Structural Change Of Protein Adsorption On Chromatographic Media And Its Thermodynamic Analysis

Study on the microscopic mechanism and process during protein chromatography areof great importance for the design and optimization of chromatographic media andbioseparation processes. The mechanism has not yet been fully clarified during extensiveapplication of hydrophobic interaction chromatography (HIC) and ion-exchangechromatography (IEC) in bioseparation. Therefore, current work focuses on the mechanismanalysis of interaction between model protein and chromatographic media. The details inthis work are summarized as follows:1. Chromatography, hydrogen and deuterium (H/D) exchange NMR, confocalscanning were combined to obtain the protein adsorption information induced by pore size.The relationship between microscopic mechanism of protein chromatography onresidue-level and macroscopic retention behavior were discussed. The result showed thatadsorption of protein on polystyrene (PST) was not sensitive to large pores geometry andapparent sensitivity only existed in the mesoporous range. With pore size increasing, thebound lysozyme presented a tendency of signifcantly decreased retention. However, incontrast to diffusion retention factor presents a reverse upward tendency. The loss ofadsorbed lysozyme residue signal was significantly higher than natural state. Lysozyme in14nm and30nm pores showed marked signals intensity loss compared with its free state,suggesting that their structures were disrupted, unfolded seriously.Protein unfolded weakerin120nm, which refected that most structures and more compactness of protein moleculewere preserved against exchange than in small pores.2. The influences of protein’s flexibility on interaction between protein and cationexchange media (SP Sepharose FF) were explored by chromatography. It was found thatproteins with high adiabatic compressibility (higher fexibility) were more strongly retained.In addition, In situ acquirement of the accurate residue details of protein onchromatographic media was investigated by H/D exchange NMR. The binding site wasdetermined by electrostatic calculations using computer simulation methods in conjunctionwith hydrogen deuterium labeled protein and NMR. The result showed that adsorbedprotein molecule lost more amide proton signals than its natural state. Furthermore, thehigher fexibility of protein, the more residue signal intensity lost. Such unfolding behavior was induced by electrostatic interactions, which should be dominant in ion exchangechromatography. However, for several distinct fragments, the protection degrees varied, andthe adsorbed lysozyme lost more signal intensity and was less protected at disorderstructures (coil, bend, and turn), but comparatively was more protected against exchange atsecondary structure domains (α-helix, β-sheet). Although such unfolding properties wassimilar to HIC, it was weaker.3. Calorimetry of three different flexible proteins adsorption on the IEC adsorbent wasanalyzed by isothermal titration microcalorimetry (ITC), and then the Gibbs free energychange ΔG, enthalpy ΔH, entropy change ΔS could be calculated from thermodynamic datacollected in ITC experiments. The result showed that adsorbed proteins (lysozyme andRibonuclease A) on IEC adsorbent experienced a great conformational change and heatrelease during adsorption, which played an important role in adsorption. Moreover, theflexible protein (lysozyme) released more heat than the rigid protein (Ribonuclease A).Theadsorption mechanism for ion exchange chromatography is an exothermal enthalpy drivenprocess.