On protein denaturation in aqueous-organic mixtures but not in pure organic solvents

FTIR spectroscopy was used to quantitatively assess the secondary structure of proteins in aqueous-organic mixtures ranging from pure water to a pure solvent. For every such solution/suspension, the alpha-helix content of the protein was independently calculated from the amide I and amide III spectral regions (which gave essentially identical results). In all cases studied (two unrelated enzymic proteins-lysozyme and subtilisin; three dissimilar water-miscible solvents-acetonitrile, tetrahydrofuran, and 1-propanol), the protein secondary structure was much more native-like in pure organic solvents than in most water-solvent mixtures, e.g., 60% (v/v) organic solvents. In fact, placing lyophilized (or crystalline) proteins in the anhydrous solvents tested had no appreciable effect on the alpha-helix content, whereas the latter declined markedly in the 60% (v/v) solvents. This behavior was found to be kinetically controlled, i.e., to be due to inherent restrictions on protein conformational mobility in anhydrous, in contrast to aqueous-organic, media.