Protein structure perturbations on chromatographic surfaces

• Published in: Journal of Chromatography A Vol. 849; no. 1; pp. 149 - 159
• Main Authors: Sane, Samir U, Cramer, Steven M, Przybycien, Todd M
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 16-07-1999; Elsevier
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods; Lactalbumin; Lysozyme; Proteins; Ribonuclease; Proteins; Protein folding; Lactalbumin; Stationary phases, LC; Raman spectrometry; Ribonuclease; Lysozyme; Chromatographic retention; Adsorption isotherm; HPLC chromatography; Esterases; Mobile phase; Conformational changes; pH; Rigid chain; Elution; Gradient; Phase composition; Enzyme; Unfolding; Secondary structure; Protein; Reversed phase chromatography; Flexible structure; Glycosidases; Hydrolases; O-Glycosidases; Cation exchanger; Bonded stationary phase
• ISSN: 0021-9673
• DOI: 10.1016/S0021-9673(99)00554-3

Amide I band Raman spectroscopy was used to quantify the secondary structure contents of proteins adsorbed on ion-exchange and reversed-phase materials. Neither ribonuclease A, a rigid protein, nor α-lactalbumin, a flexible protein, exhibited any significant secondary structural change on adsorption to an agarose-based cation-exchange support. On reversed-phase supports, however, lysozyme demonstrated a significant perturbation in secondary structure in the adsorbed state as compared to its structure in solution. For a constant concentration of adsorbed protein, the perturbed structure of adsorbed lysozyme was relatively insensitive to mobile phase conditions. However, the extent of structural change decreased as the concentration of adsorbed protein decreased. In agreement with the Raman spectroscopic characterization, reversed-phase linear gradient elution of lysozyme produced two peaks: a weakly binding peak corresponding to the native state and a strongly binding peak corresponding to the denatured state. The results presented in this paper demonstrate the utility of the Raman spectroscopic technique for in-situ characterization of protein secondary structures and their use in the molecular-level interpretation of protein retention behavior.