Structural variations in hydrophobic side chains produce two different detergents in the Triton family, Triton X-100 and Triton X-114, that have markedly different physicochemical properties (see Detergents). Early studies showed that the polyoxyethylene glycol detergent Triton X-100 has a very low critical micelle concentration (cmc of 0.2 mM and is highly efficient in solubilizing membrane proteins without dissolving much membrane lipid (1). This strongly hydrophobic detergent and the structurally related Nonidet P-40 are widely used in lysing cells and solubilizing membrane proteins. Integral membrane proteins, whose functional activity does not critically depend on membrane lipids, are successfully solubilized by using Triton X-100. Examples include the receptors for GABA (2), prostacyclin (3), prolactin (4), transferrin (5), and insulin (6). Although Triton X-100 is highly efficient in solubilizing membrane proteins, it is relatively mild and nondenaturing toward soluble, globular proteins, such as the g-globulins (see Immunoglobulin). This is underscored by the large number of current reports, in which cell lysis in a buffer containing 1% Triton X-100 or the closely related detergent Nonidet P-40, is successfully followed by immunoprecipitation of specific proteins (7-9).
Triton X-114 is prominent in recent literature for specific reasons. This detergent has a strikingly greater solubility in water at lower temperatures (0°C), whereas at higher temperatures (30°C) it separates from water to form a separate detergent phase (10). Upon the formation of a discrete layer, the detergent retains specific, detergent-solubilized proteins, so the property of phase separation has been used in protein purification studies. When membrane proteins of adrenal medullary chromaffin granules are subjected to such fractionation in Triton X-114, cholesterol- and phospholipid-associated proteins like ATPase I and glycoprotein IV are obtained as detergent-insoluble proteins following solubilization at 0°C. Next, the detergent solution is warmed and layered over a cushion of 0.25 M sucrose in buffer containing 0.06% Triton X-114 and then centrifuged (see Density Gradient Centrifugation). The resulting aqueous phase (top layer, 0.04% Triton X-114) contains a mixture of soluble proteins, chromogranin A, soluble DBH, and membrane glycoproteins III, H, J, and K. Then the glycoproteins are isolated by removing the top layer, followed by exhaustive dialysis at 4°C in a buffer containing 1% Amberlite XAD-2 (11). Based on this phase separation method and the earlier observation that detergents with low cmc values (such as Triton X-100 and Triton X-114) are relatively inefficient in solubilizing the GPI-anchored proteins, Hooper and Bashir developed a technique of differential solubilization and temperature-induced phase separation in Triton X-114 to distinguish between the GPI-anchored proteins and those anchored by a simple membrane-spanning polypeptide (12, 13). When this method is applied to pig kidney microvillar membranes, abundant in both GPI-anchored and polypeptide-linked ectoenzymes, Triton X-114 at 0°C solubilizes only the ectoenzymes harboring a polypeptide anchor, whereas the GPI-linked ectoenzymes are sedimented by low-speed centrifugation. Then the detergent-solubilized supernatant is further fractionated by phase separation at 30°C into an aqueous phase and a detergent-rich phase, which contains the polypeptide-linked ectoenzymes.