Investigation of the Retention Behavior and Structural Change of Proteins in Reversed Phase and Hydrophobic Interaction Chromatography

Abstract

The retention behavior of proteins was investigated in reversed phase chromatography (RPC) and hydrophobic interaction chromatography (HIC). The observation was performed in three viewpoints : (1) What are the differences in the retention behavior between small molecules and proteins? (2) What are the differences between RPC and HIC of proteins and the reason of the differences? (3) How can be detected the structural changes of proteins eluted from the column?

The retention behavior of proteins was able to be understood by the parameters used in describing the retention behavior of small molecules. However, applicable range of the parameters for proteins was limited and, sometimes, peculier behavior appeard in RPC and HIC for proteins; that is, the retention time of proteins showed extreme difference for the small change of organic fraction and showed the skewed U-shaped dependence on fraction of organic solvent. Even though the retention mechanism of proteins in RPC and HIC was based on the same fundamental principle relied on the hydrophobic properties of the proteins and stationary phase, the retention behavior of the proteins in RPC differed significantly from that in HIC. The difference between RPC and HIC resulted from adding organic solvent in mobile phase of RPC to elute the protein strongly bound to the ligand of the stationary phase. From the view point of thermodynamics, the driving force of transferring proteins from the mobile phase to the stationary phase was ΔS° for RPC and ΔS° for HIC.

The investigation of structural change of the proteins eluted from the column was performed with UV and photodiode array detector as a function of the change of peak shape and the retention time. The number (Z) of solvent molecules required to displace the solute from the surface was useful for measuring the structural change of the proteins by combining the thermodynamic observation. For the identification of splitted peaks of hemeproteins eluted from RPC, apoproteins were prepared and compared with the retention time of heme proteins. K_D values were measured by using size-exclusion chromatography (SEC) with the same mobile phase used in RPC and HIC. From these values the phenomena such as aggregation or division into subunits of proteins were suggested.