How does a protein unfold on a reversed-phase liquid chromatography surface?

Nuclear magnetic resonance and isotope-exchange techniques were used to study unfolding of lysozyme adsorbed to reversed-phase liquid chromatography surfaces. All surfaces resulted in significant amide exchange, indicating solvent exposure and some loss of native structure. However, none of the surfaces resulted in complete exchange. The greatest amount of structure was preserved on the C-4 silica, with the most protection in the alpha-helix domain. C-18 silica and Source RPLC resulted in much greater solvent exposure. No simple correlation was found between chromatographic retention and degree of surface unfolding. Variations in residual conformation may explain the complex retention behavior of proteins vs. small molecules. (C) 1999 Elsevier Science B.V. All rights reserved.