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

• Published in: Journal of Chromatography A Vol. 849; no. 1; pp. 135 - 148
• Main Authors: McNay, Jennifer L, Fernandez, Erik J
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 16-07-1999; Elsevier
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Hydrolases; Lysozyme; Proteins; Proteins; Nuclear magnetic resonance spectrometry; Protein folding; Isotope exchange; Lysozyme; Stationary phases, LC; Circular dichroism spectrum; Enzyme; Chromatographic retention; HPLC chromatography; Unfolding; NMR spectrometry; Egg white; Secondary structure; Conformational changes; Protein; Reversed phase chromatography; Glycosidases; Hydrolases; Helical structure; O-Glycosidases; Bonded stationary phase
• ISSN: 0021-9673
• DOI: 10.1016/S0021-9673(99)00546-4

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 α-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.