Acetonitrile-protein interactions: amino acid solubility and preferential solvation.

The solubility of amino acids and the preferential solvent interaction of hen-egg lysozyme in acetonitrile (AN)-water mixtures (<60 w/v% AN) were investigated by means of densimetric and refractometric methods at 25 degreesC. The free energy of transfer from water to aqueous AN was negative for most nonpolar side-chains of amino acids and positive for the peptide group, the extent being comparable to those for methanol and ethanol systems. Addition of AN to an aqueous solvent was thus suggested to weaken the hydrophobic interaction and to enhance the peptide-peptide hydrogen bond therein leading to the denaturation of proteins. A parallel examination by circular dichroism confirmed that the conformation of lysozyme (pH 3) remains native in aqueous AN up to 40% but changes to the helix-rich form at higher AN concentrations. At all solvent compositions up to 50% AN (pH 3), however, lysozyme was preferentially hydrated probably due to a local salting-out of the AN molecules from the charges on the protein surface, indicating the increase of the chemical potential of the protein. These results are discussed in relation to the role of AN as an eluting organic solvent in reverse-phase chromatography.