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In general, biological detergents are most commonly used to disrupt the bipolar lipid membrane of cells in order to first free and then solubilize membrane-bound proteins. Some detergents can also be used to solubilize recombinant protein, while others find their usage in the stabilization, crystallization, or denaturation of proteins. Additional applications include the extraction of DNA and RNA, the solubilization of specimens for diagnostic applications, the lysis of cells, the preparation of liposomes, the prevention of reagent and analyte precipitation from solution, and the prevention of non-specific binding in immunoassays.
The value of detergents in these applications is derived from their amphiphlic nature. Each detergent molecule is characterized by a hydrophilic "head" region and a hydrophobic "tail" region. The result of this characteristic is the formation of thermodynamically stable micelles with hydrophobic cores in aqueous media. This hydrophobic core provides an environment that allows for the dissolution of hydrophobic molecules or domains of proteins.
The concentration at which micelles begin to form, which is the maximum monomer concentration, is the critical micelle concentration (CMC). The CMC constitutes a measure of the free energy of micelle formation. The lower the CMC, the more stable the micelle and the more slowly molecules are incorporated into or removed from the micelle. The average number of monomers in a micelle is the aggregation number (AN). The CMC and AN are highly dependent on factors such as temperature, pH, ionic strength, and detergent homogeneity and purity. At low temperatures detergents will form a cloudy crystalline suspension. As the temperature increases, the crystals dissolve to form monomers if the concentration is below the CMC or micelles if the concentration is above the CMC. The temperature at which micelles form from crystals is the critical micelle temperature (CMT). The triple point, the temperature at which crystals, monomers, and micelles are in equilibrium, is the Krafft Point.
When selecting a detergent, the first consideration is usually the ionic form of the hydrophilic group, which is either anionic, cationic, zwitterionic, or non-ionic. Anionic and cationic detergents typically modify protein structure to a greater extent than the other two classes. The degree of modification varies with the individual protein and the particular detergent. Ionic detergents are also more sensitive to pH, ionic strength, and the nature of the counterion, and can interfere with charge-based analytical methods. Alternatively, most non-ionic detergents are non-denaturing, but are less effective at disrupting protein aggregation. Zwitterionic detergents uniquely offer some intermediate class properties that are superior to the other three detergent types in some applications. Offering the low-denaturing and net-zero charge characteristics of non-ionic detergents, zwitterionics also efficiently disrupt protein aggregation.
Ease of removal or exchange is often a factor in the selection of a detergent. Some of the more common removal methods include dialysis, gel filtration chromatography, hydrophobic adsorption chromatography, and protein precipitation. The CMC value associated with the detergent is a useful guide to hydrophobic binding strengths – the higher the CMC, the weaker the binding and the easier the removal. Another useful parameter is the micelle molecular weight, which indicates relative micelle size. In most cases, the smaller the micelle, the easier the removal. If protein-detergent complexes are to be separated based on the molecular size of the protein, a small micelle size is usually preferred. The micelle molecular weight is simply the aggregation number multiplied by the monomer molecular weight.
The references below are a good source for in-depth coverage of the properties and applications of detergents.
- Helenius, A., et al., Properties of Detergents. Methods Enzymol, 56, 734-749 (1979).
- Neugebauer, J.M., Detergents: an overview. Methods Enzymol, 182, 239-253 (1990).
- Hjelmeland, L.M., Solubilization of native membrane proteins. Methods Enzymol, 182, 253-264 (1990).
- Marston, F.A.O., and Hartley, D.L., Solubilization of protein aggregates. Methods Enzymol, 182, 264-276 (1990).
- Hjelmeland, L.M., Removal of detergents from membrane proteins. Methods Enzymol, 182, 277-282 (1990).
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