Detergents are amphipathic molecules which contain both hydrophobic and polar regions permitting them to act as solubilizing agents. The hydrophobic region tends to aggregate into micelles and associate with hydrocarbon and nonpolar domains, while the polar region can form hydrogen bonds with water.
Detergents and Their Properties
The main properties of detergents include:
- The Hydrophile-Lipophile Balance (HLB, a measure of the hydrophilic nature of the detergent
- The aggregation number (the number of detergent molecules within a micelle, which equals micellar MW/monomeric MW)
- The critical micellar concentration (CMC, the concentration above which micelles form)
- The Kraft Point (the temperature at which an equilibrium exists between the monomeric detergent, the insoluble crystalline state, and the micellar state; usually, this temperature is equal to the critical micellar temperature, CMT)
For non-denaturing solubilization, a HLB between 12-20 is preferred or > 20 for extrinsic protein solubilization; For non-ionic detergents, the Cloud Point is the temperature above the CMT where detergents become cloudy and form a detergent-rich phase and an aqueous layer. This is helpful to separate integral membrane proteins from hydrophilic proteins after solubilizing at low temperature first (e.g. Triton X-114 has cloud point = 23 °C).
Classes of Detergents
Detergents can be grouped into three main classes:
- Zwitterionic detergents
- Ionic detergents
- Non-ionic detergents
Zwitterionic detergents protect the native state of proteins without changing the native charge of the protein molecules. Zwitterionic detergents are extremely useful for isoelectric focusing and 2D electrophoresis, in addition to their solubilization properties. A number of synthetic zwitterionic detergents are known as sulfobetaines. Sulfobetaines keep their zwitterionic characteristics over a wide scope of pH and are frequently utilized for 2D gel electrophoresis.
Non-detergent Sulfobetaines (NDSBs) are a family of zwitterionic compounds which have hydrophilic groups that are similar to the zwitterionic detergents but with much shorter hydrophobic chains that cannot aggregate to create micelles. NDSBs may enhance the yield of membrane, nuclear and cytoskeletal proteins when utilized with detergents, although they are not considered as detergents.
The short hydrophobic groups combined with the charge neutralization of the sulfobetaine group leads to higher yields of the membrane proteins. NDSBs also help decrease aggregation and aid in refolding proteins found in bacterial expression systems and inclusion bodies. The NDSBs are zwitterionic over a large pH range, removed easily by dialysis and do not significantly absorb in the near UV range.
Ionic detergents possess a charged headgroup (cationic or anionic) and are extremely useful for the complete disruption of cells and denaturation of proteins for separation during SDS-PAGE. These can include cationic detergents like CTAP, anionic detergents like SDS, and anionic bile salts like deoxycholate.
Often called non-denaturing detergents, non-ionic detergents are helpful for the solubilization of membrane proteins because they can break lipid-protein and lipid-lipid interactions, but have a limited ability to break protein-protein interactions.
They include low CMC detergents like Triton X-100 and a NP-40 alternate which cannot be dialyzed away, the very dialyzable (CMC ~ 25 mM) alkyl glucosides such as octyl glucopyranoside, and uncharged bile salts like Big Chap (which can be dialyzed).
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