Abstract
Design and optimization of Immobilized Metal Affinity Chromatography (IMAC) processes require deep knowledge of driving factors responsible for interaction between immobilized metal and biomolecules. Based on this requirement, interactions between lactoferrin from cheese whey and IDA-Cu2+-cryogel system was investigated. Data from adsorption of lactoferrin in the system at pH 6, 7 and 8, as well as NaCl concentration from 200 to 1000 mmol L−1 were adjusted Langmuir, Freundlich, Temkin and Langmuir-Freundlich isotherm models. Although all models were able to explain the interaction lactoferrin-cryogel system, the Langmuir-Freundlich model was the most accurate one. In addition, it could explain quantitatively the cooperativity and heterogeneity of the bounds between protein and matrix. The methods used in this project are useful for both better understanding of the protein-immobilized metal interactions and developing preparative scale IMAC.
Highlights
Lactoferrin (LF) from cheese whey is a glycoprotein from the transferrin family, whose molecular mass is around 80 kDa
The TACN gel adsorption capacity was directly infuenced by pH and ionic strength, wherein at low values of pH (e.g. 5), the enzyme adsorption rised with NaCl concentration increase
At higher pH values (e.g. 9), adsorption behavior was reversed, wherein the maximum adsorption capacity was found in solutions without salt, which reinforces the complexity of phenomena behind Immobilized Metal Affinity Chromatography (IMAC) and that the differential affinity of metal ions to biomolecules in solution is dependent on the chelate ion and the composition of the mobile phase
Summary
Lactoferrin (LF) from cheese whey is a glycoprotein from the transferrin family, whose molecular mass is around 80 kDa. Given its wide application in medicine, biology, pharmaceutical and food industry, due to their biological properties, such as antiviral, antibacterial, antioxidant and immunomodulatory activities, several technologies of processing have been studied and developed for isolation of LF at high levels of purity. The majority of these processes are focused on chromatographic techniques, such as purification of lactoferrin by cation exchange [2], heparin-Sepharose affinity [3], ligand affinity [4] and heparinpoly(glycidyl methacrylate) affinity chromatography [5]. They have large pores, which allow solutions containing cell fragments and particulates to be drained throughout chromatographic columns without causing their obstruction [1]
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