(III) into the cells of most animals, is widely studied as classical examples of high affinity metal-protein binding and cellular uptake via receptor-mediated endocytosis [1,2]
These results suggest that Tf is unlikely to act as a natural carrier of any metal ion, except Fe(III), into cells but can reduce toxicity of exogenous metal ions by binding them in serum and preventing their entry into cells
Urea-PAGE is a classical technique used for the separation of nucleic acids, but its use for separation of different conformations of the same protein is relatively unknown

Summary

Introduction

The transferrin (Tf) family of proteins, which evolved for the purpose of safe and efficient transport of Fe(III) into the cells of most animals, is widely studied as classical examples of high affinity metal-protein binding and cellular uptake via receptor-mediated endocytosis [1,2]. Binding of Fe(III) to both Tf lobes results in a closed. There are subtle but biologically important differences between the two Tf lobes in the thermodynamics and kinetics of Fe(III) binding and release [4]. The ability of Tf to bind many other ( trivalent) metal ions at the Fe(III) binding site [5] and the fact that human blood Tf is normally only ~30% Fe(III) saturated [6] resulted in the hypothesis that

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Conclusion