Detergents are extensively used to solubilise different kinds of biological membranes. Unfortunately, however, most detergents alter the properties of the enzymes thus solubilized. This effect may be reversible, i.e., the detergent can be removed with recovery of the original properties of the enzyme, or it may be irreversible. As has been shown with several enzymes from the mitochondrial inner membrane, the ratio detergent./ protein is critical in this respect (1,2). A general rule is that the ratio detergent/protein should be kept at the lowest level at which the protein remains soluble during the purification procedure.
When gel chromatography is used to separate detergent-solubilized membrane proteins, a relatively high detergent concentration is required in the column to keep the sample soluble at the beginning of the run. As the protein concentrations of the separated zones are much lower than that of the starting zone, a uniform detergent level in the column may result in an unfavorable detergent/protein ratio. In the purification of the mitochondrial oligomycin-sensitive ATPase with the aid of gel chromatography this difficulty has been overcome by applying the detergent as a gradient in the column.