Chromatographic resolution of lysozyme variants.

There are seven avian lysozyme variants of nearly identical three-dimensional structure which have amino acid substitutions broadly distributed on their surface. By using these protein variants, it was possible to study the relationship between protein structure and chromatographic retention. It was determined that according to the mode of separation various regions of the proteins surface determine chromatographic retention. At one extreme, immunosorbents targeted a very small region on the protein surface. Hydrophobic interaction chromatography was an intermediate case in which one surface domain of the lysozymes controlled chromatographic behavior. At the opposite extreme, cation-exchange columns probed most of the protein surface. It was concluded that identification of random variations in protein structure will be most successfully detected by a separation mode that broadly targets the surface of a protein.