Holo/apo conversion two-dimensional urea PAGE for speciation of Fe³⁺-bound transferrin in serum

This paper presents holo/apo conversion two-dimensional urea polyacrylamide gel electrophoresis (HAC-2D urea PAGE) as a novel method for speciating Fe³⁺-bound transferrin (Tf) species in biological samples, with a combination of metal ion contaminant sweeping (MICS) technique and Fe³⁺ detection PAGE. In the HAC-2D urea MICS-PAGE approach, HAC was performed to dissociate all the Fe³⁺ ions bound to Tf from the Fe-Tf species, during a two-step urea PAGE. Using this method, Fe₂-Tf, Fe_N-Tf, and Fe_C-Tf (holo-Tf, Fe³⁺-bound Tf attached to N-lobe, and Fe³⁺-bound Tf attached C-lobe, respectively) were completely isolated based on the difference in the higher-order structure of Tf, visible as horizontally aligned spots off the diagonal. The Fe³⁺ ions bound to Tf in each gel fraction were determined using PAGE with a fluorescent probe. Without the MICS technique, which electrophoretically removes all contaminant Fe³⁺ ions from the gel medium to ensure accurate determination of the Fe³⁺ concentration, it becomes challenging to precisely measure the distribution of metalloprotein species owing to the contaminants. Finally, the distribution of each Fe-bound Tf in a standard human serum sample was successfully determined by complete separation from large amounts of coexisting proteins, and the free Fe³⁺ concentration in the serum was estimated.