12.12 : Purificación de proteínas de membrana con detergentes

Detergents are used to purify the integral proteins of the membrane. The hydrophobic portion of the detergent can replace membrane phospholipids while solubilizing the membrane proteins. When detergent monomers reach a specific concentration in a solution called critical micelle concentration (CMC), they form micelles. Above CMC, the concentration of the detergent monomers remains in equilibrium with the micelle. The number of detergent monomers present in the CMC varies for each detergent, and it depends upon the size and structure of the detergent monomer. The formation of CMC is also affected by temperature and pH. The structure and concentration of a detergent and its micelle structure determine its ability to solubilize the lipid bilayer.

For protein purification, the protein and the detergent concentration should be higher than the CMC. There should be a minimum of one micelle for each membrane protein. The detergent: lipid ratio also plays an essential role in protein purification, and it is determined using the trial and error method for each target protein. Some proteins can be extracted with a detergent-lipid ratio ranging from 0.1:1 to 1:1, but the lipid bilayer remains undisturbed. While in ratios ranging from 1:1 to 2:1, the detergent molecules solubilize the membrane, resulting in mixed micelles with lipid, protein, and detergent. If the ratio is increased to 10:1, all the lipid molecules in the lipid-protein complex will be replaced by the detergent molecule.

The choice of detergent depends on whether the purified protein should be functionally active or inactive. Detergents with a bigger polar head or longer alkyl chain are considered milder than the detergent with a smaller polar head or shorter alkyl chain. The membrane composition also affects the choice of detergents. For example, Triton X-100 acts on the bacterial inner membrane but cannot solubilize the outer membrane. In contrast, digitonin is known to work specifically on the eukaryotic plasma membrane.