[27]
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stored in 0.001 M phosphate buffer. This material gave no trouble with flow rates unless it was several months old. Acknowledgment This work was supported in part by NIH Grant GM-14603.
[27] H y d r o p h o b i c C h r o m a t o g r a p h y
By ROBERT M. KENNEDY Hydrophobic interactions are a phenomenon of great biological significance. They are one of the main forces that stabilize the three-dimensional structure of proteins. Hydrophobic interactions are involved in antibody-antigen reactions and enzyme-substrate reactions. They also contribute to the maintenance of the lipid bilayer structure of biological membranes and are involved in the binding of proteins to these membranes. Hydrophobic interactions can be exploited and used as a means of separation. Hydrophobic matrices can be constructed and used for the fractionation of mixtures containing molecules with hydrophobic moieties. We should start by saying something about the nature of hydrophobic interactions. A definition of hydrophobicity is the repulsion between a nonpolar compound and a polar environment such as water. When a single hydrophobic compound is put into water, an energetically unfavorable condition results. The hydrophobic compound forces the surrounding water into an ordered structure as if it were forming a cavity. This process occurs with a decrease in entropy. If two or more nonpolar compounds are put into water, they aggregate spontaneously. This aggregation is due to hydrophobic interactions. Hydrophobic interactions are energetically favorable because of a gain in entropy; that is, there is an increase in disorder when there are less hydrophobic sites exposed to the polar environment. Hydrophobic interactions are neither a binding of hydrophobic groups to each other, nor are they attractive forces per se. Hydrophobic interactions are forced on nonpolar compounds by the polar environment. It is the structure of the water that creates hydrophobic interactions. Given that it is the structure of water that creates hydrophobic interactions, it follows that if one changes the structure of water by dissolving METHODS IN ENZYMOLOGY, VOL. 182
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
340
PURIFICATION PROCEDURES: CHROMATOGRAPHICMETHODS
[27]
salts or organic solvents in it, then hydrophobic interactions would somehow be affected. Generally speaking, increasing ionic strength increases hydrophobic interactions. Both anions and cations can be listed in a series from those that highly favor hydrophobic interactions to those that decrease hydrophobic interactions. For anions, the series is PO43- > 5042> CH3COO- > C1- > Br- > NO3- > CIO4- > I- > SCN-, and for cations the series is NH4 + > Rb + > K + > Na + > Cs ÷ > Li + > Mg 2+ > Ca 2÷ > Ba 2÷. The strong chaotropic salts disrupt the structure of water and thus tend to decrease the strength of hydrophobic interactions; the antichaotropic salts tend to favor them. Organic solvents are also commonly used to alter the polarity of water. We will, primarily, be discussing the separation of proteins, but keep in mind that most biomolecules have a certain degree of hydrophobic character. The degree of hydrophobicity of a protein is dependent on its amino acid sequence. Certain amino acids are hydrophobic; in order of decreasing hydrophobicity they are tryptophan, norleucine, phenylalanine, tyrosine, leucine, valine, methionine, alanine. Hydrophobic interactions stabilize the tertiary and quaternary structure of proteins. In addition, many hydrophobic amino acids are exposed on the surface and it is these that give a native protein its degree of hydrophobicity. The ability of a protein to undergo hydrophobic interactions in the native state depends on its surface hydrophobic sites and surface hydrophobicity depends on an intact tertiary or quaternary structure. This is to be distinguished from the native hydrophobicity of the protein, which is dependent on its primary structure. When using hydrophobicity as a basis for separation, these differing sources of hydrophobicity in a protein, those which arise from tertiary and quaternary structure and those which arise from primary structure, can be exploited. Several general works are available which explore hydrophobic interactions in depth. 1,2 Comparison of Hydrophobic Interaction Chromatography and Reversed-Phase Chromatography Hydrophobic interaction chromatography (HIC) and reversed-phase chromatography (RPC) are two separation methods based on the interactions between the hydrophobic moieties of a sample and an insoluble, immobilized hydrophobic group (i.e., those on the matrix). It is helpful to know the basic differences between HIC and RPC. In hydrophobic interaction chromatography the matrix is hydrophilic and is substituted with 1 C. Tarfford, "The Hydrophobic Effect." Wiley, New York, 1980. 2 A. Ben-Naim, "Hydrophobic Interactions." Plenum, New York, 1980.
[2 7]
HYDROPHOBIC CHROMATOGRAPHY
341
short-chain phenyl or octyl nonpolar groups. The mobile phase is usually an aqueous salt solution. In reversed phase chromatography the matrix is silica that has been substituted with longer n-alkyl chains, usually C8 (octylsilyl) or C~8 (octadecylsilyl). The matrix is less polar than the mobile phase. The mobile phase is usually a mixture of water and a less polar organic modifier. In early work a distinction was made between methods on the basis of the polarity of the mobile phase. Normal phase systems were those in which the matrix was silica and the mobile phase was a nonpolar solvent such as hexane. Reversed-phase systems were so called because in these the mobile phase, a water solution, is more polar than the stationary phase, normally a C8 or C~8 derivatized silica. Separations on HIC matrices are usually done in aqueous salt solutions, which generally are nondenaturing conditions. Samples are loaded onto the matrix in a high-salt buffer and elution is by a descending salt gradient. Separations on reversed-phase media are usually done in mixtures of aqueous and organic solvents, which are often denaturing conditions. These two methods exploit the different sources of the hydrophobicity of proteins. HIC depends on surface hydrophobic groups and is carried out under conditions which maintain the integrity of the protein molecule. RPC depends on the native hydrophobicity of the protein and is carried out under conditions which expose nearly all hydrophobic groups to the matrix, i.e., denaturing conditions. A study done by Fausnaugh et al. compared the recovery of enzyme activity after HIC and RPC. 3 Procedures for Hydrophobic Chromatography Fortunately, although the mechanics of hydrophobic interactions are complicated, the chromatographic techniques based on hydrophobic interactions are easy to use. Choice o f Gel For an uncharacterized protein, a phenyl-substituted resin is usually the best choice to start, because strongly hydrophobic proteins will not easily be eluted from octyl-substituted resins. The phenyl ligand is intermediate in hydrophobicity between n-butyl and n-pentyl, and will bind to aromatic amino acids through zr-~" interactions. Octyl-substituted resins such as octyl-Sepharose CL-4B can be used for weakly hydrophobic proteins. It is also the medium of choice for use with membrane proteins since it still retains hydrophobic binding properties in the presence of solubilizing concentrations of detergents. 3 j. L. Fausnaugh, L. A. Kennedy, and F. E. Regnier, J. Chromatogr. 317, 141 (1985).
342
PURIFICATION PROCEDURES" CHROMATOGRAPHIC METHODS
[27]
Running the Column Binding. The binding of proteins to hydrophobic gels is influenced by: 1. The hydrophobicity of the ligand: For example, phenyl-Sepharose CL-4B is less hydrophobic than octyl-Sepharose CL-4B. 2. The ionic strength of the buffer: Those salts which cause salting out [e.g., (NH4)2SO4] also promote the binding of proteins to hydrophobic ligands. Binding to octyl- and phenyl-Sepharose CL-4B is generally negligible unless high-salt buffer solutions are used. A salt concentration just below that used for salting out the protein is normally used. 3. Temperature: It has been noted that a 20-30% reduction in binding strength is seen when the temperature is reduced from 20 to 4 °. The strength of the hydrophobic interactions will be lessened, therefore, if the experiment is done in a cold room. To ensure that sample molecules bind to HIC supports, it is usually necessary to add something to the sample that will increase the hydrophobic interactions between the sample and the matrix. In HIC this can be done in rseveral ways. One of the more common strategies is to apply the sample in a high concentration of salt [1.7 M (NH4)2SO4, 4 M KC1, 4 M NaC1] in a buffered solution, pH range from 6.5 to 8.0. Elution. Once the sample has been applied to the column and the hydrophobic species of interest has bound, unbound proteins are washed through with the starting buffer. Elution of the protein of interest can be done in several ways: 1. Reducing the concentration of salting out ions in the buffer with a negative salt gradient 2. Increasing the concentration of chaotropic ions in the buffer in a positive gradient 3. Eluting with a positive gradient of a detergent (note that the gel must be cleaned afterward due to the hydrophobic nature of detergents) or with a polarity-reducing organic solvent, usually ethylene glycol (up to 75% ethylene glycol has been used) 4. Raising the pH 5. Reducing the temperature Note that the hydrophobicity of the ligand used will affect the ease of desorption. Elution gradients can be either step or linear. Most of the elution strategies are nondenaturing. Use of detergents and lowering the polarity of the eluent are often last-resort methods used to elute a very strongly bound protein since these two procedures often denature proteins. It is often good practice to utilize two or more of these elution tech-
[28]
CHROMATOGRAPHY
ON IMMOBILIZED
REACTIVE DYES
343
niques simultaneously. The existence of a wide variety of possible elution methods is potentially very valuable for the resolution of complex mixtures, Extensive information on specific applications is available from the manufacttirers of hydrophobic media and is not presented here.
Regeneration and Storage HIC gels can be reused several times; exactly how many times depends on the quality of the buffers, sample, etc. After every chromatographic run, a wash with 6 M urea will remove tightly bound proteins. The gel can then be equilibrated with starting buffer and is immediately ready for the next run. If detergents have been used on the gel, the cleaning procedure is slightly more complicated. The following procedure is recommended by Pharmacia (Piscataway, N J) for cleaning octyl- and phenylSepharose after use with detergents. Wash the gel sequentially with 1. One bed volume of distilled water 2. One bed volume each of 25, 50, and 95% ethanol 3. Two bed volumes of n-butanol 4. One bed volume of 95, 50, and 25% ethanol 5. One bed volume of distilled water 6. Reequilibrate the gel with starting buffer to make it ready for the next experiment Chromatography is the most accepted separation tool in modern biochemistry laboratories. Each chromatographic method exploits different physical or biological properties of the molecule as a basis for separation. In this chapter we have explored the uses of hydrophobicity as a basis for two chromatographic methods, HIC and RPC. Most protein purifications require more than one chromatographic step. Hydrophobicity is an often overlooked physiochemical property of the biomolecule which can be exploited in the logical design of a protein purification scheme.
[28] C h r o m a t o g r a p h y o n I m m o b i l i z e d R e a c t i v e D y e s By EARLE STELLWAGEN Of all the fractionation procedures used in protein purification, only affinity chromatography takes advantage of the property that clearly distinguishes one protein from another, namely its function. The surfaces of virtually all proteins are designed to selectively bind one or a small numMETHODS IN ENZYMOLOGY, VOL. 182
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
HYDROPHOBICCHROMATOGRAPHY
339
stored in 0.001 M phosphate buffer. This material gave no trouble with flow rates unless it was several months old. Acknowledgment This work was supported in part by NIH Grant GM-14603.
[27] H y d r o p h o b i c C h r o m a t o g r a p h y
By ROBERT M. KENNEDY Hydrophobic interactions are a phenomenon of great biological significance. They are one of the main forces that stabilize the three-dimensional structure of proteins. Hydrophobic interactions are involved in antibody-antigen reactions and enzyme-substrate reactions. They also contribute to the maintenance of the lipid bilayer structure of biological membranes and are involved in the binding of proteins to these membranes. Hydrophobic interactions can be exploited and used as a means of separation. Hydrophobic matrices can be constructed and used for the fractionation of mixtures containing molecules with hydrophobic moieties. We should start by saying something about the nature of hydrophobic interactions. A definition of hydrophobicity is the repulsion between a nonpolar compound and a polar environment such as water. When a single hydrophobic compound is put into water, an energetically unfavorable condition results. The hydrophobic compound forces the surrounding water into an ordered structure as if it were forming a cavity. This process occurs with a decrease in entropy. If two or more nonpolar compounds are put into water, they aggregate spontaneously. This aggregation is due to hydrophobic interactions. Hydrophobic interactions are energetically favorable because of a gain in entropy; that is, there is an increase in disorder when there are less hydrophobic sites exposed to the polar environment. Hydrophobic interactions are neither a binding of hydrophobic groups to each other, nor are they attractive forces per se. Hydrophobic interactions are forced on nonpolar compounds by the polar environment. It is the structure of the water that creates hydrophobic interactions. Given that it is the structure of water that creates hydrophobic interactions, it follows that if one changes the structure of water by dissolving METHODS IN ENZYMOLOGY, VOL. 182
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
340
PURIFICATION PROCEDURES: CHROMATOGRAPHICMETHODS
[27]
salts or organic solvents in it, then hydrophobic interactions would somehow be affected. Generally speaking, increasing ionic strength increases hydrophobic interactions. Both anions and cations can be listed in a series from those that highly favor hydrophobic interactions to those that decrease hydrophobic interactions. For anions, the series is PO43- > 5042> CH3COO- > C1- > Br- > NO3- > CIO4- > I- > SCN-, and for cations the series is NH4 + > Rb + > K + > Na + > Cs ÷ > Li + > Mg 2+ > Ca 2÷ > Ba 2÷. The strong chaotropic salts disrupt the structure of water and thus tend to decrease the strength of hydrophobic interactions; the antichaotropic salts tend to favor them. Organic solvents are also commonly used to alter the polarity of water. We will, primarily, be discussing the separation of proteins, but keep in mind that most biomolecules have a certain degree of hydrophobic character. The degree of hydrophobicity of a protein is dependent on its amino acid sequence. Certain amino acids are hydrophobic; in order of decreasing hydrophobicity they are tryptophan, norleucine, phenylalanine, tyrosine, leucine, valine, methionine, alanine. Hydrophobic interactions stabilize the tertiary and quaternary structure of proteins. In addition, many hydrophobic amino acids are exposed on the surface and it is these that give a native protein its degree of hydrophobicity. The ability of a protein to undergo hydrophobic interactions in the native state depends on its surface hydrophobic sites and surface hydrophobicity depends on an intact tertiary or quaternary structure. This is to be distinguished from the native hydrophobicity of the protein, which is dependent on its primary structure. When using hydrophobicity as a basis for separation, these differing sources of hydrophobicity in a protein, those which arise from tertiary and quaternary structure and those which arise from primary structure, can be exploited. Several general works are available which explore hydrophobic interactions in depth. 1,2 Comparison of Hydrophobic Interaction Chromatography and Reversed-Phase Chromatography Hydrophobic interaction chromatography (HIC) and reversed-phase chromatography (RPC) are two separation methods based on the interactions between the hydrophobic moieties of a sample and an insoluble, immobilized hydrophobic group (i.e., those on the matrix). It is helpful to know the basic differences between HIC and RPC. In hydrophobic interaction chromatography the matrix is hydrophilic and is substituted with 1 C. Tarfford, "The Hydrophobic Effect." Wiley, New York, 1980. 2 A. Ben-Naim, "Hydrophobic Interactions." Plenum, New York, 1980.
[2 7]
HYDROPHOBIC CHROMATOGRAPHY
341
short-chain phenyl or octyl nonpolar groups. The mobile phase is usually an aqueous salt solution. In reversed phase chromatography the matrix is silica that has been substituted with longer n-alkyl chains, usually C8 (octylsilyl) or C~8 (octadecylsilyl). The matrix is less polar than the mobile phase. The mobile phase is usually a mixture of water and a less polar organic modifier. In early work a distinction was made between methods on the basis of the polarity of the mobile phase. Normal phase systems were those in which the matrix was silica and the mobile phase was a nonpolar solvent such as hexane. Reversed-phase systems were so called because in these the mobile phase, a water solution, is more polar than the stationary phase, normally a C8 or C~8 derivatized silica. Separations on HIC matrices are usually done in aqueous salt solutions, which generally are nondenaturing conditions. Samples are loaded onto the matrix in a high-salt buffer and elution is by a descending salt gradient. Separations on reversed-phase media are usually done in mixtures of aqueous and organic solvents, which are often denaturing conditions. These two methods exploit the different sources of the hydrophobicity of proteins. HIC depends on surface hydrophobic groups and is carried out under conditions which maintain the integrity of the protein molecule. RPC depends on the native hydrophobicity of the protein and is carried out under conditions which expose nearly all hydrophobic groups to the matrix, i.e., denaturing conditions. A study done by Fausnaugh et al. compared the recovery of enzyme activity after HIC and RPC. 3 Procedures for Hydrophobic Chromatography Fortunately, although the mechanics of hydrophobic interactions are complicated, the chromatographic techniques based on hydrophobic interactions are easy to use. Choice o f Gel For an uncharacterized protein, a phenyl-substituted resin is usually the best choice to start, because strongly hydrophobic proteins will not easily be eluted from octyl-substituted resins. The phenyl ligand is intermediate in hydrophobicity between n-butyl and n-pentyl, and will bind to aromatic amino acids through zr-~" interactions. Octyl-substituted resins such as octyl-Sepharose CL-4B can be used for weakly hydrophobic proteins. It is also the medium of choice for use with membrane proteins since it still retains hydrophobic binding properties in the presence of solubilizing concentrations of detergents. 3 j. L. Fausnaugh, L. A. Kennedy, and F. E. Regnier, J. Chromatogr. 317, 141 (1985).
342
PURIFICATION PROCEDURES" CHROMATOGRAPHIC METHODS
[27]
Running the Column Binding. The binding of proteins to hydrophobic gels is influenced by: 1. The hydrophobicity of the ligand: For example, phenyl-Sepharose CL-4B is less hydrophobic than octyl-Sepharose CL-4B. 2. The ionic strength of the buffer: Those salts which cause salting out [e.g., (NH4)2SO4] also promote the binding of proteins to hydrophobic ligands. Binding to octyl- and phenyl-Sepharose CL-4B is generally negligible unless high-salt buffer solutions are used. A salt concentration just below that used for salting out the protein is normally used. 3. Temperature: It has been noted that a 20-30% reduction in binding strength is seen when the temperature is reduced from 20 to 4 °. The strength of the hydrophobic interactions will be lessened, therefore, if the experiment is done in a cold room. To ensure that sample molecules bind to HIC supports, it is usually necessary to add something to the sample that will increase the hydrophobic interactions between the sample and the matrix. In HIC this can be done in rseveral ways. One of the more common strategies is to apply the sample in a high concentration of salt [1.7 M (NH4)2SO4, 4 M KC1, 4 M NaC1] in a buffered solution, pH range from 6.5 to 8.0. Elution. Once the sample has been applied to the column and the hydrophobic species of interest has bound, unbound proteins are washed through with the starting buffer. Elution of the protein of interest can be done in several ways: 1. Reducing the concentration of salting out ions in the buffer with a negative salt gradient 2. Increasing the concentration of chaotropic ions in the buffer in a positive gradient 3. Eluting with a positive gradient of a detergent (note that the gel must be cleaned afterward due to the hydrophobic nature of detergents) or with a polarity-reducing organic solvent, usually ethylene glycol (up to 75% ethylene glycol has been used) 4. Raising the pH 5. Reducing the temperature Note that the hydrophobicity of the ligand used will affect the ease of desorption. Elution gradients can be either step or linear. Most of the elution strategies are nondenaturing. Use of detergents and lowering the polarity of the eluent are often last-resort methods used to elute a very strongly bound protein since these two procedures often denature proteins. It is often good practice to utilize two or more of these elution tech-
[28]
CHROMATOGRAPHY
ON IMMOBILIZED
REACTIVE DYES
343
niques simultaneously. The existence of a wide variety of possible elution methods is potentially very valuable for the resolution of complex mixtures, Extensive information on specific applications is available from the manufacttirers of hydrophobic media and is not presented here.
Regeneration and Storage HIC gels can be reused several times; exactly how many times depends on the quality of the buffers, sample, etc. After every chromatographic run, a wash with 6 M urea will remove tightly bound proteins. The gel can then be equilibrated with starting buffer and is immediately ready for the next run. If detergents have been used on the gel, the cleaning procedure is slightly more complicated. The following procedure is recommended by Pharmacia (Piscataway, N J) for cleaning octyl- and phenylSepharose after use with detergents. Wash the gel sequentially with 1. One bed volume of distilled water 2. One bed volume each of 25, 50, and 95% ethanol 3. Two bed volumes of n-butanol 4. One bed volume of 95, 50, and 25% ethanol 5. One bed volume of distilled water 6. Reequilibrate the gel with starting buffer to make it ready for the next experiment Chromatography is the most accepted separation tool in modern biochemistry laboratories. Each chromatographic method exploits different physical or biological properties of the molecule as a basis for separation. In this chapter we have explored the uses of hydrophobicity as a basis for two chromatographic methods, HIC and RPC. Most protein purifications require more than one chromatographic step. Hydrophobicity is an often overlooked physiochemical property of the biomolecule which can be exploited in the logical design of a protein purification scheme.
[28] C h r o m a t o g r a p h y o n I m m o b i l i z e d R e a c t i v e D y e s By EARLE STELLWAGEN Of all the fractionation procedures used in protein purification, only affinity chromatography takes advantage of the property that clearly distinguishes one protein from another, namely its function. The surfaces of virtually all proteins are designed to selectively bind one or a small numMETHODS IN ENZYMOLOGY, VOL. 182
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
