Instrumental comparison of the determination of Cr³+ uptake by human transferrin

UV-VIS absorbance, inductively coupled plasma-optical emission spectroscopy (ICP-OES), and particle beam/hollow cathode-optical emission spectroscopy (PB/HC-OES) are presented as techniques for determining Cr³+ loading into transferrin (Tf), with and without Fe³+. The methods are compared based on loading percentages (i.e. 100% loading would be equal to 2 M(n+): 1 Tf) determined for Cr³+ loading into apo-transferrin. Spectral interferences and overlapping LMCT bands cause inaccurate chromium (qualitative) and iron (qualitative and quantitative) results for the UV-VIS absorbance method. The ICP-OES and PB/HC-OES methods are in good agreement providing evidence that the PB/HC-OES method is a valid technique for investigating metal-protein complexes. Maximum Cr³+ loading into apo-transferrin over a 24 h period was determined to be 26.8 3.5% by the ICP-OES method and 25.3 2.2% by the PB/HC-OES method. Loading percentages were increased to 49.7 1.9% (ICP-OES) and 55.7 3.2% (PB/HC-OES) when the metal-transferrin solution was allowed to incubate for up to 10 days. Under non-excess carbonate conditions the Cr³+ loading is elevated over a 24 h incubation time, but under physiological conditions the loading is inhibited. Equal loading of Fe³+ and Cr³+ into apo-transferrin was achieved when chromium was at a level more than 5 times in excess of iron. Inhibition of Cr³+ loading was only observed when an excess of Fe³+ was available to bind into apo-transferrin. The ability for Cr³+ to displace Fe3+ from holo-transferrin was observed as small amounts of Cr³+ were loaded into the once occupied metal binding site.