Biological membranes, composed of complex assemblies of lipids and proteins, serve as physi-cal barriers in the cell and are sites for many cellular signaling events. The majority of mem-brane lipids contain two hydrophobic hydrocar-bon tails connected to a polar head group. This architecture allows lipids to form bilayer struc-tures in which the polar head groups are exposed outwards towards the aqueous environment and the hydrophobic tails are sandwiched between the hydrophilic head groups. Integral membrane proteins are held in the membrane by hydropho-bic interactions between the hydrocarbon chains of the lipids and the hydrophobic domains of the proteins.
In order to understand the function and structure of membrane proteins, it is necessary to carefully isolate these proteins in their native form in a highly purified state. It is estimated that about one third of all membrane-associated proteins are integral membrane proteins, but their solu-bilization and purification is more challenging because most of these proteins are present at very low concentrations. Although membrane protein solubilization can be accomplished by using amphiphilic detergents, preservation of their biological and functional activities can be a challenging process as many membrane proteins are susceptible to denaturation during the isola-tion process. Detergents solubilize membrane proteins by mimicking the lipid bilayer environ-ment. Micelles formed by the aggregation of detergent molecules are analogous to the bilayer of the biological membranes. Proteins can incorporate into these micelles by hydrophobic interactions. Hydrophobic regions of membrane protein, normally embedded in the membrane lipid bilayer, are surrounded by a layer of deter-gent molecules and the hydrophilic portions are exposed to the aqueous medium. This property allows hydrophobic membrane proteins to stay in solution.Detergents are amphipathic in nature and contain a polar group at one end and long hydrophobic carbon chain at the other end. The polar group forms hydrogen bonds with water molecules, while the hydrocarbon chains aggregate via hydrophobic interactions. At low concentrations, detergent molecules exist as monomers. When the detergent monomer concentration is increased above a critical concentration, detergent molecules self associate to form thermodynamically stable, non-cova-lent aggregates known as micelles. The critical micelle concentration (CMC) is an important parameter for selecting an appropriate detergent. At the CMC, detergents begin to accumulate in the membrane. The effective CMC of a detergent can also be affected by other components of the biological system, such as lipids, proteins, pH, ionic strength, and temperature of the medium. An important point to note here is that any addition of salts to ionic detergents, such as SDS, may reduce their CMC because salt would tend to reduce the repulsion between the charged head groups. Here micelles will form at a lower concentration.
At low concentrations, detergents merely bind to the membrane by partitioning into the lipid bilayer. As the concentration of detergent increases, the membrane bilayer is disrupted and is lysed, producing lipid-protein-detergent mixed micelles. Any further increase in deter-gent concentration will produce a heterogeneous complex of detergent, lipid-detergent, and pro-tein-detergent mixed micelles. In the detergent-protein mixed micelles, hydrophobic regions of the membrane proteins are surrounded by the hydrophobic chains of micelle-forming lipids. Excessive amounts of detergents are normally used for solubilization of membrane proteins to ensure complete dissolution and provide for a large number of micelles to give one micelle per protein molecule. For further physiochemical and biochemical characterization of membrane proteins, it is often necessary to remove the unbound detergent. Excess amounts of deter-gents can be removed by hydrophobic absorp-tion on a resin, gel chromatography (based on the difference in size between protein-detergent, lipid-detergent, and homogenous detergent micelles), ion-exchange chromatography (based on the charge difference between protein-deter-gent and protein-free detergent micelles), or by dialysis. Detergents with high CMC can be read-ily removed from protein-detergent complexes by dialysis, whereas low CMC detergents dialyze away very slowly.