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By EDWARD F. ROSSOMANDO This chapter introduces ion-exchange chromatography (IEC) as a method for the purification of proteins and enzymes. While this chapter discusses the general principles for the operation of all types of IEC, the focus will be on the type called open-column IEC, as distinguished from another widely used type, high-performance liquid IEC (HPLC). This latter topic will be discussed elsewhere in this volume (see [32]). When proteins are to be purified by IEC, some problems arise because proteins have unique origins. Proteins are derived from biological sources and their extraction often requires the use of detergents and chaotropic salts for solubilization. One problem is that such solubilizers interfere with the operation of IEC. Another problem is that proteins in cells are compartmentalized and therefore separated from other proteins with proteolytic activity. Extraction results in disruption of these barriers with the exposure of proteins to proteases and the potential for degradation during the course of the purification. A final problem related to source is that, since proteins must be extracted from biological materials, the amount of protein available may be limited, such as when working with embryonic tissue. This necessitates modification of the IEC protocols normally used when larger quantities are available. Problems will also arise because proteins have unique properties. For example, proteins are ampholytes; that is, they contain both positive and negative charges; the former result from the ionization of lysine and arginine residues and the latter from aspartic and glutamic acid residues. Since the ionization of such groups is pH dependent, the net charge on a protein will be a function of the pH of its environment. Also, proteins are often "sticky," adhering to surfaces such as glass or the packing material in the IEC column. Although organic solvents, detergents, and salts can eliminate some adsorption, their addition may precipitate the proteins or, in the case of enzymes, destroy catalytic activity. Some suggestions for dealing with these problems will be discussed in this chapter. IEC is designed specifically for the separation of ionic or ionizable compounds. Similar to other types of liquid chromatography, IEC has both stationary (column packing) and mobile phases. It differs from other types of liquid chromatography in that the stationary phase carries ionizable functional groups, fixed by chemical bonding to the stationary phase. Of course, to satisfy requirements for electrical neutrality, these fixed METHODS IN ENZYMOLOGY, VOL. 182
Copyright 6) 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
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charges will carry a counterion of opposite sign. This counterion is not fixed and can be displaced. IEC is named on the basis of the sign of these displaceable charges. Thus, in anion IEC the fixed charges are positive and in cation IEC the fixed charges are negative. Principles of Operation IEC involves two separate events. These are (1) the binding of the protein to the fixed charges and (2) the elution or displacement of the protein from the fixed charges. Because retention involves an electrostatic interaction between the fixed charges and those of the protein, binding involves replacement of the nonfixed ions by the protein. Elution, in turn, involves displacement of the protein from the fixed charges by a new counterion with a greater affinity for the fixed charges than the protein, and which then becomes the new, nonfixed ion. The ability of counterions (salts) to displace proteins bound to fixed charges is a function of the difference in affinities between the fixed charges and the nonfixed charges of both the protein and the salt. Affinities in turn are affected by several variables, including the magnitude of the net charge of the protein and the concentration and type of salt used for displacement. For additional details on the mechanisms underlying these processes see Refs. 1-3. Types of Ion Exchangers Widely used solid-phase packings include cellulose, dextrans, agarose, and polystyrene. The exchange groups used include DEAE (diethylaminoethyl), a weak base, that will have a net positive charge when ionized and will therefore bind and exchange anions; and CM (carboxymethyl), a weak acid, with a negative charge when ionized that will bind and exchange cations. Another form of weak anion exchanger contains the PEI (polyethyleneimine) functional group. This material, most usually found on thin layer sheets, is useful for binding proteins at pH values above their pI. The polystyrene matrix can be obtained with quaternary ammonium G. V. Samsonov, "Ion Exchange Sorption and Preparative Chromatography of Biologically Active Molecules," pp. 99, 105. Consultants Bureau, New York, 1986. 2 C. F. Poole and S. S. Schuette, "Contemporary Practice of Chromatography." Elsevier, Amsterdam. 1984 pp. 304-312. 3 p. j. Schoenmakers, "Optimization of Chromatographic Selectivity." Elsevier, Amsterdam, 1986.
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functional groups for strong base anion exchange or with sulfonic acid functional groups for strong acid cation exchange. Intermediate and weak ion-exchange materials are also available. Studies comparing the effectiveness of exchangers of various types have been reported. For example, in one study the effectiveness of a polystyrene resin was compared to cellulose for the purification of a glucoamylase from crude extracts of Aspergillus awamori. 4 Both an anionexchange polystyrene resin (Bio-Rad AG1-X4) and DEAE-cellulose were used. The results indicated that the use of the polystyrene resin solved one problem, namely, adsorption of the protein to cellulose. In addition, the resin produced a better separation of this enzyme from another, an amylase. Recently a newer form of anion-exchange system consisting of stacks of thin, noncompressible, microporous poly(vinyl chloride)-silica sheets has been introduced (FASTCHROM, Kontes, Life Sciences Products, Vineland, NJ). When these sheets are coated with PEI they yield a positively charged, hydrophilic surface for the separation of proteins and DNA. 5 Preparation and Regeneration of Packing The column packings require washing, swelling, and conversion to the desired " f o r m " prior to use. All these procedures can be performed prior to pouring the column. Washing and Swelling. To allow the column packing to reach a size equilibrium, prepare a slurry by mixing the packing with about 10 times its volume of the buffer to be used for loading the column. The slurry should be allowed to settle for about 1 hr. The top layer of clear solution, containing the "fines," should be decanted and the washing step repeated at least once. This treatment will result in better flow rates for the column. Changing Displaceable Counterion. When obtained from the manufacturer, the fixed charges of the column packing will have associated with them a counterion. This counterion can be changed, a procedure that also should be performed prior to pouring the column. 6 This exchange can be accomplished by washing the packing in a salt solution containing the counterion of choice. However, all the packing materials have a "selectivity series" that might be likened to an affinity, and in making the displacement it is necessary to go " u p " the selectivity series. For example, the selectivity for a representative cation exchanger might be Rb + > 4 R. S. Bhella and I. Altosaar, Anal. Biochem. 140, 200 (1984). 5j. j. Piotrowskiand M. H. Scholla, BioChromatography 3, 161 (1988). 6 E. L. Johnsonand R. Stevenson, "Basic LiquidChromatography,"p. 116. Varian Associates, Palo Alto, California, 1977.
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Cs + > K + > N H 4 + > Na ÷ > H ÷ > Li +. Such packings are usually supplied in the H ÷ form. Thus, it is a simple matter to replace the H ÷ with an ion higher in the series by washing in a 1 M solution of that salt. Similar selectivity series exist for anion exchangers, and the manufacturer's literature should be consulted for this information. Size of Column: Bed Capacity. Although the length o f the column is less important, the separation does depend on the amount of column packing available since if this is insufficient, material that should have been retained will flow through. Although the amount of material that the column can bind must eventually be determined by experiment, information provided by the manufacturer on the bed capacity can narrow the quest. The manufacturer provides this information in the form of miUiequivalents per dry gram or milliequivalents per milliliter of resin bed. For cation exchangers, supplied in the hydrogen form, this would be milliequivalents of H ÷ that can be exchanged while for anion exchangers supplied in the chloride form this would be milliequivalents of CI-. The columns used for IEC need not be large. For example, columns made from Pasteur pipets have been used for the purification of the peptides derived from proenkephalin. 7 Used for this purpose, the pipet should be plugged with a small amount of glass wool, the resin prepared, and introduced into the pipet. The volume of the resin in such a column would be on the order of 0.25 cm 3. For such columns elution will be a simple matter of washing the column with about 1 ml of each of the eluents. Batch os Open Column. IEC need not be performed using a column. 8 The alternative, batch IEC, is usually performed with the slurry of the stationary phase in a vessel such as a beaker. In one study, used for the separation of lactate dehydrogenase, 9 the essential component of the batch system is the resin (Bio-Rad AG MP-1), which is added to a tube containing the enzyme (isozymal forms of human lactate dehydrogenase). After shaking and equilibration, the resin is separated from the buffer either by centrifugation, filtration, or a combination of the two. The authors note that the separation they obtained was less than desirable, which they attribute to the fact that the batch procedure is both a nonequilibrium and too rapid a process. However, sometimes the speed of the process can be an advantage such as when separations on a larger scale are required. 7 S. P. Wilson, J. Neurosci. Methods 15, 155 (1985). 8 D. Reichenberg, in "Ion Exchangers in Organic and Biochemistry" (C. Calmon and T. R. E. Kressman, eds.). Interscience, New York, 1957. 9 M. P. Menon, S. Miller, and B. S. Taylor, J. Chromatogr. 378, 450 (1986).
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If the decision has been made not tO use batch IEC, then the alternative is to use open-column IEC for the separation. The essential components of an open-column ion-exchange apparatus include the column, capped at both ends, and a reservoir for the eluants. Usually a pump is required to allow the eluents to be delivered at a constant rate; a detector, coupled to a recorder, to monitor the components in the eluent; and a fraction collector to allow for the recovery of the components. It is useful to have the fraction collector and the recorder coupled with an "event marker" such that each change of the collector is marked on the recorder. This will facilitate the correlation of detector events with the recovery of the components after separation. Optional accessories include a conductance cell to monitor " o n line" the concentration of salt in the eluent, and computerized management of data obtained from the various monitors including the detector and fraction collector for graphing purposes. In open-column IEC, the columns are usually transparent and capped at both ends. They can be obtained from a number of suppliers or made from glass or plastic tubing. Each end should have an inlet and outlet port to which tubing is attached. The column should be mounted in a vertical position and, prior to being filled with the slurry, a supporting bed should be placed inside the column against the bottom cap. This bed, the purpose of which is to prevent the resin or cellulose from running through the outlet port at the bottom, can be a plastic mesh, a sintered glass disk, or glass wool. In filling the column, a slurry of the packing is transferred from the beaker in which it had been prepared to the supporting bed. The column packing need not fill the column although it is best that the volume of buffer above the packing be kept small to minimize mixing. The column can be operated (that is, eluted) by pumping the buffer either from the top down or from the bottom up. When operating a column from the bottom to the top, there will be no "head" or layer of buffer between the cap of the column and the packing, minimizing the mixing and dilution of incoming buffers with those already present. Practical Information on IEC Operation
Preparation and Loading Sample. Perhaps one of the most important considerations in purification of proteins is the problem of their degradation, usually as a result of protease activities being brought into contact with them following the rupture of cells and their limiting membranes. Degradation can occur at any time, resulting in inactivation of enzymes or errors in characterization of the components recovered. Many precautions have been included in purification schemes to prevent degradation.
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These include working at low temperatures (4°) to slow proteolytic action and the addition to the "lysing" buffers of a mixture of protease inhibitors. Another problem in the preparation and loading of proteins for IEC is the ampholytic nature of proteins. Proteins contain both positive and negative charges, and for each protein a unique point has been defined, the isoelectric point, or pI, which is that pH at which the net charge on the protein is zero. Although an anion-exchange column would generally be used above the pL and a cation exchanger below, it is often found that proteins can bind at a variety of pH values because the magnitude of the charge in different regions of the protein may vary. Clearly, in cases where the amino acid composition is unknown, it will be necessary to try both anion and cation exchangers and various pH values to find the best conditions for separation. Also, since a change in pH can result in a change in the charge composition of the proteins, an altered elution profile can be obtained if the pH is not held constant during the elution. Thus, it is advisable not to work with the stationary phase in the H+ form and to use buffered salt solutions as eluents to maintain a constant pH during the eluti0n. Finally, loading of IEC columns is a simple matter because samples are introduced onto the column under conditions that promote binding. Loading an IEC column involves applying the sample onto the packing. Any volume can be applied as long as the total amount of protein does not exceed the binding capacity of the packing. In fact, IEC can be used for concentration of proteins. For example, proteins can be concentrated by adsorption to an anion-exchange column in low salt and eluted, in a concentrated form, with higher salt concentrations. Composition of Elution Solution. Elution of an IEC column requires a decision on the composition of the elution solution. The components of the elution solution include the buffer, the salt to be used for the displacement, and any components required for solubility and stability. The buffer and its pH should also be chosen on the basis of compatibility with the stability of the protein. Of course, the pH should be one that would allow binding. When dealing with an enzyme, the elution buffer should not result in loss of activity. Further, if the protein is to be located in the fractions on the basis of its activity, a buffer could be chosen which provides optimal conditions for the assay. This problem becomes acute with proteins that require detergents for solubilization, since at the concentrations used for solubilization some may interfere with the determination of activity. However, their removal often results in the precipitation of the protein. Therefore, a compromise often must be made by using
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sufficient detergent to prevent precipitation and sacrificing some loss of sensitivity in the assay. Some consideration of the purpose of the IEC step is necessary when choosing the displacement salt. For example, if IEC is only one step in a multistep purification scheme, then the choice of the buffer and the displacement salt should include some consideration of the subsequent steps in the scheme. For example, if the next step requires the sample from the IEC to be concentrated, then the use of a volatile salt such as ammonium carbonate is recommended. In such solvents, samples can be lyophilized or subjected to rotary evaporation to concentrate the protein without concentrating the salt. Elution Gradients. After the proteins have been adsorbed, their displacement requires the introduction of counterions into the eluent. The counterions, usually added in the form of a salt solution, can be introduced into the system in one of two ways: either discontinuously in a step gradient, or continuously with a linear gradient. Although elution of adsorbed proteins only begins following the introduction of salt, the process of elution of unadsorbed proteins will begin immediately after loading. Therefore, it is best to wash the column first with several volumes of the loading buffer. If the column effluent is monitored continuously during this washing step, it is possible to establish two points: (1) what fraction of the sample adsorbs to the column and (2) how much of the sample did not adhere and just "runs through" with the lowsalt loading buffer. Clearly, if none of the proteins is adsorbed, a change in the IEC conditions is in order. The displacement phase of the elution begins with the introduction of the salt. The salt solution should be introduced at a constant rate and a pump is best used for this purpose. The salt solution can be introduced onto the column from either the bottom up or from the top down. If a step elution is to be used the solution of salt of the next higher concentration in the step should now be introduced and should be maintained for at least two to three column volumes or until there is convincing evidence that this particular salt concentration has achieved equilibrium and has displaced all the protein that can be eluted at this particular concentration. At this point the solution of the next higher concentration can be introduced and the process repeated. The salt concentration is introduced in a stepwise manner until all the protein is eluted. The concentrations of salt in each step are usually determined by trial and error. If the salt is to be introduced as a gradient, two solutions are prepared, one of the low salt and the other of the high salt. With the aid of a mixing device, often two flasks connected by a siphon, the solution is introduced onto the column,
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beginning with the lowest concentration. By the end of the elution the solution of highest salt concentration will have been introduced. It should be noted that the resolution of the components is often affected by the magnitude and number of steps or the form of the gradient. With gradient elution the form of the gradient, steep vs shallow, should be varied to modify the resolution. For both types of elution it is advantageous to have some measure of the salt concentration in the effluent. This can be accomplished with a conductivity meter, which can be installed in line with the other detectors, or measurements of salt concentration can be obtained using a separate instrument that functions like a pH meter. The instrument has a probe which, after calibration, can be inserted into each of the fractions obtained during the column run. With the aid of a calibration curve, the readings obtained on the fractions can be converted to concentration, producing a profile of the salt concentration at each stage of the elution. Although IEC can be performed in the presence of detergents, it is best to use those that are uncharged and that do not have an absorption maximum at the wavelength used for monitoring the column (see below). For example, Triton X-100 absorbs in the ultraviolet (UV) range and will interfere with the detection of proteins at 280 nm. Detergents that do not interfere with UV detection include the type called zwitterions, sold under the name Z-314. However, with detergents, as with all solvents, it is best to test the solubility of the protein in the solvent prior to loading the column. Examples of the use of detergents with IEC have been presented elsewhere. 10 Of course, when working with proteins, particularly enzymes, in which activity must be maintained, denaturation must be avoided. This requirement often precludes the use of organic solvents as eluents. Graphical Presentation of Data. The elution of proteins from IEC columns can be conveniently followed by monitoring the eluent at 280 nm. This is because most proteins contain aromatic residues (such as tyrosine and tryptophan) which have an absorption maximum in this region. In the absence of such residues, as with the protein collagen, it is possible to monitor the effluent at 210-230 nm, the absorption region of the peptide linkage. The optical density of the effluent can be monitored on line using a spectrophotometer equipped with a flow cell or on samples from each of the fractions. In the latter case, one then plots the absorption vs the fraction number to display the elution profile of the column. The salt concentration monitored either with continuous read-out meto E. F. Rossomando, "High Performance Liquid Chromatography in Enzymatic Analysis." Wiley, New York, 1987.
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ters or by reading the salt concentration of the fractions is also included on such a plot. If enzyme activity measurements or a specific protein assay are performed, these also should be presented on the column profile. The combination of the specific assay and the protein profile gives a simple visual display of the approximate purification obtained by the column procedure. Acknowledgments I wish to thank Ms. Jane Hadjimichaelfor suggestionsconcerning the text and editorial assistance and Ms. Pamela Vachon for typing the manuscript.
[25] G e l F i l t r a t i o n
By EARLE STELLWAGEN Among the chromatographic techniques employed for protein purification, gel filtration is unique in that fractionation is based on the relative size of protein molecules. In contrast to conventional filtration, none of the proteins is retained by a gel filtration column. This feature is at once both the strength and weakness of gel filtration; a strength because the function of fragile proteins is not damaged by binding to a chromatographic support, and a weakness because the absence of such binding limits the resolution of the chromatography. Principle Gel filtration is performed using porous beads as the chromatographic support. A column constructed from such beads will have two measurable liquid volumes, the external volume, consisting of the liquid between the beads, and the internal volume, consisting of the liquid within the pores of the beads. Large molecules will equilibrate only with the external volume while small molecules will equilibrate with both the external and internal volumes. A mixture of proteins is applied in a discrete volume or zone at the top of a gel filtration column and allowed to percolate through the column. The large protein molecules are excluded from the internal volume and therefore emerge first from the column while the smaller protein molecules, which can access the internal volume, emerge later. The dimensions important to gel filtration are the diameter of the pores that access the internal volume and the hydrodynamic diameter of the METHODS IN ENZYMOLOGY, VOL. 182
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
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By EDWARD F. ROSSOMANDO This chapter introduces ion-exchange chromatography (IEC) as a method for the purification of proteins and enzymes. While this chapter discusses the general principles for the operation of all types of IEC, the focus will be on the type called open-column IEC, as distinguished from another widely used type, high-performance liquid IEC (HPLC). This latter topic will be discussed elsewhere in this volume (see [32]). When proteins are to be purified by IEC, some problems arise because proteins have unique origins. Proteins are derived from biological sources and their extraction often requires the use of detergents and chaotropic salts for solubilization. One problem is that such solubilizers interfere with the operation of IEC. Another problem is that proteins in cells are compartmentalized and therefore separated from other proteins with proteolytic activity. Extraction results in disruption of these barriers with the exposure of proteins to proteases and the potential for degradation during the course of the purification. A final problem related to source is that, since proteins must be extracted from biological materials, the amount of protein available may be limited, such as when working with embryonic tissue. This necessitates modification of the IEC protocols normally used when larger quantities are available. Problems will also arise because proteins have unique properties. For example, proteins are ampholytes; that is, they contain both positive and negative charges; the former result from the ionization of lysine and arginine residues and the latter from aspartic and glutamic acid residues. Since the ionization of such groups is pH dependent, the net charge on a protein will be a function of the pH of its environment. Also, proteins are often "sticky," adhering to surfaces such as glass or the packing material in the IEC column. Although organic solvents, detergents, and salts can eliminate some adsorption, their addition may precipitate the proteins or, in the case of enzymes, destroy catalytic activity. Some suggestions for dealing with these problems will be discussed in this chapter. IEC is designed specifically for the separation of ionic or ionizable compounds. Similar to other types of liquid chromatography, IEC has both stationary (column packing) and mobile phases. It differs from other types of liquid chromatography in that the stationary phase carries ionizable functional groups, fixed by chemical bonding to the stationary phase. Of course, to satisfy requirements for electrical neutrality, these fixed METHODS IN ENZYMOLOGY, VOL. 182
Copyright 6) 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
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charges will carry a counterion of opposite sign. This counterion is not fixed and can be displaced. IEC is named on the basis of the sign of these displaceable charges. Thus, in anion IEC the fixed charges are positive and in cation IEC the fixed charges are negative. Principles of Operation IEC involves two separate events. These are (1) the binding of the protein to the fixed charges and (2) the elution or displacement of the protein from the fixed charges. Because retention involves an electrostatic interaction between the fixed charges and those of the protein, binding involves replacement of the nonfixed ions by the protein. Elution, in turn, involves displacement of the protein from the fixed charges by a new counterion with a greater affinity for the fixed charges than the protein, and which then becomes the new, nonfixed ion. The ability of counterions (salts) to displace proteins bound to fixed charges is a function of the difference in affinities between the fixed charges and the nonfixed charges of both the protein and the salt. Affinities in turn are affected by several variables, including the magnitude of the net charge of the protein and the concentration and type of salt used for displacement. For additional details on the mechanisms underlying these processes see Refs. 1-3. Types of Ion Exchangers Widely used solid-phase packings include cellulose, dextrans, agarose, and polystyrene. The exchange groups used include DEAE (diethylaminoethyl), a weak base, that will have a net positive charge when ionized and will therefore bind and exchange anions; and CM (carboxymethyl), a weak acid, with a negative charge when ionized that will bind and exchange cations. Another form of weak anion exchanger contains the PEI (polyethyleneimine) functional group. This material, most usually found on thin layer sheets, is useful for binding proteins at pH values above their pI. The polystyrene matrix can be obtained with quaternary ammonium G. V. Samsonov, "Ion Exchange Sorption and Preparative Chromatography of Biologically Active Molecules," pp. 99, 105. Consultants Bureau, New York, 1986. 2 C. F. Poole and S. S. Schuette, "Contemporary Practice of Chromatography." Elsevier, Amsterdam. 1984 pp. 304-312. 3 p. j. Schoenmakers, "Optimization of Chromatographic Selectivity." Elsevier, Amsterdam, 1986.
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functional groups for strong base anion exchange or with sulfonic acid functional groups for strong acid cation exchange. Intermediate and weak ion-exchange materials are also available. Studies comparing the effectiveness of exchangers of various types have been reported. For example, in one study the effectiveness of a polystyrene resin was compared to cellulose for the purification of a glucoamylase from crude extracts of Aspergillus awamori. 4 Both an anionexchange polystyrene resin (Bio-Rad AG1-X4) and DEAE-cellulose were used. The results indicated that the use of the polystyrene resin solved one problem, namely, adsorption of the protein to cellulose. In addition, the resin produced a better separation of this enzyme from another, an amylase. Recently a newer form of anion-exchange system consisting of stacks of thin, noncompressible, microporous poly(vinyl chloride)-silica sheets has been introduced (FASTCHROM, Kontes, Life Sciences Products, Vineland, NJ). When these sheets are coated with PEI they yield a positively charged, hydrophilic surface for the separation of proteins and DNA. 5 Preparation and Regeneration of Packing The column packings require washing, swelling, and conversion to the desired " f o r m " prior to use. All these procedures can be performed prior to pouring the column. Washing and Swelling. To allow the column packing to reach a size equilibrium, prepare a slurry by mixing the packing with about 10 times its volume of the buffer to be used for loading the column. The slurry should be allowed to settle for about 1 hr. The top layer of clear solution, containing the "fines," should be decanted and the washing step repeated at least once. This treatment will result in better flow rates for the column. Changing Displaceable Counterion. When obtained from the manufacturer, the fixed charges of the column packing will have associated with them a counterion. This counterion can be changed, a procedure that also should be performed prior to pouring the column. 6 This exchange can be accomplished by washing the packing in a salt solution containing the counterion of choice. However, all the packing materials have a "selectivity series" that might be likened to an affinity, and in making the displacement it is necessary to go " u p " the selectivity series. For example, the selectivity for a representative cation exchanger might be Rb + > 4 R. S. Bhella and I. Altosaar, Anal. Biochem. 140, 200 (1984). 5j. j. Piotrowskiand M. H. Scholla, BioChromatography 3, 161 (1988). 6 E. L. Johnsonand R. Stevenson, "Basic LiquidChromatography,"p. 116. Varian Associates, Palo Alto, California, 1977.
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Cs + > K + > N H 4 + > Na ÷ > H ÷ > Li +. Such packings are usually supplied in the H ÷ form. Thus, it is a simple matter to replace the H ÷ with an ion higher in the series by washing in a 1 M solution of that salt. Similar selectivity series exist for anion exchangers, and the manufacturer's literature should be consulted for this information. Size of Column: Bed Capacity. Although the length o f the column is less important, the separation does depend on the amount of column packing available since if this is insufficient, material that should have been retained will flow through. Although the amount of material that the column can bind must eventually be determined by experiment, information provided by the manufacturer on the bed capacity can narrow the quest. The manufacturer provides this information in the form of miUiequivalents per dry gram or milliequivalents per milliliter of resin bed. For cation exchangers, supplied in the hydrogen form, this would be milliequivalents of H ÷ that can be exchanged while for anion exchangers supplied in the chloride form this would be milliequivalents of CI-. The columns used for IEC need not be large. For example, columns made from Pasteur pipets have been used for the purification of the peptides derived from proenkephalin. 7 Used for this purpose, the pipet should be plugged with a small amount of glass wool, the resin prepared, and introduced into the pipet. The volume of the resin in such a column would be on the order of 0.25 cm 3. For such columns elution will be a simple matter of washing the column with about 1 ml of each of the eluents. Batch os Open Column. IEC need not be performed using a column. 8 The alternative, batch IEC, is usually performed with the slurry of the stationary phase in a vessel such as a beaker. In one study, used for the separation of lactate dehydrogenase, 9 the essential component of the batch system is the resin (Bio-Rad AG MP-1), which is added to a tube containing the enzyme (isozymal forms of human lactate dehydrogenase). After shaking and equilibration, the resin is separated from the buffer either by centrifugation, filtration, or a combination of the two. The authors note that the separation they obtained was less than desirable, which they attribute to the fact that the batch procedure is both a nonequilibrium and too rapid a process. However, sometimes the speed of the process can be an advantage such as when separations on a larger scale are required. 7 S. P. Wilson, J. Neurosci. Methods 15, 155 (1985). 8 D. Reichenberg, in "Ion Exchangers in Organic and Biochemistry" (C. Calmon and T. R. E. Kressman, eds.). Interscience, New York, 1957. 9 M. P. Menon, S. Miller, and B. S. Taylor, J. Chromatogr. 378, 450 (1986).
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If the decision has been made not tO use batch IEC, then the alternative is to use open-column IEC for the separation. The essential components of an open-column ion-exchange apparatus include the column, capped at both ends, and a reservoir for the eluants. Usually a pump is required to allow the eluents to be delivered at a constant rate; a detector, coupled to a recorder, to monitor the components in the eluent; and a fraction collector to allow for the recovery of the components. It is useful to have the fraction collector and the recorder coupled with an "event marker" such that each change of the collector is marked on the recorder. This will facilitate the correlation of detector events with the recovery of the components after separation. Optional accessories include a conductance cell to monitor " o n line" the concentration of salt in the eluent, and computerized management of data obtained from the various monitors including the detector and fraction collector for graphing purposes. In open-column IEC, the columns are usually transparent and capped at both ends. They can be obtained from a number of suppliers or made from glass or plastic tubing. Each end should have an inlet and outlet port to which tubing is attached. The column should be mounted in a vertical position and, prior to being filled with the slurry, a supporting bed should be placed inside the column against the bottom cap. This bed, the purpose of which is to prevent the resin or cellulose from running through the outlet port at the bottom, can be a plastic mesh, a sintered glass disk, or glass wool. In filling the column, a slurry of the packing is transferred from the beaker in which it had been prepared to the supporting bed. The column packing need not fill the column although it is best that the volume of buffer above the packing be kept small to minimize mixing. The column can be operated (that is, eluted) by pumping the buffer either from the top down or from the bottom up. When operating a column from the bottom to the top, there will be no "head" or layer of buffer between the cap of the column and the packing, minimizing the mixing and dilution of incoming buffers with those already present. Practical Information on IEC Operation
Preparation and Loading Sample. Perhaps one of the most important considerations in purification of proteins is the problem of their degradation, usually as a result of protease activities being brought into contact with them following the rupture of cells and their limiting membranes. Degradation can occur at any time, resulting in inactivation of enzymes or errors in characterization of the components recovered. Many precautions have been included in purification schemes to prevent degradation.
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These include working at low temperatures (4°) to slow proteolytic action and the addition to the "lysing" buffers of a mixture of protease inhibitors. Another problem in the preparation and loading of proteins for IEC is the ampholytic nature of proteins. Proteins contain both positive and negative charges, and for each protein a unique point has been defined, the isoelectric point, or pI, which is that pH at which the net charge on the protein is zero. Although an anion-exchange column would generally be used above the pL and a cation exchanger below, it is often found that proteins can bind at a variety of pH values because the magnitude of the charge in different regions of the protein may vary. Clearly, in cases where the amino acid composition is unknown, it will be necessary to try both anion and cation exchangers and various pH values to find the best conditions for separation. Also, since a change in pH can result in a change in the charge composition of the proteins, an altered elution profile can be obtained if the pH is not held constant during the elution. Thus, it is advisable not to work with the stationary phase in the H+ form and to use buffered salt solutions as eluents to maintain a constant pH during the eluti0n. Finally, loading of IEC columns is a simple matter because samples are introduced onto the column under conditions that promote binding. Loading an IEC column involves applying the sample onto the packing. Any volume can be applied as long as the total amount of protein does not exceed the binding capacity of the packing. In fact, IEC can be used for concentration of proteins. For example, proteins can be concentrated by adsorption to an anion-exchange column in low salt and eluted, in a concentrated form, with higher salt concentrations. Composition of Elution Solution. Elution of an IEC column requires a decision on the composition of the elution solution. The components of the elution solution include the buffer, the salt to be used for the displacement, and any components required for solubility and stability. The buffer and its pH should also be chosen on the basis of compatibility with the stability of the protein. Of course, the pH should be one that would allow binding. When dealing with an enzyme, the elution buffer should not result in loss of activity. Further, if the protein is to be located in the fractions on the basis of its activity, a buffer could be chosen which provides optimal conditions for the assay. This problem becomes acute with proteins that require detergents for solubilization, since at the concentrations used for solubilization some may interfere with the determination of activity. However, their removal often results in the precipitation of the protein. Therefore, a compromise often must be made by using
[24]
ION-EXCHANGECHROMATOGRAPHY
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sufficient detergent to prevent precipitation and sacrificing some loss of sensitivity in the assay. Some consideration of the purpose of the IEC step is necessary when choosing the displacement salt. For example, if IEC is only one step in a multistep purification scheme, then the choice of the buffer and the displacement salt should include some consideration of the subsequent steps in the scheme. For example, if the next step requires the sample from the IEC to be concentrated, then the use of a volatile salt such as ammonium carbonate is recommended. In such solvents, samples can be lyophilized or subjected to rotary evaporation to concentrate the protein without concentrating the salt. Elution Gradients. After the proteins have been adsorbed, their displacement requires the introduction of counterions into the eluent. The counterions, usually added in the form of a salt solution, can be introduced into the system in one of two ways: either discontinuously in a step gradient, or continuously with a linear gradient. Although elution of adsorbed proteins only begins following the introduction of salt, the process of elution of unadsorbed proteins will begin immediately after loading. Therefore, it is best to wash the column first with several volumes of the loading buffer. If the column effluent is monitored continuously during this washing step, it is possible to establish two points: (1) what fraction of the sample adsorbs to the column and (2) how much of the sample did not adhere and just "runs through" with the lowsalt loading buffer. Clearly, if none of the proteins is adsorbed, a change in the IEC conditions is in order. The displacement phase of the elution begins with the introduction of the salt. The salt solution should be introduced at a constant rate and a pump is best used for this purpose. The salt solution can be introduced onto the column from either the bottom up or from the top down. If a step elution is to be used the solution of salt of the next higher concentration in the step should now be introduced and should be maintained for at least two to three column volumes or until there is convincing evidence that this particular salt concentration has achieved equilibrium and has displaced all the protein that can be eluted at this particular concentration. At this point the solution of the next higher concentration can be introduced and the process repeated. The salt concentration is introduced in a stepwise manner until all the protein is eluted. The concentrations of salt in each step are usually determined by trial and error. If the salt is to be introduced as a gradient, two solutions are prepared, one of the low salt and the other of the high salt. With the aid of a mixing device, often two flasks connected by a siphon, the solution is introduced onto the column,
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PURIFICATION PROCEDURES" CHROMATOGRAPHIC METHODS
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beginning with the lowest concentration. By the end of the elution the solution of highest salt concentration will have been introduced. It should be noted that the resolution of the components is often affected by the magnitude and number of steps or the form of the gradient. With gradient elution the form of the gradient, steep vs shallow, should be varied to modify the resolution. For both types of elution it is advantageous to have some measure of the salt concentration in the effluent. This can be accomplished with a conductivity meter, which can be installed in line with the other detectors, or measurements of salt concentration can be obtained using a separate instrument that functions like a pH meter. The instrument has a probe which, after calibration, can be inserted into each of the fractions obtained during the column run. With the aid of a calibration curve, the readings obtained on the fractions can be converted to concentration, producing a profile of the salt concentration at each stage of the elution. Although IEC can be performed in the presence of detergents, it is best to use those that are uncharged and that do not have an absorption maximum at the wavelength used for monitoring the column (see below). For example, Triton X-100 absorbs in the ultraviolet (UV) range and will interfere with the detection of proteins at 280 nm. Detergents that do not interfere with UV detection include the type called zwitterions, sold under the name Z-314. However, with detergents, as with all solvents, it is best to test the solubility of the protein in the solvent prior to loading the column. Examples of the use of detergents with IEC have been presented elsewhere. 10 Of course, when working with proteins, particularly enzymes, in which activity must be maintained, denaturation must be avoided. This requirement often precludes the use of organic solvents as eluents. Graphical Presentation of Data. The elution of proteins from IEC columns can be conveniently followed by monitoring the eluent at 280 nm. This is because most proteins contain aromatic residues (such as tyrosine and tryptophan) which have an absorption maximum in this region. In the absence of such residues, as with the protein collagen, it is possible to monitor the effluent at 210-230 nm, the absorption region of the peptide linkage. The optical density of the effluent can be monitored on line using a spectrophotometer equipped with a flow cell or on samples from each of the fractions. In the latter case, one then plots the absorption vs the fraction number to display the elution profile of the column. The salt concentration monitored either with continuous read-out meto E. F. Rossomando, "High Performance Liquid Chromatography in Enzymatic Analysis." Wiley, New York, 1987.
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GEL FILTRATION
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ters or by reading the salt concentration of the fractions is also included on such a plot. If enzyme activity measurements or a specific protein assay are performed, these also should be presented on the column profile. The combination of the specific assay and the protein profile gives a simple visual display of the approximate purification obtained by the column procedure. Acknowledgments I wish to thank Ms. Jane Hadjimichaelfor suggestionsconcerning the text and editorial assistance and Ms. Pamela Vachon for typing the manuscript.
[25] G e l F i l t r a t i o n
By EARLE STELLWAGEN Among the chromatographic techniques employed for protein purification, gel filtration is unique in that fractionation is based on the relative size of protein molecules. In contrast to conventional filtration, none of the proteins is retained by a gel filtration column. This feature is at once both the strength and weakness of gel filtration; a strength because the function of fragile proteins is not damaged by binding to a chromatographic support, and a weakness because the absence of such binding limits the resolution of the chromatography. Principle Gel filtration is performed using porous beads as the chromatographic support. A column constructed from such beads will have two measurable liquid volumes, the external volume, consisting of the liquid between the beads, and the internal volume, consisting of the liquid within the pores of the beads. Large molecules will equilibrate only with the external volume while small molecules will equilibrate with both the external and internal volumes. A mixture of proteins is applied in a discrete volume or zone at the top of a gel filtration column and allowed to percolate through the column. The large protein molecules are excluded from the internal volume and therefore emerge first from the column while the smaller protein molecules, which can access the internal volume, emerge later. The dimensions important to gel filtration are the diameter of the pores that access the internal volume and the hydrodynamic diameter of the METHODS IN ENZYMOLOGY, VOL. 182
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