Hydrophobic interaction chromatography selectivity changes among three stable proteins: conformation does not play a major role.

Interesting retention and selectivity changes have been noted for a number of proteins in hydrophobic interaction chromatography (HIC). In this study, we investigated the degree to which conformational changes may be responsible for selectivity changes of stable proteins. Hydrogen-deuterium isotope exchange detected by mass spectrometry was used to investigate changes in solvent accessibility during adsorption on HIC media. Lysozyme was determined to exhibit EX2 hydrogen exchange kinetics both in solution and adsorbed to Butyl Sepharose 4 Fast Flow and Phenyl Sepharose 6 Fast Flow high sub surfaces. A small, but significant, increase in solvent accessibility was observed upon adsorption. Similar approaches were used to analyze solvent accessibility of three stable proteins with melting temperatures above 50 degrees C exhibiting significant selectivity changes on Butyl Sepharose and Toyopearl Butyl 650M. While all three proteins (lysozyme, chymotrypsinogen A, and ovalbumin) exhibited enhanced exchange while adsorbed, no differences in solvent accessibility on the different adsorbents were observed. More detailed studies of lysozyme showed no significant changes in labeling prior or during elution. These results demonstrate that HIC surfaces examined here do not dramatically alter the structure of these stable proteins and that differences in conformation are not responsible for the selectivity changes observed. Thus, other factors such as different preferred binding orientations or variations between the media pore structure, size, and/or surface chemistry must be responsible.