Solute and mobile phase contributions to retention in hydrophobic interaction chromatography of proteins

Abstract

Hydrophobic interaction chromatography utilizes high salt concentration mobile phases to induce an interaction between a weakly hydrophobic matrix and exposed hydrophobic amino acids of a native protein. Proteins with a hydrophilic exterior have shorter retention times on a hydrophobic interaction column than do proteins with more hydrophobic exteriors. To examine the effect of amino acid substitutions on protein retention, lysozyme isolated from related bird species was chromatographed on a hydrophobic interaction column at increasing ammonium sulfate concentrations. Chromatographic retention deviated only when amino acid substitutions occurred on the surface of lysozyme opposite the catalytic cleft. This area may constitute a contact surface area and extends from Residue 41 to 102 and from 75 to the α-helical region starting with Residue 89. Retention was analyzed by plotting log k′ versus the molal concentration of ammonium sulfate. The slope did not deviate significantly for each of the bird lysozymes, indicating a similar contact surface area. However, there was significant deviation in the intercept of each of the lysozyme lines, which probably reflects the strength of the hydrophobic interaction. The intercept increased as the lysozyme became more hydrophobic. Hydrophilic amino acid substitutions affected retention as much as hydrophobic ones. The ionization state of histidine residues within the contact area between lysozyme and the column surface also influenced retention. An uncharged histidine residue increased retention, while a decrease in retention was seen with a charged histidine residue. The amino acid substitutions did not appear to affect the size of the hydrophobic contact surface area, but rather the strength of the hydrophobic interaction. The effect of salt composition on protein retention indicated that factors other than surface tension could influence retention. These factors appear to include protein hydration and specific interactions between the protein and the salt ions. Of these, the latter may or may not result in an alteration in protein structure. The magnitude of the effect of salt composition was found to be dependent upon the protein.