The selective extraction of iron-binding glycoprotein lactoferrin via a “deferrization-restoring” SPE strategy

Abstract

Here we report a “deferrization-restoring” solid-phase-extraction (SPE) strategy for the selective extraction of iron-binding glycoprotein lactoferrin (Lf) from complex matrices. Lf is first conducted with deferrization process to emerge apo-lactoferrin (apo-Lf) with Fe vacancy domains, then Tb3+ functionalized SiO2 microspheres (Tb@SiO2) are fabricated and adopted as adsorbent for the selective adsorption of apo-Lf based on the coordination between the Fe vacancy domains in apo-Lf and Tb3+ on the surface of Tb@SiO2 microspheres. The adsorption behavior of apo-Lf fits the Langmuir model, corresponding to a theoretical adsorption capacity of 262.5 mg g−1. The retained apo-Lf could be exactly recovered by using a 0.01 mol L−1 FeCl3 solution. The exploiting of FeCl3 as eluting reagent not only able to strip the adsorbed apo-Lf efficiently, i.e., a recovery of 88.4% is achieved, but also able to restore the protein structure to Lf, attributed to the stronger coordination ability of Fe3+ to apo-Lf than Tb3+. Circular dichroism assay indicates that nearly no difference is observed between the restored Lf via this “deferrization-restoring” SPE strategy and Lf standard. This strategy is practically applied to the selective extraction of Lf from milk whey, achieving high-purity as demonstrated by SDS-PAGE assays.