Metal retention in human transferrin: Consequences of solvent composition in analytical sample preparation methods

Abstract

The analysis of metal-binding proteins requires careful sample manipulation to ensure that the metal–protein complex remains in its native state and the metal retention is preserved during sample preparation or analysis. Chemical analysis for the metal content in proteins typically involves some type of liquid chromatography/electrophoresis separation step coupled with an atomic (i.e., inductively coupled plasma-optical emission spectroscopy or -mass spectrometry) or molecular (i.e., electrospray ionization-mass spectrometry) analysis step that requires altered-solvent introduction techniques. UV-VIS absorbance is employed here to monitor the iron content in human holo-transferrin (Tf) under various solvent conditions, changing polarity, pH, ionic strength, and the ionic and hydrophobic environment of the protein. Iron loading percentages (i.e. 100% loading equates to 2 Fe³⁺ : 1 Tf) were quantitatively determined to evaluate the effect of solvent composition on the retention of Fe³⁺ in Tf. Maximum retention of Fe³⁺ was found in buffered (20 mM Tris) solutions (96 ± 1%). Exposure to organic solvents and deionized H₂O caused release of ∼23–36% of the Fe³⁺ from the binding pocket(s) at physiological pH (7.4). Salt concentrations similar to separation conditions used for ion exchange had little to no effect on Fe³⁺ retention in holo-Tf. Unsurprisingly, changes in ionic strength caused by additions of guanidine HCl (0–10 M) to holo-Tf resulted in unfolding of the protein and loss of Fe³⁺ from Tf; however, denaturing and metal loss was found not to be an instantaneous process for additions of 1–5 M guanidinium to Tf. In contrast, complete denaturing and loss of Fe³⁺ was instantaneous with ≥6 M additions of guanidinium, and denaturing and loss of iron from Tf occurred in parallel proportions. Changes to the hydrophobicity of Tf (via addition of 0–14 M urea) had less effect on denaturing and release of Fe³⁺ from the Tf binding pocket compared to changes in ionic strength.