Frost, Membrane Lecture

Commonly Used Detergents

Detergents are polar lipids that are soluble in water. The presence of both a hydrophobic and hydrophilic portion makes these compounds very useful for lysis of lipid membranes, solubilization of antigens, and washing of immune complexes. Types of Detergents A large number of detergents are available for scientific use. These are usually categorized according to the type of hydrophilic group they contain - anionic, cationic, amphoteric, or nonionic (see Table 1). In general, nonionic and amphoteric detergents are less denaturing for proteins than ionic detergents. Sodium cholate and deoxycholate are the least denaturing of the ionic detergents of commonly used detergents. Two properties of detergents are important in their consideration for biological studies: the critical micelle concentration (CMC) and the micelle molecular weight. The CMC is the concentration at which monomers of detergent molecules combine to form micelles. Each detergent micelle has a characteristic molecular weight. Detergents with a high micelle molecular weight, such as nonionic detergents, are difficult to remove from samples by dialysis. The CMC and the micelle molecular weight vary depending on the buffer, salt concentration, pH and temperature. In general, adding salt will lower the CMC and raise the micelle size. TABLE 1 PHYSICAL PROPERTIES OF COMMONLY USED DETERGENTS Detergent Anionic SDS Cholate Deoxycholate Cationic C16TAB Amphoteric LysoPC CHAPS Zwittergent 3-14 Nonionic Octylglucoside Digitonin C12E8 Lubrol Triton X-100 Nonidet P-40 Tween 80 Monomer, Da mw 288 430 432 365 495 615 364 292 1,229 542 582 650 650 1,310 Micelle, Da mw 18,000 4,300 4,200 62,000 92,000 6,150 30,000 8,000 70,000 65,000 64,000 90,000 90,000 76,000 CMC % (w/v) 0.23 0.60 0.21 0.04 0.0004 0.49 0.011 0.73 ----0.005 0.006 0.021 0.017 0.002 CMC Molarity 8.0 x 10-3 1.4 x 10-2 5.0 x 10-3 1 x 10-3 7 x 10-6 1.4 x 10-3 3.0 x 10-4 2.3 x 10-2 ---8.7 x 10-5 1.0 x 10-4 3.0 x 10-4 3.0 x 10-4 1.2 x 1015

TABLE 2 CHEMICAL PROPERTIES OF COMMONLY USED DETERGENTS Ionic Detergents SDS CHO DOC C16 + + Dialyzable + Ion exchangeable + Complexes ions Strong A280 Assay Interference + Cold Precipitates High Cost + Availability Toxicity + Ease of Purification + Radiolabeled Defined Composition Auto-oxidation + + + + + + + + + + + + + + + + + + + + + + + + + +/+ + + + + + + + +/+ + +/+ + + + + + + + + + + + +/+ +/+ +/+/+ + + + + + + + + Nonionic Detergents LYS CHA ZWI OGL DIG C12 LUB TNX NP40 T80 +/+ +/+/+ -

Property Strongly denaturing

Choice of Detergents Ionic detergents are very good solubilizing agents, but they tend to denature proteins by destroying native threedimensional structures. This denaturing ability is useful for SDS-PAGE but is detrimental where native structure is important for functional activity. It should be noted that antibodies usually retain their binding activity at 0.1% SDS or less. Nonionic and mildly ionic detergents are less denaturing and can often be used to solubilize membrane proteins while retaining protein-protein interactions. The following detergents have detrimental properties for some procedures: 1. Phenol containing detergents (ie., Triton X-100 and NP40) have a high absorbance at 280nm and thus interfere with protein monitoring at that OD. Lubrol may be substituted. Phenol-containing detergents also induce precipitation in the Folin (Lowry) protein assay. They can also be iodinated...and thus should not be used if iodinating proteins.

2. High micellar molecular weight interferes with gel filtration and not easily removed by dialysis. 3. Sodium cholate and sodium deoxycholate are insoluble below pH 7.5. Above an ionic strength of 0.1%SDS they will often crystalize. 4. Ionic detergents interfere with nondenaturing electrophoresis and isoelectric focusing.