Before the classical techniques of biochemical purification and analysis can be applied to membrane proteins, they must be converted into a water-soluble form. In a few limited and special cases, solubilization of membrane proteins may be achieved by detachment from the membrane using proteolytic cleavage, or relatively minor changes in ionic conditions. In almost all other cases, however, the solubilization of membrane proteins in intact and native form can only be achieved by the use of detergents. Successful isolation of membrane antigens requires an understanding of the forces that hold membranes together, and of the mechanism of action of detergents.
The cell membrane consists of an essentially fluid bilayer of lipids, arranged with their hydrophobic portions facing inwards and their polar head-groups interacting with the aqueous environment (Singer and Nicolson, 1972). In discussions of membranes, the lumen of intracellular organelles such as the endoplasmic reticulum and Golgi apparatus is considered to be topographically extracellular. Membrane lipids exhibit varying degrees of asymmetry in their disposition; most of the carbohydrate of glycolipids and glycoproteins lies on the extracellular face, while phosphatidyl ethanolamine lies mainly on the cytoplasmic face (Rothman and Lenard, 1977). Lateral diffusion of lipids is rapid, but 'flip-flop' movement across the membrane is less common. Recent evidence suggests that the asymmetry of membrane lipids is maintained by 'flippase' enzymes (reviewed by Higgins, 1994).
In marked contrast to lipids, the asymmetrical disposition of membrane proteins is absolute. This asymmetry is a consequence of the fact that most membrane proteins are inserted into the membrane during their synthesis, and once inserted, the energy required to move a polar region of protein across a nonpolar lipid bilayer is so great that the process rarely occurs.
Membrane proteins are held in or on the membrane by two distinct mechanisms. Some are attached by electrostatic or other noncovalent interactions, and may be released by relatively small changes in pH or ionic strength. This class is known as peripheral membrane proteins (Singer and Nicolson, 1972). Once released, peripheral membrane proteins are usually soluble in water in the absence of detergents. Most, but not all, peripheral membrane proteins do not span or penetrate the lipid bilayer. A well-known example of a peripheral membrane protein is 02-microglobulin, which is a subunit of the major histocompatibility antigens, and is noncovalently attached to the extracellular portion of their heavy chains. Isolated (^-microglobulin is soluble in water in the absence of detergents.
The other major class of membrane proteins has been termed integral because their removal and solubilization requires disruption of the membrane with detergents. Integral membrane proteins generally possess at least one uninterrupted stretch of around 20-25 uncharged amino acids. This region spans or penetrates the lipid bilayer, and its extreme hydrophobicity ensures that the protein remains firmly embedded in the membrane.
10 Analysis of Antigens Type I and Type II membrane proteins
Integral membrane proteins may be further subdivided. Many integral membrane proteins possess a single transmembrane sequence. These may be divided into type I membrane proteins, which have a cleavable jV-terminal signal sequence and a transmembrane sequence that is usually situated close to the C terminus. Type //membrane proteins have a noncleavable hydrophobic transmembrane region close to the N terminus, which serves as a combined signal/anchor sequence. Examples of type I membrane proteins include the histocompatibility antigens, glycophorin and membrane immunoglobulin. Examples of type II membrane proteins include the transferrin receptor, the asialoglycoprotein receptor, and many ecto-enzymes and glycosyl transferases.
Many integral membrane proteins span the membrane more than once, and often many times. Either terminus may be inside or outside the cell. Proteins with multiple transmembrane domains include a large family of G-protein-coupled receptors such as rhodopsin, the coloured visual pigments, and receptors for many small molecules, as well as many pumps and channels.
Clycophosphatidylinositol (CPI)-Iinked membrane proteins and glycolipids
A further class of membrane proteins is attached to the membrane via a glycosyl phosphatidyl inosityl (GPI) anchor (Ferguson and Williams, 1988). Examples include the T cell antigen Thy-1, erythrocyte cholinesterase and the trypanosomal variant coat antigen. GPI-linked proteins often require special conditions for solubilization. They may not be adequately solubilized by non-ionic detergents such as Triton X-100 (see below), but may be fully soluble in deoxycholate (Ferguson and Williams, 1988). Recent work has shown that GPI-linked proteins are associated with substantial quantities of glycolipids and cholesterol that are not soluble in nonionic detergents, and it has been speculated that these proteins may be localized to special glycolipid microdomains in the membrane (Brown, 1992; Fiedler, 1994; Kurzchalia et al., 1995; Casey, 1995). The major lipophosphoglycan of Leishmania is also anchored in the membrane via a GPI anchor (McConville et al, 1987).
10.7.2 Solubilization of Membrane Proteins by Detergents
Membrane solubilization by detergents has been reviewed in detail (Helenius and Simons, 1975; Helenius et al, 1979). The essential points are as follows. Detergents are amphiphilic molecules which exist in aqueous solution as monomers and micelles. The micelles consist of aggregates of 2-100
monomers, with their hydrophobic portions buried in the centre and their hydrophilic portions at the surface. At low detergent concentrations, most detergent molecules exist as monomers. With increasing detergent concentration, the concentration of monomers rises until at a poorly defined concentration called the critical micelle concentration, the monomer concentration ceases to rise, and further increase in detergent concentration results from an increase in micelle concentration. The micelle size is largely independent of detergent concentration, but is influenced to a variable extent by the type of detergent, the salt concentration and the pH.
Solubilization of integral membrane proteins occurs by replacement of the planar lipid bilayer with a micelle of detergent (Helenius and Simons, 1975). The detergent micelle binds to the hydrophobic transmembrane sequence, with the hydrophilic part of the detergent facing outwards. Nonionic and weakly ionic detergents interact much more weakly or not at all with the hydrophilic cytoplasmic or extracellular portions of membrane proteins and the majority of water-soluble proteins.
It is the monomer concentration which determines the solubilizing power. Thus, the concentration of detergent should always exceed the critical micelle concentration, so that the monomer concentration is as high as possible (Helenius et al., 1979). The use of lower concentrations is fraught with the risk of incomplete solubilization and a wide variety of resultant artefacts. Concentrations much higher than 10-20 times the critical micelle concentration are best avoided because there is no increase in solubilizing power, and the effects of impurities in the detergent may become significant (Ashani and Catravas, 1980). Highly purified grades of Triton X-100 suitable for membrane research are available from Calbiochem and Boehringer.
The other major requirement for solubilization concerns the total mass of detergent. Membrane lipid and detergent may be regarded as competing with each other for the hydrophobic regions of membrane proteins, so the total mass of detergent should be at least 10 times the total mass of cell lipid. This requirement is easily satisfied in most analytical experiments, in which the mass of membrane lipid may be considered negligible. However, the total mass of detergent should be carefully considered when large-scale purifications are planned.
Removal of detergent from membrane proteins usually results in uncontrolled aggregation and precipitation. It is therefore essential that buffers should contain an adequate concentration of detergent at all stages during their isolation and handling.
The detergents that have been found useful for membrane solubilization may be divided into three broad groups: nonionic, weakly ionic and strongly ionic.
As a general rule, the order given correlates with increasing solubilizing power, but also with increasing disruption of protein-protein interactions and denaturation.
A great deal of knowledge about use of detergents has been obtained empirically, and there is a need for individualization of choice of detergent for solubilization of a particular membrane protein. Although hundreds of different detergents might be considered, the majority of membrane proteins are adequately solubilized by Triton X-100 or the closely related Nonidet P-40. In some cases, other detergents will need to be tried. Detergents such as the zwitterionic sulfobetaines (Gonenne and Ernst, 1978) and CHAPS (Hjelmeland, 1980) have been claimed to be especially effective, although the question of whether increased solubilization power can be achieved without a concomitant increase in denaturing ability has not yet been resolved.
Some detergents such as digitonin and octyl glucoside are said to be particularly gentle in that they cause minimal disruption to protein-protein interactions, and allow the isolation of macromolecular complexes in a form that is thought to mirror the form that exists in the cell (Oettgen et al, 1986; Nakamura and Rodbell, 1990; Cambier et al, 1994; Kim et al, 1994; Terashima et al, 1994).
The most widely used detergent in membrane solubilization is Triton X-100. Nonidet P-40 is virtually identical in structure. Triton X-100 has a very low critical micelle concentration (c.3 x 10^ M or 0.02%). It is a very effective membrane solubilizer, usually has minimal effect on protein-protein interactions, and leaves the nucleus intact. Sometimes, protein-protein interactions are affected, and the use of digitonin or octyl glucoside may be necessary to isolate macromolecular complexes in an intact form (see above). Antigen-antibody interactions are usually unaffected.
A typical protocol for solubilization is as follows. To 1-5 x 107 cells, add 0.5-2.0 ml 0.5% Triton X-100 in PBS, and mix gently. Leave on ice for 15-60 min, then remove nuclei and debris by centrifugation. (In many cases, solubilization is virtually instantaneous but it is customary to leave the mixture on ice for 30-60 min, just in case it is not). For many applications, a low-speed centrifugation (say 3000 g for 10 min) is sufficient. For rigorous demonstration that solubilization has been achieved, it is necessary to centrifuge at 100 000 g for 30-60 min.
Removal of Triton X-100 by dialysis is extremely slow owing to its low critical micelle concentration and alternative detergent removal methods using hydrophobic beads (Holloway, 1973) may result in drastic loss of membrane protein. Octyl glucoside may often be substituted for Triton X-100 (Baron and Thompson, 1975). It has a very high critical micelle concentration (c.25 mM)
and is easily removed by dialysis, because the dialysable monomer concentration is high (Helenius et al, 1979).
Triton X-100 or Nonidet P-40 sometimes disrupt protein-protein interactions. This has been particularly well documented in the case of certain subunits of the T and B cell receptors for antigen (Oettgen et al., 1986; Cambier et al., 1994; Kim et al, 1994; Terashima et al., 1994). In these cases, associations between subunits that are not seen when the cells are solubilized in Triton X-100 may be apparent when the cells are solubilized in digitonin.
Digitonin is prepared as a 2% w/v stock solution by adding the solid detergent to boiling water, stirring for 2 min, allowed to stand at room temperature for a week and then filtered (Bridges, 1977; Oettgen et al., 1986). It is used at a typical final concentration of 1% (w/v).
A second class of detergents in common use are the bile salts. The most widely used is sodium deoxycholate, which is more effective than Triton X-100 in sol-ubilizing certain proteins, such as the Thy-1 antigen (Barclay et al., 1975). It is possible that the reason is related to 'domains' of unusual lipids surrounding GPI anchors (Ferguson and Williams, 1988; Casey, 1995).
Deoxycholate has a greater ability than Triton X-100 or digitonin to disrupt protein-protein interactions, and may sometimes denature proteins. It will lyse the nucleus, causing release of DNA. The pAj, or deoxycholate is 6.2, and it forms an insoluble gel at a pH of 7.2 or lower. Deoxycholate is also precipitated by divalent cations. Unlike Triton X-100, deoxycholate does not absorb light appreciably at 280 nm.
Antigen-antibody interactions are usually, but not always preserved. Herrmann and Mescher (1979) found that the monoclonal anti-H-2Kk antibody 11-4.1 failed to bind antigen in the presence of deoxycholate. It appears that deoxycholate causes a significant conformational change in the antigen (Herrmann et al, 1982).
SDS is an example of a strong ionic detergent. It is highly denaturing, and very effective at disrupting protein-protein interactions, especially when combined with heat. Provided they are not heated, the subunits of proteins such as the class II histocompatibility antigens remain attached to each other in the presence of SDS (Springer et al., 1977). Few proteins cannot be solubilized in sodium dodecyl sulfate; those that cannot include keratins and other proteins that have extensive covalent cross-linking between subunits.
Solubilization of cells in SDS or deoxycholate will result in lysis of the nucleus and release of DNA, which can make the sample very viscous and difficult to handle, particularly for loading onto SDS gels for Western blots; however, the viscosity can be reduced by shearing the DNA (Section 10.10.1).
Removal of detergent and concentration of dilute protein solutions for analysis by SDS Polyacrylamide gel electrophoresis
Removal of sodium dodecyl sulfate from proteins is difficult, but can be achieved by adding nine volumes of acetone or methanol, holding at -20°C for 1 h, and centrifuging at 13 000 g for 10 min. The SDS will remain in solution.
Alternatively, the protein may be precipitated with methanol/chloroform (Wessel and Flügge, 1984). An aliquot (0.4 ml) of methanol is added to 0.1 ml of the protein solution and the samples are vortexed and centrifuged (10 s at 9000 g). To the pellet is added 0.1 ml chloroform, and the samples are vortexed and centrifuged again. For samples containing a large amount of detergent or lipid, 0.2 ml chloroform may be used. Then, 0.3 ml water is added, and the sample vortexed and centrifuged for 1 min at 9000 g. The upper phase is discarded, and a further 0.3 ml methanol added to the lower phase and the interphase with the precipitated protein. After mixing again, the sample is centrifuged at 9000 g for 2 min to pellet the protein. The supernatant is discarded, and the pellet is dried by a stream of air. The method gives high recoveries, even when only 2-3 pg of protein is used (Wessel and Flügge, 1984).
Another procedure, involving extraction of the protein into phenol followed by ether and drying under reduced pressure, allows quantitative recovery of extremely dilute proteins (10 ng/ml) from solutions containing detergents or salts (Sauvé et al., 1995). Proteins can sometimes be renatured by transfer into 6 M guanidine-hydrochloride containing ImM dithiothreitol, followed by dilution into physiological buffer (Hager and Burgess, 1980).
Most antigen-antibody bonds are disrupted by SDS, although occasionally it is possible to carry out immunoprecipitation procedures if a large excess of Triton X-100 is included. Under these conditions, the SDS is incorporated into Triton X-100 micelles, lowering its effective concentration. Antigens denatured by SDS are capable of eliciting surprisingly strong antibody responses when injected into animals (Stumph et al., 1974; Tijan et al., 1975; Carroll et al., 1978; Lane and Robbins, 1978; Granger and Lazarides, 1979; see Section 15.1.4).
10.7.4 Enrichment of Membrane Proteins by Fractionation in Triton X-114
When heated, aqueous solutions of the Triton series of detergents become cloudy owing to the formation of large micellar aggregates. For Triton X-100, the 'cloud point' occurs at about 65°C. For Triton X-114, which is very similar in its solubilizing powers, the cloud point in 150 mM NaCl is at about 22°C. If cells are solubilized in Triton X-114 and the detergent is heated to 37°C, the detergent micelles may be centrifuged to the bottom of the tube as an oily droplet containing about 11% detergent, which will contain the great majority of the membrane proteins (Fig. 10.4). The hydrophilic water-soluble cytoplasmic proteins will stay in the detergent-depleted upper phase (Bordier, 1981,1988; Brusca and Radolf, 1994; Fig. 10.5). This method of fractionation of membrane proteins is simple, inexpensive and effective. It provides a typical enrichment factor for membrane proteins of about 20-fold, which presumably reflects their abundance as a fraction of total cellular protein (Brusca and Radolf, 1994). Although the method is very reliable, an occasional membrane protein behaves anomalously, for reasons that are not well understood (Maher and Singer, 1985; Brusca and Radolf, 1994). Fractionation in Triton X-114 is also highly effective in removal of endotoxin (LPS) from water-soluble proteins (Aida and Pabst, 1990).
Integral membrane proteins
Dilute to original volume with cold PBS
Dilute to original volume with cold PBS
Integral membrane proteins
Fig. 10.4. Phase separation in Triton X-114.
Fig. 10.5. Separation of membrane IgM and secretory IgM in Triton X-114.
Fig. 10.5. Separation of membrane IgM and secretory IgM in Triton X-114.
The preparation of stock solutions for Triton X-114 fractionation is given in Table 10.2.
(1) Solubilize cells (107) in 0.5 ml 0.5% Triton X-114 in PBS, on ice, for 30-60 min. As a rough guide, for 100 jxl packed cells, use at least 1-2 ml of 0.5% Triton X-114. It is vital that the total mass of detergent (i.e. the concentration x volume) is more than 10 times the mass of membrane lipid in the cells, or there will not be adequate solubilization. If the protein concentration in the lysate is too high, there may be nonspecific aggregation and contamination of the detergent phase by insoluble cytoskeletal proteins. The addition of a small amount of bromophenol blue to the cell lysate will colour the detergent phase blue, making it easier to see.
Because the procedure requires fairly lengthy incubations at 37°C, it is a good idea to add protease inhibitors to the lysis buffer. One possibility is to add a cocktail including 2 mM PMSF (freshly made from the powder), 1 mM iodoacetamide, 1 mM EDTA, and 5 ng/ml leupeptin.
(2) Spin out nuclei in Eppendorf centrifuge at 4°C for 10 min, or use the Sorvall centrifuge (SS34 rotor at 14000 rpm for 20 min). Add 10 jd of bromophenol blue (stock solution 1 mg/ml in water). The bromophenol
Table J0.2 Preparation of stock solutions for fractionation in Triton X-114
A. For sucrose cushion; 6% sucrose, 0.06%TX-114 in PBS
10 ml PBS 0.6 g sucrose
10 ml PBS
C. Pre-condensed 11.4% TX-114 in PBS See text
Triton X-114 may be purchased from FLUKA AG, CH-9470 Buchs, Switzerland (Catalogue No. 93421; 250 ml bottles). It is recommended that the Triton X-114 be pre-condensed to remove any small amounts of detergent with chain lengths different from that of the pure detergent (Bordier, 1981). Add 2 ml TX-114 to 100 ml PBS in a measuring cylinder. Add 1.6 mg butylated hydroxytoluene as an anti-oxidant. Mix well by inversion until homogeneous. Put cylinder in 37°C room overnight. Next day, two layers will be visible. The lower layer (10-20 ml) is enriched in detergent, and the upper layer is depleted in detergent. Remove and discard the upper layer by suction using water pump. Make lower layer up to 100 ml with PBS. Mix well, and repeat pre-condensation twice, as above. Save lower phase, which consists of 11.4% Triton X-114. The 11 % solution of Triton X-114 is very viscous, but will be easier to pipette using a Pipetman tip which has had the end cut off with scissors before use, to create a larger orifice. The addition of a trace of bromophenol blue to the stock TX-114 solution allows easy visibility during pre-condensation and use.
blue will partition into the detergent phase, making the oily droplet easier to see.
(3) In an ice bucket, have a tube ready containing solution A (sucrose cushion; volume equal to volume of lysate).
(4) Take cleared lysate from step 2, and carefully load over the sucrose cushion.
(5) Place tube in 37°C water bath 3 min. Contents will go cloudy, especially upper part.
(If the volume is larger than about 1 ml, longer times may be needed for the solution to reach the desired temperatures.)
(6) Spin in an Eppendorf centrifuge at room temperature for 2-3 min. (If you are quick, the solution will not cool in the short times used).
(7) Transfer supernatant above sucrose cushion into fresh tube on ice.
(8) Remove the sucrose cushion by gentle suction, taking care to leave the blue oily droplet at the bottom. (The detergent-rich oily drop in the bottom is very small, estimated at 50-100 nl).
(9) Resuspend the oily drop containing integral membrane proteins in ice-cold PBS, to volume of original lysate. The oily droplet is difficult to resuspend and may require vigorous mixing. It may be resuspended in a smaller volume, giving a higher detergent concentration, and concentrating the membrane proteins.
(10) (Optional). Repeat steps 4-7 and pool second detergent-depleted supernatant with first, on ice.
(11) (Optional). To the pooled depleted supernates, add 50 (j.1 of 11.4% TX-114. Mix, and place in 37°C bath for 3 min. Spin as above. Discard oily pellet. This step is to deplete the aqueous phase of any contaminating membrane proteins. The supernatant will contain the water-soluble proteins. Membrane proteins isolated by Triton X-114 fractionation contain large amounts of the detergent, which will prevent proper running of SDS-poly-acrylamide gels. The detergent can be removed by the procedures given in Section 10.7.3.
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