Selective separation of ferric and non-ferric forms of human transferrin by capillary micellar electrokinetic chromatography

• Methanol addition revealed the presence of monoferric forms and minimized iron release.
• Optimization was based on the experimental design and surface response methodology.
• We monitored gradual saturation of Tf performing off-line and on-line reactions.
• Electrophoretically-mediated mixing was more effective than diffusion-mediated one.
• Short-end injection strategy decreased total time of separation down to 4 min.

The previously published method allowing the separation of non-ferric (iron-free) and ferric (iron-saturated) forms of human serum transferrin via capillary electrophoresis has been further developed. Using a surface response methodology and a three-factorial Doehlert design we have established a new optimized running buffer composition: 50 mM Tris–HCl, pH 8.5, 22.5% (v/v) methanol, 17.5 mM SDS. As a result, two previously unobserved monoferric forms of protein have been separated and identified, moreover, the loss of ferric ions from transferrin during electrophoretic separation has been considerably reduced by methanol, and the method selectivity has been yet increased resulting in a total separation of proteins exerting only subtle or none difference in mass-to-charge ratio. The new method has allowed us to monitor the gradual iron saturation of transferrin by mixing the iron-free form of protein with the buffers with different concentrations of ferric ions. It revealed continuously changing contribution of monoferric forms, characterized by different affinities of two existing iron binding sites on N- and C-lobes of protein, respectively. Afterwards, the similar experiment has been conducted on-line, i.e. inside the capillary, comparing the effectiveness of two possible modes of the reactant zones mixing: diffusion mediated and electrophoretically mediated ones. Finally, the total time of separation has been decreased down to 4 min, taking the advantage from a short-end injection strategy and maintaining excellent selectivity.