Application Of Terbium Functionalized Silica In The Separation And Purification Of Lactoferrin

Lactoferrin is a secreted protein with the function of natural defense,sterilization and anti-tumor.As an indispensable protein in the human body,it is significant of its separation and purification.Iron in lactoferrin could be removed by metal chelator,getting apo-lactoferrin(apo-Lf).Apo-Lf could be used as a drug due to its antimicrobial,anti-tumor and other important functions.According to the iron binding feature of lactoferrin,a solid-phase extraction procedure for the selective isolation of lactoferrin(Lf)from complex matrices was developed.Tb@SiO2 was prepared through silane coupling agent.Tb@SiO2 was characterized by FT-IR,XRD,TEM and EDS,and the results showed that the spherical material was synthesized successfully.Furthermore,the mass fraction of Tb was determined to be 4.5%by ICP-OES.Tb@SiO2 indeed exhibited favorable adsorption performance towards apo-Lf(iron free form of Lf)as designed mechanism,and a solid-phase extraction procedure was established for efficient and highly selective isolation of apo-Lf from the bovine milk.The adsorption behavior of apo-Lf fited 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,on the basis of Fe3+binding much stronger with Lf than Tb3+,providing a recovery of 88.4%.Lf in milk whey was translated into apo-Lf by removing its ferric ion,and the treated milk whey was used to separate and purify the apo-lf.Tb@SiO2 was further applied for the selective isolation of apo-Lf from the treated milk whey,achieving high-purity as demonstrated by SDS-PAGE assays.It virtually illustrates no change of circular dichroism spectra in the conformation of apo-Lf after the process of adsorption/desorption.The obtained apo-Lf could be further recovered its iron level under the condition of pH 7.0 and the presence of CO32-.It virtually illustrated no change of circular dichroism spectra in the conformation of the obtained lactoferrin from apo-Lf and the standard lactoferrin,and the iron level was determined to be the same of the two Lf,indicating that the apo-Lf could be converted to lactoferrin again.