title: Monitoring guanidinium-induced structural changes in ribonuclease proteins using Raman spectroscopy and 2D correlation analysis.
abstract: Assessing the stability of proteins by comparing their unfolding profiles is a very important characterization and quality control step for any biopharmaceutical, and this is usually measured by fluorescence spectroscopy. In this paper we propose Raman spectroscopy as a rapid, noninvasive alternative analytical method and we shall show this has enhanced sensitivity and can therefore reveal very subtle protein conformational changes that are not observed with fluorescence measurements. Raman spectroscopy is a powerful nondestructive method that has a strong history of applications in protein characterization. In this work we describe how Raman microscopy can be used as a fast and reliable method of tracking protein unfolding in the presence of a chemical denaturant. We have compared Raman spectroscopic data to the equivalent samples analyzed using fluorescence spectroscopy in order to validate the Raman approach. Calculations from both Raman and fluorescence unfolding curves of [D]50 values and Gibbs free energy correlate well with each other and more importantly agree with the values found in the literature for these proteins. In addition, 2D correlation analysis has been performed on both Raman and fluorescence data sets in order to allow further comparisons of the unfolding behavior indicated by each method. As many biopharmaceuticals are glycosylated in order to be functional, we compare the unfolding profiles of a protein (RNase A) and a glycoprotein (RNase B) as measured by Raman spectroscopy and discuss the implications that glycosylation has on the stability of the protein.
authors: School of Chemistry, Manchester Institute of Biotechnology, University of Manchester, Manchester, UK.
authors: Victoria L
authors: VL
authors: Brewster
authors: Lorna
authors: L
authors: Ashton
authors: Royston
authors: R
authors: Goodacre
mesh_terms: D019791:Guanidine / Q000737:chemistry*; D008958:Models, Molecular; D011487:Protein Conformation; D055550:Protein Stability; D058767:Protein Unfolding; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*; D012260:Ribonucleases / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013059:Spectrum Analysis, Raman / Q000379:methods*; D013816:Thermodynamics
chemical_list: D012260:Ribonucleases; C029600:ribonuclease B; D012259:Ribonuclease, Pancreatic; D019791:Guanidine