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Detergent Properties
Detergent Properties Darrell R. McCaslin University of Wisconsin, Madison, Wisconsin, USA
A detergent is formed when a hydrophilic group with an affinity for water and a hydrophobic group with an aversion to water are spatially segregated within a molecule’s chemical skeleton so as to create a polarity. In aqueous solution, detergents generally exist as a mixture of monomers in equilibrium with a fairly monodisperse population of detergent aggregates known as micelles. In the micelle, the hydrophobic groups are packed together to create a hydrophobic core with the attached hydrophilic groups projecting out from the surface of this core and protecting it from contact with water. As such, detergents have only limited biological functions such as the well-known role in digestion and possibly to some extent in membrane fusion events; however, they play necessary if not essential roles in the isolation, manipulation, and characterization of the constituents of biological membranes.
Chemical Structure of Detergents The chemical structures of detergents which have been employed in biological studies are quite varied and continues to grow as investigators attempt to develop new entities that will permit the facile isolation of membrane constituents while enhancing their stability once isolated. To reach this goal, various combinations of hydrophobic and hydrophilic groups have been utilized and yield a variety of physical and chemical properties.
Ring-Based Hydrocarbons The naturally occurring bile salts synthesized from cholesterol and utilized in digestive processes are the prototypes for detergents based on ring systems. Figure 1 shows cholic acid, one of the abundant bile salts which is converted to deoxycholate (DOC) by removing the hydroxyl at the arrow. Identifying the spatial segregation of the hydrophilic and hydrophobic groups in these detergents may require careful inspection of their three-dimensional chemical structure. In cholic acid, the carboxyl group is an obvious hydrophilic group, but the effects of the hydroxyl groups located on the rings must also be considered. Viewing the steroid ring nucleus in the molecule as defining a plane, the spatial arrangement of the hydroxyls creates a hydrophilic face on one side and a hydrophobic face on the other side of this plane. The structure of DOC suggests the presence of only a hydrophilic edge along the ring plane, making the whole structure more hydrophobic than cholic acid, which may explain why DOC is generally a more aggressive solubilizer than cholic acid.
HYDROPHILIC GROUPS (HEADGROUPS ) Ionic
The hydrocarbon chain is the most easily recognized hydrophobic group and when present is often referred to as the molecule’s hydrophobic tail (Figure 1). These chains are most often saturated hydrocarbons (Figure 1) and are available in many lengths. The shorter the chain the less well defined the detergent properties tend to be, whereas longer tails become essentially insoluble. Unsaturation and more complex branching structures have been explored as well as chains incorporating phenyl rings. The presence of the ultraviolet lightabsorbing phenyl should generally be avoided as it can complicate various spectroscopic techniques.
Positively (e.g., amino) and negatively (e.g., carboxyl) charged groups have been utilized as hydrophilic groups. Single-tailed ionic detergents tend to denature all proteins, with the anionic ones being more aggressive at denaturation than cationic molecules. One of the most familiar examples is the anionic detergent, sodium dodecyl sulfate, NaDodSO4 (Figure 1). It is the denaturing effect of NaDodSO4 on proteins that led to the development of one of the most widely exploited analytical tools of biochemistry, denaturing gel electrophoresis, which permits one to easily assess the number of components present in a sample as well as their approximate molecular weights. Interestingly, it is the monomeric form of the ionic detergent that drives the denaturation process. The bulk of the lipids which form the basic membrane framework are amphiphiles with two hydrophobic
Encyclopedia of Biological Chemistry, Volume 1. q 2004, Elsevier Inc. All Rights Reserved.
577
HYDROPHOBIC GROUPS (TAILS ) Simple Hydrocarbon Chains
578
DETERGENT PROPERTIES
Commercial applications of detergents have driven the development of a large class of nonionic detergents with polyoxyethylene (POE) headgroups. A chemically homogeneous example is C12E8 (Figure 1), where a 12-C tail (C12) is attached to a polymer with eight repeats of –CH2CH2O – (E8) and terminates with a hydroxyl group. The ether groups of this chain provide sites for hydrogen bonding with water offsetting the hydrophobic nature of the intervening ethylene groups. Most commercially available POE-based detergents have headgroups consisting of a single chain although the length is often heterogeneous; however, highly branched structures have been created by attachment of several POE chains to a central moiety such as sorbitol (e.g., Tweens). The POE chains are subject to peroxidation and breakdown and solutions should only be used when relatively freshly prepared; moreover, when using commercial sources one should be aware that antioxidants are sometimes included.
CONNECTING HEAD
FIGURE 1 Selected chemical structures of detergents. Removal of the hydroxyl near the arrow on cholic acid coverts the molecule to deoxycholic acid.
tails esterified to zwitterionic headgroups (contains both positive and negative charges). While the lipids themselves generally form vesicular structures when dispersed in water, removal of one of the tails creates a detergent such as the lysophosphatidylcholine in Figure 1. Such lysolipids and other zwitterionic detergents (e.g., sulfobetaines) have been used as well as highly dipolar groups (e.g., amine oxide) that lack a formal charge.
Nonionic Any uncharged group of atoms capable of accepting or donating hydrogen bonds to water can in principle become a nonionic headgroup. The headgroup must be big enough so that the detergent dissolves in solution (as a mixture of monomer and micelles) rather than forming a separate phase. Thus, simple alkyl alcohols tend to phase separate for all but the shortest hydrophobic tails and have no useful detergent properties. Carbohydrate groups are useful headgroups and one of the most frequently encountered is b-octyl glucoside (OG) in Figure 1. Octyl glucoside in the a-anomeric configuration is essentially insoluble, demonstrating how a simple change in chemical structure can dramatically alter detergent properties. More elaborate carbohydrate structures have also been explored.
AND
TAIL
Linkages between the hydrophobic and hydrophilic groups are most often either an ether or ester linkage. The latter can be susceptible to hydrolysis, especially in biological preparations, which in turn could generate an ionic group with denaturing properties.
Detergent Properties CRITICAL MICELLE CONCENTRATION (CMC) The most common concentration-dependent behavior for detergents is illustrated in Figure 2. The monomer concentration increases until the critical micelle concentration (CMC) above which an equilibrium is established between the monomer and an increasing concentration of micelles. The monomer concentration increases very little after reaching the CMC and thus is in effect the highest possible concentration of monomeric detergent. All detergent in excess of the CMC is incorporated into micelles (dashed line in Figure 2). The concentration of micelles increases linearly with total detergent concentration above the CMC, but at a slower rate since a large number of molecules are incorporated into each micelle. Some detergents exhibit more complex behavior, such as secondary association of micelles or even new phases as the total concentration continues to increase. As illustrated in Figure 2, the CMC is actually a narrow range of concentration over which the formation of micelles becomes dominant. Nonetheless, a single number is usually defined as the CMC and determined by plotting some physical observable whose response differs markedly above and below the CMC against the total
DETERGENT PROPERTIES
579
AGGREGATION NUMBER
FIGURE 2 Concentration-dependent behavior of a detergent. The calculations are based on the theory of micelle formation developed by Tanford. An aggregation number of 50 was used and the equilibrium constant for micellization was chosen to yield the critical micelle concentration (CMC) indicated.
concentration of detergent. The CMC is then taken as the intersection of lines extrapolated from the nearly linear regions above and below the transition as shown by the dotted lines in Figure 2. Using different observables or concentration ranges for the extrapolation can lead to somewhat different values for the CMC. One of the simplest methods to measure a CMC concentration ranges or for the extrapolation. is by solubilization of heme, which is essentially insoluble below the CMC and increases as the concentration of micelles increases and would be similar to the dashed line in Figure 2. CMCs range from nanomolar to several millimolar. For single-tailed detergents, the CMC decreases with increasing length of tail and with smaller headgroups. The CMC of nonionics is generally lower than that of ionics with the same length of tail. Ionic headgroups are more susceptible to solution variables than the nonionics. Ionic headgroupsthatcan betitratedaresensitive topHandmay precipitate when the headgroup is in neutral form (both cholic acid and DOC are examples). The CMCs of ionics are also influenced by the type and concentration of counterions present, e.g., using potassium instead of sodium for dodecyl sulfate, results in an insoluble salt. In general, higher salt concentrations will decrease the CMC of an ionic detergent which is a consequence of reducing the headgroup repulsion in the micelle. Even the POEbased nonioncs may be influenced by presence of salts as the ether groups are capable of complexing ions. The solubilization of other components in the micelle (e.g., mixed micelles with lipids) will generally decrease the CMC (i.e., the maximum monomer detergent concentration is decreased). Published values of CMCs are useful guides in experimental design, but should generally be confirmed under the actual experimental conditions.
Micelles are formed by self-association of many detergent molecules into a single noncovalent structure. Since the number of molecules in each micelle is somewhat variable, micelle size is characterized by the aggregation number, which is the average number of molecules per micelle and can be determined by standard hydrodynamic methods (e.g., sedimentation equilibrium). The aggregation number is needed to calculate the micelle concentration, which is the total detergent concentration in excess of the CMC divided by the aggregation number. The CMC transition becomes sharper as the aggregation number increases. The CMC is generally strongly dependent on the size of the hydrophobic moiety, but repulsion between headgroups is the dominant factor for the aggregation number. Thus, nonionics tend to have larger aggregation numbers than ionics of the same tail length. The aggregation number for ionic detergents can be strongly influenced by both the type and concentration of counterions present as well as pH since these factors can dramatically change the electrostatic repulsion between the charged headgroups. Longer hydrophobic tails tend to have larger aggregation numbers. Detergents with high CMCs tend to have more variability in their micelle size distribution. At high concentrations and under certain solution conditions (e.g., elevated temperature) larger structures may be formed which in some cases are new micellar phases and in others simply secondary aggregation of smaller micelles.
TEMPERATURE EFFECTS As a thermodynamic equilibrium, the formation of micelles can be influenced by temperature. At low enough temperatures, solid detergent will exist in equilibrium with monomeric detergent; as the temperature increases, the monomer concentration increases until it reaches the CMC at the critical micelle temperature. Above this temperature, solid detergent will begin to go into solution as micelles. The temperature at which solid, micelles, and monomer at the CMC coexist is called the Kraft point and for most detergents is the same as the critical micelle temperature. An often observed Kraft point, which is near room temperature, is that of sodium dodecyl sulfate and upon cooling one sees precipitation of detergent. A second temperature effect observed at higher temperature, especially with nonionics containing POE, is the cloud point. At this temperature and above the CMC, the solution will turn turbid due to the formation of much larger aggregates. Both the critical micelle temperature and cloud point have been exploited in purification of membrane components.
580
DETERGENT PROPERTIES
Micelle Structure SINGLE- TAILED DETERGENTS In a micelle, the hydrophobic tails are sequestered into a central core structure with the hydrophilic groups projecting out from the surface of this core into the aqueous surrounding. The tails in the core are not fully extended but are quite flexible, some even lying along the surface of the core rather than within it. The surface of the core should be regarded as having a rippled texture with some tail methylene groups protruding above it. Experimental and theoretical arguments suggest that for most detergent micelles the overall shape is best described as an oblate ellipsoid, although for small aggregation numbers the shape cannot be readily distinguished from spherical. For C12E8 micelles (Figure 1), theoretical calculations constrained by hydrodynamic measurements yield an oblate ellipsoid with dimensions shown in Figure 3. One dimension must be less than the 3.4 nm length of two fully extended 12C chains since the core cannot contain a void. For the same length of tail, the overall dimensions of the micelle will depend on the nature of the headgroup. For POE-based detergents, the headgroup is in a random-coil configuration and consequently occupies a very large region of the space surrounding the core, as illustrated in Figure 3. Figure 3, while a convenient visualization of micelle structure, cannot convey the highly dynamic processes occurring. Detergent molecules are rapidly exchanged among micelles; whole micelles disappear and reform with slightly different aggregation numbers. The hydrophobic core acts for the most part like a simple liquid hydrocarbon with the tails constantly flexing. POE headgroups occupy a great deal of space around the core and are constantly changing their conformation as well. Finally, it should be obvious that the type and size of headgroup can generate dramatically different chemical environments to which molecules
solubilized by detergents are exposed; moreover, the solution composition near a micelle may be quite different from that of the bulk aqueous solution.
RING- BASED DETERGENTS Bile salts usually have relatively high CMCs and quite small aggregation numbers (4– 10). The small aggregation number does not permit micelles as just described. The micelles are more heterogeneous and are probably best viewed as small assemblies with the hydrophobic surfaces facing each other, presenting their hydrophilic faces to the surrounding solution. The size and shape of these micelles are quite sensitive to the concentration and types of ions, and potentially pH.
Detergents as Tools in Membrane Studies MEMBRANE SOLUBILIZATION Initially when a membrane is exposed to a detergent, monomers partition into the bilayer, so one can imagine that a high CMC might be advantageous (e.g., OG has a very high CMC (. 15 mM) and is an efficient solubilizer). As more detergent intercalates into the membrane’s bilayer, one reaches a stage where fragmentation occurs, resulting in large mixed micelles containing detergent, lipids, and proteins in various ratios. Continuing to add detergent will eventually disperse the membrane components to the point that any given micelle contains no more than a few lipids, a protein, or the detergent alone. One can apply isolation and enrichment techniques for specific targets to this solubilized mix. The choice of detergent can hinder or help in isolation, e.g., a small aggregation number can facilitate separation of a large protein-containing micelle from those containing only lipid components. It is possible, and perhaps even desirable, to exchange detergents, so that one detergent might be used to speed initial membrane solubilization and another for final isolation of a stable target. It is important to recognize that it is the concentration of micelles that needs to be controlled when one is studying membrane components solubilized by detergents, and generally one should maintain a ratio of several micelles to each solubilized component to avoid the possibility of spurious associations.
MEMBRANE PROTEINS FIGURE 3 Shape of micelles formed by typical single-tailed detergents. The model shown is based on data and calculations for C12E8. The dimensions shown are in nm.
A detergent can be found that can reasonably mimic the hydrophobic core of the native membrane. However, the membrane provides a physical constraint to the embedded protein, in that the protein cannot expand
DETERGENT PROPERTIES
laterally in the plane of the membrane nor can it pull itself through the bilayer; in a detergent solution, such a physical constraint is greatly relaxed and the protein in effect has a much expanded conformational space including conformations where activity will be lost possibly irreversibly. Low CMCs and large aggregation numbers may partially compensate for the loss of the membrane’s physical constraints. Moreover, the native environment of the membrane provides a multitude of interactions with a variety of lipid headgroups, tails, and with other proteins and these interactions may differ from one side of the membrane to the other. The roles of these interactions in maintaining an active protein are not well understood and they are radically changed upon solubilization. Studies as a function of micelle concentration should be performed, as there is always a possibility of adventitious interactions, due merely to the fact that too little detergent is present.
RECONSTITUTION INTO A BILAYER
OF
PROTEIN
Many of the functions of membrane proteins are vectorial in nature, and one must eventually reconstitute the protein into a lipid bilayer where these functions can be probed. Starting with mixed micelles of protein in detergent and lipids in detergent, one must effect a controlled removal of the detergent, permitting the formation of an artificial membrane bearing the protein. This is accomplished largely by trial and error. A high CMC permits the use of dialysis since the monomer should pass through the dialysis membrane. Hydrophobic beads to which the detergent absorbs have also proven effective in reconstitution studies.
SUMMARY Obviously single-tailed ionic detergents should generally be avoided because of their potential to denature any protein. While zwitterionic headgroups can be identical or at least similar to those of native lipids, to date they have not proven any more useful than others. High CMCs may be appropriate choices for initial solubilization and eventual reconstitution. But long tails and large headgroups (usually accompanied by a low CMC) may enhance stability by restricting the conformational space accessible to the protein in detergent solution. The requirements for efficient solubilization and for the maintenance of activity may to a large extent be antagonistic. The possibility of using one detergent to solubilize and subsequently exchanging into another to enhance stability for
581
isolation and study should always be considered. Finally, there is no perfect detergent for all situations nor are there hard-and-fast rules for choosing a detergent for a specific task. In the end the choice is still largely a matter of trial and error.
SEE ALSO
THE
FOLLOWING ARTICLES
MDR Membrane Proteins † Membrane Transport, General Concepts
GLOSSARY aggregation number Number of detergent molecules in the average micelle. CMC (critical micelle concentration) Concentration of detergent in aqueous solution above which micelles begin to form, essentially the maximum concentration of detergent in monomeric form. detergent A compound having spatially segregated hydrophilic and hydrophobic regions and which, when dissolved in water above the CMC, self-associates to form micelles. hydrophilic group Structure having an affinity for water with strong favorable noncovalent bonds with water molecules. hydrophobic group Structure exhibiting an aversion to water and preference for hydrocarbon-type liquids. micelle Structure formed by the noncovalent and highly cooperative self-association of a detergent molecule so as to form a hydrophobic core from which the hydrophilic groups project into the aqueous surroundings.
FURTHER READING Gravito, R. M., and Ferguson-Miller, S. (2001). Detergents as tools in membrane biochemistry. J. Biol. Chem. 276, 32403–32406. Helenius, A., and Simons, K. (1975). Solubilization of membranes by detergents. Biochim. Biophys. Acta 415, 29–79. Helenius, A., McCaslin, D. R., Fries, E., and Tanford, C. (1979). Properties of detergents. Methods Enzymol. 56, 734–749. Tanford, C. (1980). The Hydrophobic Effect: Formation of Micelles and Biological Membrane. Wiley, New York. Tanford, C., and Reynolds, J. A. (1976). Characterization of membrane proteins in detergent solutions. Biochim. Biophys. Acta 457, 133 –170. White, S. H., Ladokhin, A. S., Jayasinghe, S., and Hristova, K. (2001). How membranes shape protein structure. J. Biol. Chem. 276, 32395–32398.
BIOGRAPHY Dr. Darrell R. McCaslin holds a B.S. with Honors in biochemistry from Oklahoma State University and a Ph.D. in physical biochemistry from Duke University. He has held positions at Duke University Medical Center and Rutgers University in Newark. Presently, he is the Director of Operations for the Biophysical Instrument Facility at the University of Wisconsin in Madison. In addition to overseeing the operations of the facility and training investigators in the use of the instrumentation, he collaborates on a variety of characterization problems.
A detergent is formed when a hydrophilic group with an affinity for water and a hydrophobic group with an aversion to water are spatially segregated within a molecule’s chemical skeleton so as to create a polarity. In aqueous solution, detergents generally exist as a mixture of monomers in equilibrium with a fairly monodisperse population of detergent aggregates known as micelles. In the micelle, the hydrophobic groups are packed together to create a hydrophobic core with the attached hydrophilic groups projecting out from the surface of this core and protecting it from contact with water. As such, detergents have only limited biological functions such as the well-known role in digestion and possibly to some extent in membrane fusion events; however, they play necessary if not essential roles in the isolation, manipulation, and characterization of the constituents of biological membranes.
Chemical Structure of Detergents The chemical structures of detergents which have been employed in biological studies are quite varied and continues to grow as investigators attempt to develop new entities that will permit the facile isolation of membrane constituents while enhancing their stability once isolated. To reach this goal, various combinations of hydrophobic and hydrophilic groups have been utilized and yield a variety of physical and chemical properties.
Ring-Based Hydrocarbons The naturally occurring bile salts synthesized from cholesterol and utilized in digestive processes are the prototypes for detergents based on ring systems. Figure 1 shows cholic acid, one of the abundant bile salts which is converted to deoxycholate (DOC) by removing the hydroxyl at the arrow. Identifying the spatial segregation of the hydrophilic and hydrophobic groups in these detergents may require careful inspection of their three-dimensional chemical structure. In cholic acid, the carboxyl group is an obvious hydrophilic group, but the effects of the hydroxyl groups located on the rings must also be considered. Viewing the steroid ring nucleus in the molecule as defining a plane, the spatial arrangement of the hydroxyls creates a hydrophilic face on one side and a hydrophobic face on the other side of this plane. The structure of DOC suggests the presence of only a hydrophilic edge along the ring plane, making the whole structure more hydrophobic than cholic acid, which may explain why DOC is generally a more aggressive solubilizer than cholic acid.
HYDROPHILIC GROUPS (HEADGROUPS ) Ionic
The hydrocarbon chain is the most easily recognized hydrophobic group and when present is often referred to as the molecule’s hydrophobic tail (Figure 1). These chains are most often saturated hydrocarbons (Figure 1) and are available in many lengths. The shorter the chain the less well defined the detergent properties tend to be, whereas longer tails become essentially insoluble. Unsaturation and more complex branching structures have been explored as well as chains incorporating phenyl rings. The presence of the ultraviolet lightabsorbing phenyl should generally be avoided as it can complicate various spectroscopic techniques.
Positively (e.g., amino) and negatively (e.g., carboxyl) charged groups have been utilized as hydrophilic groups. Single-tailed ionic detergents tend to denature all proteins, with the anionic ones being more aggressive at denaturation than cationic molecules. One of the most familiar examples is the anionic detergent, sodium dodecyl sulfate, NaDodSO4 (Figure 1). It is the denaturing effect of NaDodSO4 on proteins that led to the development of one of the most widely exploited analytical tools of biochemistry, denaturing gel electrophoresis, which permits one to easily assess the number of components present in a sample as well as their approximate molecular weights. Interestingly, it is the monomeric form of the ionic detergent that drives the denaturation process. The bulk of the lipids which form the basic membrane framework are amphiphiles with two hydrophobic
Encyclopedia of Biological Chemistry, Volume 1. q 2004, Elsevier Inc. All Rights Reserved.
577
HYDROPHOBIC GROUPS (TAILS ) Simple Hydrocarbon Chains
578
DETERGENT PROPERTIES
Commercial applications of detergents have driven the development of a large class of nonionic detergents with polyoxyethylene (POE) headgroups. A chemically homogeneous example is C12E8 (Figure 1), where a 12-C tail (C12) is attached to a polymer with eight repeats of –CH2CH2O – (E8) and terminates with a hydroxyl group. The ether groups of this chain provide sites for hydrogen bonding with water offsetting the hydrophobic nature of the intervening ethylene groups. Most commercially available POE-based detergents have headgroups consisting of a single chain although the length is often heterogeneous; however, highly branched structures have been created by attachment of several POE chains to a central moiety such as sorbitol (e.g., Tweens). The POE chains are subject to peroxidation and breakdown and solutions should only be used when relatively freshly prepared; moreover, when using commercial sources one should be aware that antioxidants are sometimes included.
CONNECTING HEAD
FIGURE 1 Selected chemical structures of detergents. Removal of the hydroxyl near the arrow on cholic acid coverts the molecule to deoxycholic acid.
tails esterified to zwitterionic headgroups (contains both positive and negative charges). While the lipids themselves generally form vesicular structures when dispersed in water, removal of one of the tails creates a detergent such as the lysophosphatidylcholine in Figure 1. Such lysolipids and other zwitterionic detergents (e.g., sulfobetaines) have been used as well as highly dipolar groups (e.g., amine oxide) that lack a formal charge.
Nonionic Any uncharged group of atoms capable of accepting or donating hydrogen bonds to water can in principle become a nonionic headgroup. The headgroup must be big enough so that the detergent dissolves in solution (as a mixture of monomer and micelles) rather than forming a separate phase. Thus, simple alkyl alcohols tend to phase separate for all but the shortest hydrophobic tails and have no useful detergent properties. Carbohydrate groups are useful headgroups and one of the most frequently encountered is b-octyl glucoside (OG) in Figure 1. Octyl glucoside in the a-anomeric configuration is essentially insoluble, demonstrating how a simple change in chemical structure can dramatically alter detergent properties. More elaborate carbohydrate structures have also been explored.
AND
TAIL
Linkages between the hydrophobic and hydrophilic groups are most often either an ether or ester linkage. The latter can be susceptible to hydrolysis, especially in biological preparations, which in turn could generate an ionic group with denaturing properties.
Detergent Properties CRITICAL MICELLE CONCENTRATION (CMC) The most common concentration-dependent behavior for detergents is illustrated in Figure 2. The monomer concentration increases until the critical micelle concentration (CMC) above which an equilibrium is established between the monomer and an increasing concentration of micelles. The monomer concentration increases very little after reaching the CMC and thus is in effect the highest possible concentration of monomeric detergent. All detergent in excess of the CMC is incorporated into micelles (dashed line in Figure 2). The concentration of micelles increases linearly with total detergent concentration above the CMC, but at a slower rate since a large number of molecules are incorporated into each micelle. Some detergents exhibit more complex behavior, such as secondary association of micelles or even new phases as the total concentration continues to increase. As illustrated in Figure 2, the CMC is actually a narrow range of concentration over which the formation of micelles becomes dominant. Nonetheless, a single number is usually defined as the CMC and determined by plotting some physical observable whose response differs markedly above and below the CMC against the total
DETERGENT PROPERTIES
579
AGGREGATION NUMBER
FIGURE 2 Concentration-dependent behavior of a detergent. The calculations are based on the theory of micelle formation developed by Tanford. An aggregation number of 50 was used and the equilibrium constant for micellization was chosen to yield the critical micelle concentration (CMC) indicated.
concentration of detergent. The CMC is then taken as the intersection of lines extrapolated from the nearly linear regions above and below the transition as shown by the dotted lines in Figure 2. Using different observables or concentration ranges for the extrapolation can lead to somewhat different values for the CMC. One of the simplest methods to measure a CMC concentration ranges or for the extrapolation. is by solubilization of heme, which is essentially insoluble below the CMC and increases as the concentration of micelles increases and would be similar to the dashed line in Figure 2. CMCs range from nanomolar to several millimolar. For single-tailed detergents, the CMC decreases with increasing length of tail and with smaller headgroups. The CMC of nonionics is generally lower than that of ionics with the same length of tail. Ionic headgroups are more susceptible to solution variables than the nonionics. Ionic headgroupsthatcan betitratedaresensitive topHandmay precipitate when the headgroup is in neutral form (both cholic acid and DOC are examples). The CMCs of ionics are also influenced by the type and concentration of counterions present, e.g., using potassium instead of sodium for dodecyl sulfate, results in an insoluble salt. In general, higher salt concentrations will decrease the CMC of an ionic detergent which is a consequence of reducing the headgroup repulsion in the micelle. Even the POEbased nonioncs may be influenced by presence of salts as the ether groups are capable of complexing ions. The solubilization of other components in the micelle (e.g., mixed micelles with lipids) will generally decrease the CMC (i.e., the maximum monomer detergent concentration is decreased). Published values of CMCs are useful guides in experimental design, but should generally be confirmed under the actual experimental conditions.
Micelles are formed by self-association of many detergent molecules into a single noncovalent structure. Since the number of molecules in each micelle is somewhat variable, micelle size is characterized by the aggregation number, which is the average number of molecules per micelle and can be determined by standard hydrodynamic methods (e.g., sedimentation equilibrium). The aggregation number is needed to calculate the micelle concentration, which is the total detergent concentration in excess of the CMC divided by the aggregation number. The CMC transition becomes sharper as the aggregation number increases. The CMC is generally strongly dependent on the size of the hydrophobic moiety, but repulsion between headgroups is the dominant factor for the aggregation number. Thus, nonionics tend to have larger aggregation numbers than ionics of the same tail length. The aggregation number for ionic detergents can be strongly influenced by both the type and concentration of counterions present as well as pH since these factors can dramatically change the electrostatic repulsion between the charged headgroups. Longer hydrophobic tails tend to have larger aggregation numbers. Detergents with high CMCs tend to have more variability in their micelle size distribution. At high concentrations and under certain solution conditions (e.g., elevated temperature) larger structures may be formed which in some cases are new micellar phases and in others simply secondary aggregation of smaller micelles.
TEMPERATURE EFFECTS As a thermodynamic equilibrium, the formation of micelles can be influenced by temperature. At low enough temperatures, solid detergent will exist in equilibrium with monomeric detergent; as the temperature increases, the monomer concentration increases until it reaches the CMC at the critical micelle temperature. Above this temperature, solid detergent will begin to go into solution as micelles. The temperature at which solid, micelles, and monomer at the CMC coexist is called the Kraft point and for most detergents is the same as the critical micelle temperature. An often observed Kraft point, which is near room temperature, is that of sodium dodecyl sulfate and upon cooling one sees precipitation of detergent. A second temperature effect observed at higher temperature, especially with nonionics containing POE, is the cloud point. At this temperature and above the CMC, the solution will turn turbid due to the formation of much larger aggregates. Both the critical micelle temperature and cloud point have been exploited in purification of membrane components.
580
DETERGENT PROPERTIES
Micelle Structure SINGLE- TAILED DETERGENTS In a micelle, the hydrophobic tails are sequestered into a central core structure with the hydrophilic groups projecting out from the surface of this core into the aqueous surrounding. The tails in the core are not fully extended but are quite flexible, some even lying along the surface of the core rather than within it. The surface of the core should be regarded as having a rippled texture with some tail methylene groups protruding above it. Experimental and theoretical arguments suggest that for most detergent micelles the overall shape is best described as an oblate ellipsoid, although for small aggregation numbers the shape cannot be readily distinguished from spherical. For C12E8 micelles (Figure 1), theoretical calculations constrained by hydrodynamic measurements yield an oblate ellipsoid with dimensions shown in Figure 3. One dimension must be less than the 3.4 nm length of two fully extended 12C chains since the core cannot contain a void. For the same length of tail, the overall dimensions of the micelle will depend on the nature of the headgroup. For POE-based detergents, the headgroup is in a random-coil configuration and consequently occupies a very large region of the space surrounding the core, as illustrated in Figure 3. Figure 3, while a convenient visualization of micelle structure, cannot convey the highly dynamic processes occurring. Detergent molecules are rapidly exchanged among micelles; whole micelles disappear and reform with slightly different aggregation numbers. The hydrophobic core acts for the most part like a simple liquid hydrocarbon with the tails constantly flexing. POE headgroups occupy a great deal of space around the core and are constantly changing their conformation as well. Finally, it should be obvious that the type and size of headgroup can generate dramatically different chemical environments to which molecules
solubilized by detergents are exposed; moreover, the solution composition near a micelle may be quite different from that of the bulk aqueous solution.
RING- BASED DETERGENTS Bile salts usually have relatively high CMCs and quite small aggregation numbers (4– 10). The small aggregation number does not permit micelles as just described. The micelles are more heterogeneous and are probably best viewed as small assemblies with the hydrophobic surfaces facing each other, presenting their hydrophilic faces to the surrounding solution. The size and shape of these micelles are quite sensitive to the concentration and types of ions, and potentially pH.
Detergents as Tools in Membrane Studies MEMBRANE SOLUBILIZATION Initially when a membrane is exposed to a detergent, monomers partition into the bilayer, so one can imagine that a high CMC might be advantageous (e.g., OG has a very high CMC (. 15 mM) and is an efficient solubilizer). As more detergent intercalates into the membrane’s bilayer, one reaches a stage where fragmentation occurs, resulting in large mixed micelles containing detergent, lipids, and proteins in various ratios. Continuing to add detergent will eventually disperse the membrane components to the point that any given micelle contains no more than a few lipids, a protein, or the detergent alone. One can apply isolation and enrichment techniques for specific targets to this solubilized mix. The choice of detergent can hinder or help in isolation, e.g., a small aggregation number can facilitate separation of a large protein-containing micelle from those containing only lipid components. It is possible, and perhaps even desirable, to exchange detergents, so that one detergent might be used to speed initial membrane solubilization and another for final isolation of a stable target. It is important to recognize that it is the concentration of micelles that needs to be controlled when one is studying membrane components solubilized by detergents, and generally one should maintain a ratio of several micelles to each solubilized component to avoid the possibility of spurious associations.
MEMBRANE PROTEINS FIGURE 3 Shape of micelles formed by typical single-tailed detergents. The model shown is based on data and calculations for C12E8. The dimensions shown are in nm.
A detergent can be found that can reasonably mimic the hydrophobic core of the native membrane. However, the membrane provides a physical constraint to the embedded protein, in that the protein cannot expand
DETERGENT PROPERTIES
laterally in the plane of the membrane nor can it pull itself through the bilayer; in a detergent solution, such a physical constraint is greatly relaxed and the protein in effect has a much expanded conformational space including conformations where activity will be lost possibly irreversibly. Low CMCs and large aggregation numbers may partially compensate for the loss of the membrane’s physical constraints. Moreover, the native environment of the membrane provides a multitude of interactions with a variety of lipid headgroups, tails, and with other proteins and these interactions may differ from one side of the membrane to the other. The roles of these interactions in maintaining an active protein are not well understood and they are radically changed upon solubilization. Studies as a function of micelle concentration should be performed, as there is always a possibility of adventitious interactions, due merely to the fact that too little detergent is present.
RECONSTITUTION INTO A BILAYER
OF
PROTEIN
Many of the functions of membrane proteins are vectorial in nature, and one must eventually reconstitute the protein into a lipid bilayer where these functions can be probed. Starting with mixed micelles of protein in detergent and lipids in detergent, one must effect a controlled removal of the detergent, permitting the formation of an artificial membrane bearing the protein. This is accomplished largely by trial and error. A high CMC permits the use of dialysis since the monomer should pass through the dialysis membrane. Hydrophobic beads to which the detergent absorbs have also proven effective in reconstitution studies.
SUMMARY Obviously single-tailed ionic detergents should generally be avoided because of their potential to denature any protein. While zwitterionic headgroups can be identical or at least similar to those of native lipids, to date they have not proven any more useful than others. High CMCs may be appropriate choices for initial solubilization and eventual reconstitution. But long tails and large headgroups (usually accompanied by a low CMC) may enhance stability by restricting the conformational space accessible to the protein in detergent solution. The requirements for efficient solubilization and for the maintenance of activity may to a large extent be antagonistic. The possibility of using one detergent to solubilize and subsequently exchanging into another to enhance stability for
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isolation and study should always be considered. Finally, there is no perfect detergent for all situations nor are there hard-and-fast rules for choosing a detergent for a specific task. In the end the choice is still largely a matter of trial and error.
SEE ALSO
THE
FOLLOWING ARTICLES
MDR Membrane Proteins † Membrane Transport, General Concepts
GLOSSARY aggregation number Number of detergent molecules in the average micelle. CMC (critical micelle concentration) Concentration of detergent in aqueous solution above which micelles begin to form, essentially the maximum concentration of detergent in monomeric form. detergent A compound having spatially segregated hydrophilic and hydrophobic regions and which, when dissolved in water above the CMC, self-associates to form micelles. hydrophilic group Structure having an affinity for water with strong favorable noncovalent bonds with water molecules. hydrophobic group Structure exhibiting an aversion to water and preference for hydrocarbon-type liquids. micelle Structure formed by the noncovalent and highly cooperative self-association of a detergent molecule so as to form a hydrophobic core from which the hydrophilic groups project into the aqueous surroundings.
FURTHER READING Gravito, R. M., and Ferguson-Miller, S. (2001). Detergents as tools in membrane biochemistry. J. Biol. Chem. 276, 32403–32406. Helenius, A., and Simons, K. (1975). Solubilization of membranes by detergents. Biochim. Biophys. Acta 415, 29–79. Helenius, A., McCaslin, D. R., Fries, E., and Tanford, C. (1979). Properties of detergents. Methods Enzymol. 56, 734–749. Tanford, C. (1980). The Hydrophobic Effect: Formation of Micelles and Biological Membrane. Wiley, New York. Tanford, C., and Reynolds, J. A. (1976). Characterization of membrane proteins in detergent solutions. Biochim. Biophys. Acta 457, 133 –170. White, S. H., Ladokhin, A. S., Jayasinghe, S., and Hristova, K. (2001). How membranes shape protein structure. J. Biol. Chem. 276, 32395–32398.
BIOGRAPHY Dr. Darrell R. McCaslin holds a B.S. with Honors in biochemistry from Oklahoma State University and a Ph.D. in physical biochemistry from Duke University. He has held positions at Duke University Medical Center and Rutgers University in Newark. Presently, he is the Director of Operations for the Biophysical Instrument Facility at the University of Wisconsin in Madison. In addition to overseeing the operations of the facility and training investigators in the use of the instrumentation, he collaborates on a variety of characterization problems.