Hydrophobic interaction chromatography in alkaline pH

Abstract

Hydrophobic interaction chromatography is a very powerful protein purification technique which is dependent on strong salting-out salts to increase the hydrophobic interactions between the protein and the ligand. Ammonium sulfate is the salt most commonly used for this purpose, but it cannot be used at very alkaline pH. Monosodium glutamate was therefore tested as a salt for hydrophobic interaction chromatography at pH 9.5. When ribonuclease A, ovalbumin, and β-lactoglobulin were individually applied to a phenyl superose column in 2 m monosodium glutamate, all three proteins bound to the column and could be subsequently eluted by decreasing the salt concentration. Using this salt, it was possible to separate commercially obtained β-lactoglobulin into authentic protein and contaminants and to purify the individual proteins from a mixture of ovalbumin and β-lactoglobulin. These results demonstrate that monosodium glutamate is a useful salt for hydrophobic interaction chromatography. Guanidine and sodium sulfate and sodium aspartate were also examined at the same pH, demonstrating that they also resulted in the binding and elution of the proteins examined.