Mechanism of protein hydrophobic chromatography. Protein unfolding and its contribution to effective hydrophobicity

Several different monomeric proteins with either one or two domains were used to study the effect of protein unfolding on effective hydrophobicity of proteins. Protein unfolding was inhibited by cross-linking with either toluene di-isocyanate or glutaraldehyde. The native enzyme was much more readily bound to the hydrophobic resin than the cross-linked species. The ligand, 3-phosphoglycerate, significantly decreased the amount of phosphoglycerate kinase able to bind to octyl-Sepharose. Sucrose also caused a statistically significant decrease in protein binding to the hydrophobic resin. These studies support the concept that the degree of protein unfolding influences effective hydrophobicity as measured by retention on octyl-Sepharose.