Protein structure perturbations on chromatographic surfaces

Amide I band Raman spectroscopy was used to quantify the secondary structur e contents of proteins adsorbed on ion-exchange and reversed-phase material s. Neither ribonuclease A, a rigid protein, nor a-lactalbumin, a flexible p rotein, exhibited any significant secondary structural change on adsorption to an agarose-based cation-exchange support. On reversed-phase supports, h owever, lysozyme demonstrated a significant perturbation in secondary struc ture in the adsorbed state as compared to its structure in solution. For a constant, concentration of adsorbed protein, the perturbed structure of ads orbed lysozyme was relatively insensitive to mobile phase conditions. Howev er, the extent of structural change decreased as the concentration of adsor bed protein decreased. In agreement with the Raman spectroscopic characteri zation, reversed-phase linear gradient elution of lysozyme produced two pea ks: 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 fo r in-situ characterization of protein secondary structures and their use in the molecular-level interpretation of protein retention behavior. (C) 1999 Elsevier Science B.V. All rights reserved.