Study On The Electrostatic Interactions In Ion Exchange Chromatography With Molecular Simulation

The chromatography technique is one of the most effective methods for biomolecule purification, which can provide high separation resolution. The ion exchange chromatography (IEC) which based on the electrostatic interactions has been widely used for large-scale protein separation. Some separation models were set up, and can describe and analyze the individual process of protein separation with IEC. However the understanding of molecular mechanistic behind the separation behavior is quite limited, which certainly hinder the prediction of protein separation during IEC. In the present work the computer-aided method with molecule simulation was introduced to study the electrostatic interaction between protein and ion exchanger in IEC. The ligand surface of adsorbent was simulated under appropriate simplification, and the protein-ligand binding energy was calculated with some molecule simulation programs. A good relation between the simulation calculation and the retention behavior in IEC could be established.Two protein, lysozyme and chymotrypsinogen A, were chosen as model protein, and the cation exchangers of SP Sepharose FF, CM Sepharose FF, Toyopearl SP 650M, Toyopearl CM 650M were used as typical adsorbents. The structure information of protein was obtained from the public resource of Protein Data bank. Some programs, including MCCE, Delphi and Pymol, were used for the molecule simulation calculation. The MCCE program was used to calcualte the influences of pH on the protonation probability of amino acid on protein molecule. With the Delphi program the influence of salt to the electrostatic potential around protein could be calculated.Firstly, based on the analysis of pore radius and ligand distribution, the net of charged points were used to construct a simulated system of protein- adsorbent porous surface of ion exchangers. The electrostatic interaction energy between protein and simulated cation-exchange surface were calculated with Delphi program which based on the Poission-Boltzmarm equation. The influences of interaction orientation,separation distance, pH and salt concentration on the electrostatic interactions were analyzed. A good relation was found between the equilibrium constant from retention experiments and the interaction energy calculated from computer simulation. Secondly, in order to describe the differences of varying ligands, the atomic structure and charge distribution of ligand was considered. The electrostatic interaction energy between protein and simulated cation-exchange surface were calculated with the Delphi program using the linearized and non-linearized Poission-Boltzmann. The results indicated that the minimum binding energies were consistent with the trends of protein retention during IEC. However, some results were out of rules.The titration cures of ion exchanger were also studied at different salt concentration. With the equilibrium constant pk and the neutralization degree x, the charged situation of ion exchanger at different pH and salt concentration could be characterized. The results are useful for describing the change of the ligand charge with solution conditions, which certainly help the computer molecule simulation in a more rational way.