Infrared techniques for quantifying protein structural stability

Biopharmaceutical and biotechnology companies and regulatory agencies require novel methods to determine the structural stabilities of proteins and the integrity of protein–protein, protein–ligand, and protein–membrane interactions that can be applied to a variety of sample states and environments. Infrared spectroscopy is a favorable method for a number of reasons: it is adequately sensitive to minimal sample amounts and is not limited by the molecular weight of the sample; yields spectra that are simple to evaluate; does not require protein modifications, a special supporting matrix, or internal standard; and is applicable to soluble and membrane proteins. In this paper, we investigate the application of infrared spectroscopy to the quantification of protein structural stability by measuring the extent of amide hydrogen/deuterium exchange in buffers containing D 2O for proteins in solution and interacting with ligands and lipid membranes. We report the thermodynamic stability of several protein preparations, including chick egg-white lysozyme, trypsin bound by benzamidine inhibitors, and cytochrome c interacting with lipid membranes of varying net-negative surface charge density. The results demonstrate that infrared spectroscopy can be used to compare protein stability as determined by amide hydrogen/deuterium exchange for a variety of cases.