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to the design and synthesis of new reactive dyes outside of the commercial dye industry. It was anticipated that the blue dye could better occupy the coenzyme site if the anthraquinone and the triazine rings were further separated by insertion of an ethylene bridge as shown in Fig. le, This immobilized dye, 24 in contrast to all other immobilized dyes and biomolecules, is able to resolve the purified alcohol dehydrogenase into two components of different activity, with the low activity form having a covalent modification on a lysine side chain. It is anticipated that additional designed dyes will increase the capability of immobilized dyes in protein purification. The chemistry employed in the synthesis of reactive dyes which is necessary for preparation of designed dyes is described in detail in two texts. 25,26 Finally, it should be noted that the solid fluorocarbons developed by du Pont de Nemours & Co. (Wilmington, DE) afford a promising new matrix for immobilized dye chromatography. A reactive dye is first subjected to a perfluoralkylation and then the perfluoroalkylated dye is essentially irreversibly adsorbed to a fluorocarbon surface. 27 Accordingly, this matrix should prevent slow bleeding of dye into proteins purified by immobilized dye chromatography. Most aspects of immobilized reactive dye-protein interaction have been reviewed by several authors 1.2,7,8,18and the concerned investigator is urged to pursue them for access to more detailed information. 24 C. R. Lowe, S. J. Burton, J. C. Pearson, and Y. D. Clonis, J. Chromatogr. 376, 121 (1986). 22 W. F. Beech, "Fibre-Reactive Dyes." Logos Press, London, 1970. ~6 K. Venkataraman, "The Chemistry of Synthetic Dyes," Vol. 6, Academic Press, New York, 1972. 27 j. V. Eveleigh, Abstr. Biotechnol. Microsymp. Macromol. Interact. Affinity Chromatogr. Technol., Mogilany, Pol. (1988).
[29] A f f i n i t y C h r o m a t o g r a p h y : G e n e r a l M e t h o d s
By STEVEN OSTROVE Affinity chromatography is one of the most powerful procedures that can be applied to protein purification. Over the years there have been many good books on the subject and many reviews of the theory of affinity chromatography.l,2 This chapter will not be a further review of the I j. Turkova, ed., "Affinity Chromatography." Elsevier, New York, 1978. 2 p. Mohr and K. Pommerening, "Affinity Chromatography: Practical and Theoretical Aspects." Dekker, New York, 1985.
METHODS IN ENZYMOLOGY,VOL. 182
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theory behind this technique nor will it review all of the proteins that have been separated by this technique since there are so many. Discussion will center on practical ideas and general considerations behind choosing a matrix, coupling a ligand, and some of the more common problems often encountered with the implementation of this procedure. Suggestions in this chapter should allow for the easier and more productive use of affinity chromatography. As the specific purification system for the sample of interest is developed, expect modification of these procedures. This chapter serves only as a guide to performing separations of biomolecules by affinity chromatography. Areas where the manufacturer of the affinity product can be contacted for specific recommendations on the use of the product are indicated throughout. Refer specific questions to the manufacturer. Affinity chromatography as a biospecific technique began only about 20 years ago even though it had been used as an experimental separation procedure for many years. 3 This procedure takes advantage of one or more biological properties of the molecule(s) being purified. These interactions are not due to the general properties of the molecule such as isoelectric point (pl), hydrophobicity, or size. This highly specific method of separation utilizes the specific reversible interactions between biomolecules. Some of the biological properties that can be exploited to effect a separation include specific shapes (that "recognize" other molecules such as receptors or enzymes), specific changes in conformation after changes in pH, or certain subareas or regions of the molecule that can interact or bind to other molecules (e.g., epitopes of antibodies). When developing a separation scheme keep in mind that the sample of interest is not the only component in the sample mixture that can be bound to an affinity matrix (gel). One affinity matrix may be specific for the sample of interest while others may be more specific for other components in the mixture (contaminating proteins). Just as the sample of interest can be bound to an affinity matrix, the contaminating proteins may also be specifically bound. An affinity gel could be chosen to bind the contaminating proteins, allowing the sample of interest to pass through the gel in the wash volume. This method of separation could result in a great saving of time. Matrix Choice of the proper matrix is a very important step in any chromatographic process. A good matrix for affinity chromatography should have the following properties: 3 p. Cuatrecasas and M. Wilchek, Biochem. Biophys. Res. Commun. 33, 235 (1968).
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1. Hydrophilic: Reduce the nonspecific interactions. 2. Large pores: Allow all areas of the matrix to be available to most of the molecules in the mixture. Some matrices allow binding only to the outer surface. This latter type of matrix is useful in separating very large molecules, cells, or viruses. 3. Rigid: The matrix must withstand the pressures of packing and solvent flow during elution or washing. 4. Inert: The matrix should not contribute to the separation. 5. Chemical stability: The matrix must be stable to all solvents used in the separation. Base the choice of an affinity gel on both the ligand and the sample. There are two major types of affinity gels: group-specific gels and covalent coupling gels. The former are usually supplied ready to use. Table I provides examples of ligands that are group specific in action and can be used to isolate whole families of biomolecules which share common properties. Covalent coupling gels (Table II) require more chemistry and some specific considerations. First, consider the length and type of the spacer arm; second, the coupling chemistry.
TABLE I LIGAND SPECIFICITY
Ligand NAD, NADP
Lectins Poly(U) Poly(A)
Histones Protein A Protein G
Specificity Dehydrogenases Polysaccharides Poly(A) Poly(U) DNA Fc antibody
Antibodies
Lysine
rRNA, dsDNA, plasminogen
Arginine Heparin Blue F3G-A Red HE-3B Orange A Benzamidine Green A Gelatin Polymyxin
Fibronectin, prothrombin Lipoproteins, DNA, RNA
2' ,5'-ADP
NADP ÷
Calmodulin Boronate Blue B
cis-Diols, tRNA, plasminogen
NAD ÷ NADP ÷
Lactate dehydrogenase Serine proteases CoA proteins, HSA, dehydrogenases Fibronectin Endotoxins Kinases Kinases, dehydrogenases, nucleic acid-binding proteins
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TABLE II COUPLING CHEMISTRY
Linkage
Ligand group
CNBr Thiolpropyl
NH2 SH
Thio Epoxy
SH NH2 OH SH NH2 COOH NH2
Tresyl Aminohexyl Carboxyihexyl
Spacer length
Equivalentto about 13 carbons Equivalentto about 11 carbons 6 6
Active pH
Specificity
8-10 9-11
Proteins,peptides Sulfhydryls
9-13 9 10 11 8-10
Sulfhydryls Proteins,peptides Carbohydrates Sulfhydryls Proteins,peptides Amino acids, proteins Carboxylic acids
Solvents The solvent system chosen for the entire affinity chromatography separation is also a critical factor to a good separation. The solvent should not degrade the sample. Unfortunately, avoiding denaturing solvents is not always possible. For example, separation of an antibody (IgG)-antigen complex requires some very harsh conditions. Dissociation at a low pH or use of a strong chaotropic agent are the most commonly used methods. Minimizing the time of contact with these agents is vitally important. One method used to reduce the contact time with harsh reagents (e.g., low pH) is to add Tris base (dry) to the collecting tubes. This will rapidly increase the pH and help to protect the sample. Try to choose an elution buffer specific for the sample (e.g., a buffer containing an analog to the sample). The elution buffer should release the sample safely and rapidly. Again, the buffer should not denature the sample, nor cause any change in its specific activity or function. Optimization of sample binding and elution conditions is usually by trial and error. When choosing a buffer system try to avoid using one that has a pKa at or near the pI of the sample. This will help prevent precipitation of the sample on the column. However, when starting a separation read the literature, as it will often provide a good starting point. Even a related separation can serve as a starting point for selecting the separation conditions. Spacer Arms Choosing a gel with or without a spacer arm depends on the ligand, the sample, and the binding chemistry. A spacer arm is used to keep the
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TABLE III SPACER LENGTH CONSIDERATIONS
Ligand
Protein
Best spacer arm
Small Small Large Large
Small Large Small Large
Short Long None None
ligand away from the matrix so that the active site of the ligand is available to the sample. This is especially important with small ligands. As a general rule if the ligand is large and the sample is small (low molecular weight) this spacing may be unnecessary. With samples of high molecular weight a spacer arm can be used to limit steric hindrance and increase the availability of the active site (Table III). A wide variety of spacer arm lengths are available. If unsure of the required spacer arm length, start with one that is equivalent to about six carbon atoms. This seems to be a good length for many affinity applications. 4 Shorter arms give less flexibility so the ligand will not " w a v e " around in the medium. Predicate the spacer arm length on the amount of steric hindrance deemed tolerable. As spacers are evaluated, remember that the spacer molecule itself can cause steric hindrance by blocking adjacent active sites on the gel; thus, longer is not always better. Gel Preparation After choosing the affinity gel type, prepare the resin (gel) for use. The manufacturer will usually supply the instructions needed to prepare the gel correctly. However, short of those instructions, following these general guidelines will help ensure a successful preparation. First, calculate the amount of gel that needs to be packed into a column (or flask for batch work) by the capacity of the gel for the sample. That is, x units of gel bind y units of sample. Next, calculate the volume: (total sample/sample units) × gel volume per sample unit. This value should be multiplied by a factor of 2-3 and this factored amount of gel should be used. For example, if I ml of protein A-Sepharose binds 20 mg IgG and you are using 40 mg of sample (with contaminants), divide the total (40 mg) by the gel capacity (20 rag) and multiply by the gel volume 4 C. R. Lowe, Biochem. J. 133, 499 (1973).
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(1 ml). Multiply this figure by 2-3, giving a final gel volume of 4-6 ml, for best results. If the gel is supplied in a preswollen state, reconstituting the gel is unnecessary to obtain the full swollen volume. A wash is all that is usually required. The swollen gel is typically supplied in glycerin or similar material which is used to help in the gel preparation and to stabilize the ligand or activated coupling complex. Wash on a sintered glass filter of medium grade (#3) or on a membrane-type filter that has a low protein-binding capacity. This allows easy removal of the washed gel with a minimum of loss due to sticking to the filter. A wash ratio of about 200 : 1 (buffer to gel) works well. For the safest wash buffer use either distilled water or the starting buffer (unless otherwise directed). By definition the starting buffer is that buffer used to initially prepare the matrix for the addition of the sample. It creates an environment on the gel so that the sample will bind specifically to the attached ligand. If the gel needs to be swollen to regain full working volume, then use a swelling buffer prior to washing. This buffer is often a low concentration phosphate buffer (0.1 M) at or near neutral pH. Swelling times vary between 15 min and 1 hr. After swelling, wash the gel either in the buffer solution used for swelling, distilled water, or starting buffer. Since washing and swelling buffers are generally pulled through the gel under a low vacuum, it is critical that the gel does not become dry at this stage. Following the reswelling and the wash, the ligand can be bound to the gel or loaded into a column if no ligand is to be added (i.e., groupspecific gels). Coupling or Linkage Chemistry Before using a covalent coupling gel, the ligand-binding (linkage) chemistry needs to be decided. There are a variety of linking groups available, such as cyanogen bromide (CNBr), tresyl, epoxy, and triazine. The linkage chemistry may be available either in an activated or nonactivated form. Activated means " r e a d y " to use without additional chemical activation steps (washing is still necessary). The nonactivated gels require some additional chemical activation step, such as carbodiimide treatment, to prepare them for binding the ligand. Leaching, or loss of ligand, after binding is inevitable. The trick is to minimize this loss. CNBr-type linkages commonly leach more than do tresyl or epoxy linkages. Other types of ligand linkages are also possible by using C~---C, C ~ O , and other available bonds. Nucleic acids and sugars can also be bound through their amine or hydroxyl groups. Table II lists some examples of
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the active groups that can be used to link ligands to the matrix, and the types of sample that can bind to these ligands. Coupling the Ligand Next, couple the ligand to the matrix, assuming that you have a specific ligand that needs to be attached. Consider the steric effects of the ligand, the spacer arm, and the sample. High concentrations of small ligands can block some active sites on the matrix, causing a lower binding efficiency. Large ligands can also block adjacent sites, again resulting in lower binding efficiency. Spacer arms and ligands can also cause some blockage of adjacent sites as the ligand " w a v e s " back and forth. As a general rule use about 10 mg ligand/ml of gel. This amount applies also to proteins having an average size of (50 kDa). A lower amount should be applied for larger molecules such as IgG (5 mg/ml), or IgM (1 mg/ml), or molecules with low dissociation constant (KD) values. Mix the ligand and the matrix together using a rotating motion. Avoid magnetic stirrers at all times since they can damage the matrix. The volume ratio of binding buffer (with ligand) to gel matrix should be about 2:1 for best results. Carefully control the pH, ionic strength, and ion content during this stage of coupling. Coupling times of 2-4 hr at room temperature or up to 16 hr in the cold (4°) are commonly used. The choice of time and temperature is determined by the stability of the ligand and the amount of time available. The time available is important since there should be no processing interruptions from the time the gel is activated until the excess ligand is washed out. This minimizes loss of coupling activity. Once again users should consult the manufacturer's instructions for the optimum coupling conditions. Coupling of the ligand to the matrix can be by a single point or multipoint attachment. An example of single-point attachment is the binding of a single primary amine via CNBr coupling. This type of linkage offers the best flexibility to the ligand and thus the most accessibility of the active site to the sample. Single-point attachment is possible only if secondary and tertiary amines are blocked. Multipoint attachments are stronger than single-point attachments and are less likely to leach during the run. However, this type of coupling often causes blockage of the active site of the ligand. Blocking Unreacted Groups After incubation of the ligand with the matrix, remove the excess ligand and block the unreacted sites on the matrix. When coupling a
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ligand to the matrix some sites on the matrix remain unreacted. These unreacted sites are potential sites for nonspecific interactions with the sample or contaminants in the solution. Blocking these sites is most easily accomplished with reagents that have an opposite charge or can be covalently linked to the matrix. For example, when a carboxyl group (COO-) is used to couple a ligand, such as an amino group (NH2), use a Tris or ethanolamine solution (0.1-I.0 M) as the blocking agent. When NH2 groups are the coupling sites for ligand containing COO-, acetic acid can be used as the blocking agent. The concentration of the blocking agent should be in excess of the total reactive site concentration on the matrix. This assures complete blockage of all unreacted sites. Normally, a 5- to 10-fold excess over the ligand concentration is sufficient. Control of the pH of the blocking agent is another critical factor important to good affinity separations. A pH that is either too high or too low may prevent complete blocking or even destroy the matrix or the bound ligand. The blocking reaction is usually done at room temperature for 2-4 hr, but can also be done in the cold (4°) for longer periods of time. Wash out the excess blocking agent and equilibrate the column with 5 to 10 column volumes of starting buffer before sample application. The coupling process can be summarized as follows: 1. 2. 3. 4. 5. 6. 7.
Swell the matrix in swelling buffer (15-60 min). Wash the matrix (200: 1, buffer:gel). Add ligand and incubate with mixing (2-4 hr, 2 : 1 buffer : gel). Wash out unused ligand and buffer (200: 1, buffer:gel). Block unreacted sites on the matrix (2-4 hr, room temperature). Wash the matrix (200: 1, buffer:gel). Use or store the gel under appropriate conditions (4-8°).
Monitoring Coupling Efficiency The extent of ligand coupling determines both the efficiency of the separation and the amount of purified sample that can be prepared. The amount of ligand bound can be determined in several ways: 1. Measure the difference in UV absorption before and after coupling: a. A2s0 is best for proteins; however, other wavelengths specific for other ligands which can be coupled should be chosen. For example, A4o5 for heme groups can be used. This technique is best accomplished when the concentration of ligand is not very high, since a small amount of ligand binding will not be detected. In
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addition, avoid buffer components that absorb at this wavelength [e.g., dithiothreitol (DTT)], or high concentrations of Tris buffer. b. Perform a colorimetric protein assay (see [6]). c. Perform a fluorescence detection assay (more sensitive than the colorimetric assays). Dissolve a portion of the gel containing the ligand and do a protein assay, amino acid analysis, or assay for total nitrogen. 3. Perform an activity test (a small binding experiment). 4. Perform a radiolabeled ligand or RIA test (assumes that the radiolabeled ligand binds at the same rate, and to the same extent, as nonradiolabeled ligand). .
Binding the Sample The binding of proteins to a ligand, through the carboxyl or the amino groups available, is based on the specific affinity of the protein for a particular ligand. As indicated previously, the ligand should not be coupled in such a way as to block or interfere with the availability of the active site on either the ligand or the protein. Binding between the ligand and the protein is generally noncovalent. Although the binding is specific, the forces involved are general chemical interactions, such as hydrogen bonds. The buffer conditions that are used to load the protein on to the column are often phosphate or Tris buffers (0.1-0.2 M) containing salts such as sodium chloride (0.5 M). The choice of buffer and concentration is predicated on minimizing nonspecific interactions and maximization of the specific attraction between the ligand and the protein. Load the sample in the normal downward direction such that it will bind to the upper half to upper third of the matrix. Flow Rates Different flow rates are used in the various stages of every affinity chromatography run: (1) the loading of the sample, (2) the wash step to remove nonspecifically bound material, (3) the elution phase, where the protein of interest is removed from the gel, and (4) the regeneration of the matrix for the next run. The flow rate commonly used for loading of the sample is often about 10 cm/hr. The notation cm/hr refers to linear flow rate of the buffer. To calculate the volume flow rate, which is the rate that is used for the run, multiply the linear flow rate by the cross-sectional area of the column. The flow rate used is dependent upon the kinetics of the binding of the
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desired protein to the ligand. Factors such as temperature, concentration, and the KD influence the interaction of the protein and ligand. If the protein has a high affinity for the ligand (KD < 10-6), the flow can be faster since the protein still can bind effectively. On the other hand, if the affinity is low, use a much slower flow rate. During the wash step the flow rate can be considerably faster since the wash serves to remove nonspecifically adhering material (assumed for this discussion not to be of interest). The flow rate at this stage can be increased to about 20 to 50 cm/hr in order to effect a rapid cleaning of the matrix. However, if the protein of interest is loosely bound to the ligand, a lower flow rate is better. Perhaps most important is the flow rate during the elution phase. This flow could also be faster then the loading rate. Elution flow rates depend on the strength of the elution buffer as related to the affinity of the sample. The goal is to use a buffer that will easily strip the desired protein from the ligand without damaging it, the ligand, or the matrix. The elution flow rate can be as high as the wash flow, but is always lower than the flow rate used for packing the matrix. The flow rate during reequilibration can also be very rapid. At this point, only the coupled ligand should be left on the matrix. Therefore, flow rates up to the packing rate can be used to save time. Flow rates should not exceed about 80% of the flow rate used to pack the resin. This avoids compression during the chromatography run. Another factor that determines the maximum flow rate is the stability of the matrix. In order to avoid gel compression and deformation of the beads do not exceed the maximum flow rate recommended for the matrix. Also, try to avoid turbulence and high shear rates due to rapid buffer flow in the matrix when loading or eluting. The manufacturer can generally provide information on the best flow rates for all steps. Nonspecific Interactions Nonspecific interactions, if a problem, can usually be avoided by using a salt concentration between 0.1 to 0.5 M since in this range nonspecific ionic attractions are greatly reduced. These salt concentrations are usually not so high as to make hydrophobic interactions between the protein and the matrix or ligand a problem. As always, the manufacturer of the matrix usually can supply the specific information needed to prevent nonspecific interactions. Other methods that may be used to decrease nonspecific interactions include the addition of agents such as glycerol up to a concentration of about 10% (no higher due to increased viscosity of the buffer, resulting in
[29]
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higher back pressures and lower flow rates). Low levels of detergent (below the critical micelle concentration) are also useful in reducing nonspecific interactions. Detergents, however, can interfere with both ligand and protein binding, and not every ligand can be safely used with all detergents. For example, a ligand may dissociate or denature in the presence of detergents and some may interact with the active site of the ligand, lowering its affinity for the protein. Specific vs. Nonspecific Elution Specific elution of the protein of interest is always the best method to use in affinity chromatography (see Table IV). This type of elution is the result of a competitive action of the eluent for the ligand. An eluent is chosen that has a greater affinity for the ligand than the protein so that it will displace the protein from the ligand. The eluent can then be removed by a more stringent cleaning of the matrix. An example of a specific eluent is the use of c~-methylglucoside to elute samples from concanavalin A (ConA)-Sepharose. If there are no known specific eluents for the protein of interest then nonspecific elution may be used (e.g., elution using a salt gradient, changing the pH or temperature). Design conditions so that the protein of interest is eluted separately from the majority of contaminating proteins. One procedure is to raise the eluting buffer concentration to a level just below that at which the desired protein starts to be eluted, followed by an
TABLE IV ELUT1ON CONDITIONS Ligand
Eluent
Protein A
Acetic acid Glycine c~-D-Methylmannoside Borate buffer C~-D-Methylglucoside Temperature Salt Salt Urea Arginine pH NAD ÷, NADP ÷ Salt
ConA
Lysine Blue dye Gelatin 5'-AMP
Specific
Nonspecific X
X X X X X X X X X X X
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increasing shallow gradient. This results in sharper peaks and greater purity. If there are any proteins remaining bound to the matrix after the protein of interest has been completely eluted, the elution buffer strength can be increased rapidly to remove this remaining material. Elution is most often done in the "forward" direction, i.e., the same direction as sample application. Ideally, the sample should bind to the upper third to half of the column. Molecules with the highest affinity for the ligand will bind near the top of the column, while the remainder will bind further down the column as the affinity decreases. If the protein of interest is bound near the top of the column then the rest of the proteins can be more easily eluted in the forward direction, leaving the protein of interest on the column. Even if this protein moves down during the preliminary elution it will not come off the column. At this point if the eluent flow can be reversed and a strong eluent used, the sample can also be eluted off the top of the column in a sharper peak and in a shorter time, thus limiting exposure of the protein to potentially harsh conditions. Such a flow reversal can be accomplished by turning the column upside down, or using valves to allow the eluent to flow from the bottom of the column to the top. When reversing the direction of flow in a column always make sure that flow adaptor are used to prevent the loss of the matrix through the top of the column. The use of reversed elution to yield a more concentrated sample is valid only in certain situations. These occur when the desired protein binds more strongly than the other proteins and when it is bound to the upper half of the matrix. In all other cases it is still best to elute the sample in the forward direction. Measurement of the elution profile is usually done by monitoring parameters such as the Aaso or fluorescence. Specific assays for the protein of interest, such as enzyme activity, can yield information on the concentration and the condition of the separated material, and should be used whenever possible. Detection of any ligand that has leached off the matrix is usually difficult and requires specific assays. Radioimmunoassays (RIA) for ligand or matrix material are often useful in these situations. Electrophoresis (i.e., S D S - P A G E and immunoelectrophoresis) may also be used to determine protein purity, activity, and the extent of leaching. Regeneration Thoroughly clean resins prior to their reuse. Regeneration means that any material that remains on the resin must be removed and the gel reequilibrated with starting buffer. If all the material is not removed, and the ligand not properly prepared, the efficiency of the gel will be impaired.
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This will result in less material binding in successive runs and a concomitant loss in resolution. With proper care an affinity resin can be used multiple times. The actual number of uses depends on the sample, the ligand, and the elution conditions. Clean the gel with either a higher concentration of the specific eluent or by using a high concentration of a nonspecific agent such as sodium chloride (e.g., 0.5-1 M). Increasing the salt concentration is usually effective in removing nonspecifically bound material as well as some of the specifically bound sample that may be left on the resin. Take care not to damage the bound ligand or to alter its activity. In some cases high salt levels cause proteins to change their conformation. 5 If the ligand is a protein, its active site may be altered, causing it to lose some or all of its binding capacity or affinity for the sample. Some procedures are gentle enough for almost all gels. A general regeneration scheme (recommended by Pharmacia LKB Biotechnology, Piscataway, NJ) 6 follows. However, if the manufacturer provides a specific method for regeneration, then follow their advice: 1. Wash with 10 column volumes of Tris-C1 (0.1 M, pH 8.5) containing 0.5 M NaCI. 2. Follow with 10 column volumes of sodium acetate (0.1 M, pH 4.5) containing 0.5 M NaCI. 3. Reequilibrate with 10 column volumes of starting buffer. Any regeneration procedure requires buffer volumes up to 10 times the column volume. This assures that all areas of the resin have been reached and cleaned. Be sure those cations or anions needed for ligand stability are added to the regeneration buffer. These ions are usually present in the start and elution buffers, but are often overlooked in the regeneration buffer. For example, ConA requires Ca 2÷ and Mg 2÷ or Mn 2÷ at concentrations of 110 mM to maintain its tertiary structure. Resins are usually stable to most regeneration buffers, but if in doubt check with the resin manufacturer. Sterilization If the sample is to be kept sterile, the affinity column and gel must also be sterilized. Take special care throughout the entire process to assure the maintenance of this sterility. The gel can often be sterilized by autoclav5 p. H. von Hippel and T. Schleich, in "Structure and Stability of Biological Macromolecules" (S. N. Timasheff and G. D. Fasman, eds.). Dekker, New York, 1969. 6 Pharmacia LKB, "Affinity Chromatography: Principles and Methods," p. 88. Piscataway, New Jersey, 1983.
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ing, and is more easily accomplished before ligand attachment. The ligand can be filter sterilized before coupling to the resin. Coupling can be done under aseptic conditions such as with sterile buffers, air, and vessels. Depyrogenation of the matrix with agents such as sodium hydroxide is also best done prior to ligand attachment. If sterilization is necessary after the ligand is attached, take care to avoid altering the ligand or the linkage to the matrix. Autoclaving is not usually feasible at this stage since proteins and most other biological material are denatured under these conditions. Possible solutions to the sterilization of sensitive gels include radiation treatment or chemical sterilization. One gentle method for the sterilization of a sensitive gel-ligand system follows7: 1. Equilibrate the column with 2% chlorhexidine diacetate and 0.2% benzoyl alcohol. 2. Let stand for 4 days. 3. Wash with sterile buffer; a neutral phosphate buffer or the start buffer can be used. 4. Reequilibrate with the chlorhexidine diacetate (2%) and benzoyl alcohol (0.2%). 5. Rewash with the sterile buffer. 6. Store in 0.5% chlorhexidine diacetate and 0.05% benzoyl alcohol. Gel Storage Storage of the gel after preparation is usually quite easy. The actual conditions used for proper storage are dependent on the ligand that is bound to the matrix. In general, 4° is the preferred temperature. This lowers the possibility of bacterial growth and does not harm either the matrix or the ligand. Avoid freezing since this may rupture the matrix. It is best not to store a gel in the middle of the coupling process. This is especially true with CNBr gels, since they will lose activity rapidly at the pH used for activation. Clean the gel before storage by removing all residual material that is known to adhere to the column. This will allow for easier reuse of the matrix. In general, store the gel at temperatures below 8 ° but not frozen. Store all affinity resins in the presence of antibacterial agents such as chlorhexidine digluconate (or acetate), sodium azide, 20% ethanol, and thimerosal (do not use this with SH-active ligands, e.g., thiolpropyl-Sepharose). Base the choice of the antibacterial agent on the stability of the ligand to 7 S. S. Block, ed., "Disinfection, Sterilization, and Preservation." Lea & Fabiger, Philadelphia, Pennsylvania, 1977.
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that agent and the charge characteristics of the gel-ligand combination. Is it anionic, cationic, or neutral? Is it temperature sensitive? Is it subject to degradation by enzymatic action? To maintain the same level of activity after storage as existed previously, choose a bacteriostatic agent that will not bind to the gel matrix or ligand, and one that is easily washed out when the gels are reused (e.g., ethanol). Carefully remove all of the storage solution prior to reuse to prevent denaturation of the sample. Do not freeze the gel at any time. This will disrupt the matrix and can lead to fine particles that can interfere with the buffer flow. Again, follow the manufacturer's advice for proper storage.
[30] A f f i n i t y C h r o m a t o g r a p h y : S p e c i a l i z e d T e c h n i q u e s
By STEVEN OSTROVE and SHELLY WEISS This chapter discusses some specialized affinity chromatography techniques: cell affinity chromatography, metal chelate affinity chromatography, covalent affinity chromatography, and other binding techniques and the scaling up of affinity chromatography. It will be a guide in the use of these techniques and give a start in understanding the reasons behind their use. In addition, some of the possible problems and danger areas associated with these techniques are described. Not all of the specific methodologies available for separation by affinity chromatography will be reviewed in this chapter, nor will it provide an exhaustive list of examples for each technique. As you read this chapter, and try to use the techniques, however, you will find new and different ways to accomplish your separation task. Certain assumptions need to be made before we begin: First, that you are aware of general affinity chromatography procedures; second, that you know how some parameters such as temperature, pH, ionic strength, and flow rates affect affinity separations (see [29] in this volume). Cell Affinity Chromatography Isolating cells by affinity chromatography requires some special considerations due to the size and sensitivities of the living cell. Cells can be separated by affinity chromatography in two ways: either by binding the cell directly to the matrix as one binds a protein, or by binding a protein or METHODS IN ENZYMOLOGY, VOL. 182
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
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to the design and synthesis of new reactive dyes outside of the commercial dye industry. It was anticipated that the blue dye could better occupy the coenzyme site if the anthraquinone and the triazine rings were further separated by insertion of an ethylene bridge as shown in Fig. le, This immobilized dye, 24 in contrast to all other immobilized dyes and biomolecules, is able to resolve the purified alcohol dehydrogenase into two components of different activity, with the low activity form having a covalent modification on a lysine side chain. It is anticipated that additional designed dyes will increase the capability of immobilized dyes in protein purification. The chemistry employed in the synthesis of reactive dyes which is necessary for preparation of designed dyes is described in detail in two texts. 25,26 Finally, it should be noted that the solid fluorocarbons developed by du Pont de Nemours & Co. (Wilmington, DE) afford a promising new matrix for immobilized dye chromatography. A reactive dye is first subjected to a perfluoralkylation and then the perfluoroalkylated dye is essentially irreversibly adsorbed to a fluorocarbon surface. 27 Accordingly, this matrix should prevent slow bleeding of dye into proteins purified by immobilized dye chromatography. Most aspects of immobilized reactive dye-protein interaction have been reviewed by several authors 1.2,7,8,18and the concerned investigator is urged to pursue them for access to more detailed information. 24 C. R. Lowe, S. J. Burton, J. C. Pearson, and Y. D. Clonis, J. Chromatogr. 376, 121 (1986). 22 W. F. Beech, "Fibre-Reactive Dyes." Logos Press, London, 1970. ~6 K. Venkataraman, "The Chemistry of Synthetic Dyes," Vol. 6, Academic Press, New York, 1972. 27 j. V. Eveleigh, Abstr. Biotechnol. Microsymp. Macromol. Interact. Affinity Chromatogr. Technol., Mogilany, Pol. (1988).
[29] A f f i n i t y C h r o m a t o g r a p h y : G e n e r a l M e t h o d s
By STEVEN OSTROVE Affinity chromatography is one of the most powerful procedures that can be applied to protein purification. Over the years there have been many good books on the subject and many reviews of the theory of affinity chromatography.l,2 This chapter will not be a further review of the I j. Turkova, ed., "Affinity Chromatography." Elsevier, New York, 1978. 2 p. Mohr and K. Pommerening, "Affinity Chromatography: Practical and Theoretical Aspects." Dekker, New York, 1985.
METHODS IN ENZYMOLOGY,VOL. 182
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theory behind this technique nor will it review all of the proteins that have been separated by this technique since there are so many. Discussion will center on practical ideas and general considerations behind choosing a matrix, coupling a ligand, and some of the more common problems often encountered with the implementation of this procedure. Suggestions in this chapter should allow for the easier and more productive use of affinity chromatography. As the specific purification system for the sample of interest is developed, expect modification of these procedures. This chapter serves only as a guide to performing separations of biomolecules by affinity chromatography. Areas where the manufacturer of the affinity product can be contacted for specific recommendations on the use of the product are indicated throughout. Refer specific questions to the manufacturer. Affinity chromatography as a biospecific technique began only about 20 years ago even though it had been used as an experimental separation procedure for many years. 3 This procedure takes advantage of one or more biological properties of the molecule(s) being purified. These interactions are not due to the general properties of the molecule such as isoelectric point (pl), hydrophobicity, or size. This highly specific method of separation utilizes the specific reversible interactions between biomolecules. Some of the biological properties that can be exploited to effect a separation include specific shapes (that "recognize" other molecules such as receptors or enzymes), specific changes in conformation after changes in pH, or certain subareas or regions of the molecule that can interact or bind to other molecules (e.g., epitopes of antibodies). When developing a separation scheme keep in mind that the sample of interest is not the only component in the sample mixture that can be bound to an affinity matrix (gel). One affinity matrix may be specific for the sample of interest while others may be more specific for other components in the mixture (contaminating proteins). Just as the sample of interest can be bound to an affinity matrix, the contaminating proteins may also be specifically bound. An affinity gel could be chosen to bind the contaminating proteins, allowing the sample of interest to pass through the gel in the wash volume. This method of separation could result in a great saving of time. Matrix Choice of the proper matrix is a very important step in any chromatographic process. A good matrix for affinity chromatography should have the following properties: 3 p. Cuatrecasas and M. Wilchek, Biochem. Biophys. Res. Commun. 33, 235 (1968).
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1. Hydrophilic: Reduce the nonspecific interactions. 2. Large pores: Allow all areas of the matrix to be available to most of the molecules in the mixture. Some matrices allow binding only to the outer surface. This latter type of matrix is useful in separating very large molecules, cells, or viruses. 3. Rigid: The matrix must withstand the pressures of packing and solvent flow during elution or washing. 4. Inert: The matrix should not contribute to the separation. 5. Chemical stability: The matrix must be stable to all solvents used in the separation. Base the choice of an affinity gel on both the ligand and the sample. There are two major types of affinity gels: group-specific gels and covalent coupling gels. The former are usually supplied ready to use. Table I provides examples of ligands that are group specific in action and can be used to isolate whole families of biomolecules which share common properties. Covalent coupling gels (Table II) require more chemistry and some specific considerations. First, consider the length and type of the spacer arm; second, the coupling chemistry.
TABLE I LIGAND SPECIFICITY
Ligand NAD, NADP
Lectins Poly(U) Poly(A)
Histones Protein A Protein G
Specificity Dehydrogenases Polysaccharides Poly(A) Poly(U) DNA Fc antibody
Antibodies
Lysine
rRNA, dsDNA, plasminogen
Arginine Heparin Blue F3G-A Red HE-3B Orange A Benzamidine Green A Gelatin Polymyxin
Fibronectin, prothrombin Lipoproteins, DNA, RNA
2' ,5'-ADP
NADP ÷
Calmodulin Boronate Blue B
cis-Diols, tRNA, plasminogen
NAD ÷ NADP ÷
Lactate dehydrogenase Serine proteases CoA proteins, HSA, dehydrogenases Fibronectin Endotoxins Kinases Kinases, dehydrogenases, nucleic acid-binding proteins
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TABLE II COUPLING CHEMISTRY
Linkage
Ligand group
CNBr Thiolpropyl
NH2 SH
Thio Epoxy
SH NH2 OH SH NH2 COOH NH2
Tresyl Aminohexyl Carboxyihexyl
Spacer length
Equivalentto about 13 carbons Equivalentto about 11 carbons 6 6
Active pH
Specificity
8-10 9-11
Proteins,peptides Sulfhydryls
9-13 9 10 11 8-10
Sulfhydryls Proteins,peptides Carbohydrates Sulfhydryls Proteins,peptides Amino acids, proteins Carboxylic acids
Solvents The solvent system chosen for the entire affinity chromatography separation is also a critical factor to a good separation. The solvent should not degrade the sample. Unfortunately, avoiding denaturing solvents is not always possible. For example, separation of an antibody (IgG)-antigen complex requires some very harsh conditions. Dissociation at a low pH or use of a strong chaotropic agent are the most commonly used methods. Minimizing the time of contact with these agents is vitally important. One method used to reduce the contact time with harsh reagents (e.g., low pH) is to add Tris base (dry) to the collecting tubes. This will rapidly increase the pH and help to protect the sample. Try to choose an elution buffer specific for the sample (e.g., a buffer containing an analog to the sample). The elution buffer should release the sample safely and rapidly. Again, the buffer should not denature the sample, nor cause any change in its specific activity or function. Optimization of sample binding and elution conditions is usually by trial and error. When choosing a buffer system try to avoid using one that has a pKa at or near the pI of the sample. This will help prevent precipitation of the sample on the column. However, when starting a separation read the literature, as it will often provide a good starting point. Even a related separation can serve as a starting point for selecting the separation conditions. Spacer Arms Choosing a gel with or without a spacer arm depends on the ligand, the sample, and the binding chemistry. A spacer arm is used to keep the
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TABLE III SPACER LENGTH CONSIDERATIONS
Ligand
Protein
Best spacer arm
Small Small Large Large
Small Large Small Large
Short Long None None
ligand away from the matrix so that the active site of the ligand is available to the sample. This is especially important with small ligands. As a general rule if the ligand is large and the sample is small (low molecular weight) this spacing may be unnecessary. With samples of high molecular weight a spacer arm can be used to limit steric hindrance and increase the availability of the active site (Table III). A wide variety of spacer arm lengths are available. If unsure of the required spacer arm length, start with one that is equivalent to about six carbon atoms. This seems to be a good length for many affinity applications. 4 Shorter arms give less flexibility so the ligand will not " w a v e " around in the medium. Predicate the spacer arm length on the amount of steric hindrance deemed tolerable. As spacers are evaluated, remember that the spacer molecule itself can cause steric hindrance by blocking adjacent active sites on the gel; thus, longer is not always better. Gel Preparation After choosing the affinity gel type, prepare the resin (gel) for use. The manufacturer will usually supply the instructions needed to prepare the gel correctly. However, short of those instructions, following these general guidelines will help ensure a successful preparation. First, calculate the amount of gel that needs to be packed into a column (or flask for batch work) by the capacity of the gel for the sample. That is, x units of gel bind y units of sample. Next, calculate the volume: (total sample/sample units) × gel volume per sample unit. This value should be multiplied by a factor of 2-3 and this factored amount of gel should be used. For example, if I ml of protein A-Sepharose binds 20 mg IgG and you are using 40 mg of sample (with contaminants), divide the total (40 mg) by the gel capacity (20 rag) and multiply by the gel volume 4 C. R. Lowe, Biochem. J. 133, 499 (1973).
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(1 ml). Multiply this figure by 2-3, giving a final gel volume of 4-6 ml, for best results. If the gel is supplied in a preswollen state, reconstituting the gel is unnecessary to obtain the full swollen volume. A wash is all that is usually required. The swollen gel is typically supplied in glycerin or similar material which is used to help in the gel preparation and to stabilize the ligand or activated coupling complex. Wash on a sintered glass filter of medium grade (#3) or on a membrane-type filter that has a low protein-binding capacity. This allows easy removal of the washed gel with a minimum of loss due to sticking to the filter. A wash ratio of about 200 : 1 (buffer to gel) works well. For the safest wash buffer use either distilled water or the starting buffer (unless otherwise directed). By definition the starting buffer is that buffer used to initially prepare the matrix for the addition of the sample. It creates an environment on the gel so that the sample will bind specifically to the attached ligand. If the gel needs to be swollen to regain full working volume, then use a swelling buffer prior to washing. This buffer is often a low concentration phosphate buffer (0.1 M) at or near neutral pH. Swelling times vary between 15 min and 1 hr. After swelling, wash the gel either in the buffer solution used for swelling, distilled water, or starting buffer. Since washing and swelling buffers are generally pulled through the gel under a low vacuum, it is critical that the gel does not become dry at this stage. Following the reswelling and the wash, the ligand can be bound to the gel or loaded into a column if no ligand is to be added (i.e., groupspecific gels). Coupling or Linkage Chemistry Before using a covalent coupling gel, the ligand-binding (linkage) chemistry needs to be decided. There are a variety of linking groups available, such as cyanogen bromide (CNBr), tresyl, epoxy, and triazine. The linkage chemistry may be available either in an activated or nonactivated form. Activated means " r e a d y " to use without additional chemical activation steps (washing is still necessary). The nonactivated gels require some additional chemical activation step, such as carbodiimide treatment, to prepare them for binding the ligand. Leaching, or loss of ligand, after binding is inevitable. The trick is to minimize this loss. CNBr-type linkages commonly leach more than do tresyl or epoxy linkages. Other types of ligand linkages are also possible by using C~---C, C ~ O , and other available bonds. Nucleic acids and sugars can also be bound through their amine or hydroxyl groups. Table II lists some examples of
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the active groups that can be used to link ligands to the matrix, and the types of sample that can bind to these ligands. Coupling the Ligand Next, couple the ligand to the matrix, assuming that you have a specific ligand that needs to be attached. Consider the steric effects of the ligand, the spacer arm, and the sample. High concentrations of small ligands can block some active sites on the matrix, causing a lower binding efficiency. Large ligands can also block adjacent sites, again resulting in lower binding efficiency. Spacer arms and ligands can also cause some blockage of adjacent sites as the ligand " w a v e s " back and forth. As a general rule use about 10 mg ligand/ml of gel. This amount applies also to proteins having an average size of (50 kDa). A lower amount should be applied for larger molecules such as IgG (5 mg/ml), or IgM (1 mg/ml), or molecules with low dissociation constant (KD) values. Mix the ligand and the matrix together using a rotating motion. Avoid magnetic stirrers at all times since they can damage the matrix. The volume ratio of binding buffer (with ligand) to gel matrix should be about 2:1 for best results. Carefully control the pH, ionic strength, and ion content during this stage of coupling. Coupling times of 2-4 hr at room temperature or up to 16 hr in the cold (4°) are commonly used. The choice of time and temperature is determined by the stability of the ligand and the amount of time available. The time available is important since there should be no processing interruptions from the time the gel is activated until the excess ligand is washed out. This minimizes loss of coupling activity. Once again users should consult the manufacturer's instructions for the optimum coupling conditions. Coupling of the ligand to the matrix can be by a single point or multipoint attachment. An example of single-point attachment is the binding of a single primary amine via CNBr coupling. This type of linkage offers the best flexibility to the ligand and thus the most accessibility of the active site to the sample. Single-point attachment is possible only if secondary and tertiary amines are blocked. Multipoint attachments are stronger than single-point attachments and are less likely to leach during the run. However, this type of coupling often causes blockage of the active site of the ligand. Blocking Unreacted Groups After incubation of the ligand with the matrix, remove the excess ligand and block the unreacted sites on the matrix. When coupling a
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ligand to the matrix some sites on the matrix remain unreacted. These unreacted sites are potential sites for nonspecific interactions with the sample or contaminants in the solution. Blocking these sites is most easily accomplished with reagents that have an opposite charge or can be covalently linked to the matrix. For example, when a carboxyl group (COO-) is used to couple a ligand, such as an amino group (NH2), use a Tris or ethanolamine solution (0.1-I.0 M) as the blocking agent. When NH2 groups are the coupling sites for ligand containing COO-, acetic acid can be used as the blocking agent. The concentration of the blocking agent should be in excess of the total reactive site concentration on the matrix. This assures complete blockage of all unreacted sites. Normally, a 5- to 10-fold excess over the ligand concentration is sufficient. Control of the pH of the blocking agent is another critical factor important to good affinity separations. A pH that is either too high or too low may prevent complete blocking or even destroy the matrix or the bound ligand. The blocking reaction is usually done at room temperature for 2-4 hr, but can also be done in the cold (4°) for longer periods of time. Wash out the excess blocking agent and equilibrate the column with 5 to 10 column volumes of starting buffer before sample application. The coupling process can be summarized as follows: 1. 2. 3. 4. 5. 6. 7.
Swell the matrix in swelling buffer (15-60 min). Wash the matrix (200: 1, buffer:gel). Add ligand and incubate with mixing (2-4 hr, 2 : 1 buffer : gel). Wash out unused ligand and buffer (200: 1, buffer:gel). Block unreacted sites on the matrix (2-4 hr, room temperature). Wash the matrix (200: 1, buffer:gel). Use or store the gel under appropriate conditions (4-8°).
Monitoring Coupling Efficiency The extent of ligand coupling determines both the efficiency of the separation and the amount of purified sample that can be prepared. The amount of ligand bound can be determined in several ways: 1. Measure the difference in UV absorption before and after coupling: a. A2s0 is best for proteins; however, other wavelengths specific for other ligands which can be coupled should be chosen. For example, A4o5 for heme groups can be used. This technique is best accomplished when the concentration of ligand is not very high, since a small amount of ligand binding will not be detected. In
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addition, avoid buffer components that absorb at this wavelength [e.g., dithiothreitol (DTT)], or high concentrations of Tris buffer. b. Perform a colorimetric protein assay (see [6]). c. Perform a fluorescence detection assay (more sensitive than the colorimetric assays). Dissolve a portion of the gel containing the ligand and do a protein assay, amino acid analysis, or assay for total nitrogen. 3. Perform an activity test (a small binding experiment). 4. Perform a radiolabeled ligand or RIA test (assumes that the radiolabeled ligand binds at the same rate, and to the same extent, as nonradiolabeled ligand). .
Binding the Sample The binding of proteins to a ligand, through the carboxyl or the amino groups available, is based on the specific affinity of the protein for a particular ligand. As indicated previously, the ligand should not be coupled in such a way as to block or interfere with the availability of the active site on either the ligand or the protein. Binding between the ligand and the protein is generally noncovalent. Although the binding is specific, the forces involved are general chemical interactions, such as hydrogen bonds. The buffer conditions that are used to load the protein on to the column are often phosphate or Tris buffers (0.1-0.2 M) containing salts such as sodium chloride (0.5 M). The choice of buffer and concentration is predicated on minimizing nonspecific interactions and maximization of the specific attraction between the ligand and the protein. Load the sample in the normal downward direction such that it will bind to the upper half to upper third of the matrix. Flow Rates Different flow rates are used in the various stages of every affinity chromatography run: (1) the loading of the sample, (2) the wash step to remove nonspecifically bound material, (3) the elution phase, where the protein of interest is removed from the gel, and (4) the regeneration of the matrix for the next run. The flow rate commonly used for loading of the sample is often about 10 cm/hr. The notation cm/hr refers to linear flow rate of the buffer. To calculate the volume flow rate, which is the rate that is used for the run, multiply the linear flow rate by the cross-sectional area of the column. The flow rate used is dependent upon the kinetics of the binding of the
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desired protein to the ligand. Factors such as temperature, concentration, and the KD influence the interaction of the protein and ligand. If the protein has a high affinity for the ligand (KD < 10-6), the flow can be faster since the protein still can bind effectively. On the other hand, if the affinity is low, use a much slower flow rate. During the wash step the flow rate can be considerably faster since the wash serves to remove nonspecifically adhering material (assumed for this discussion not to be of interest). The flow rate at this stage can be increased to about 20 to 50 cm/hr in order to effect a rapid cleaning of the matrix. However, if the protein of interest is loosely bound to the ligand, a lower flow rate is better. Perhaps most important is the flow rate during the elution phase. This flow could also be faster then the loading rate. Elution flow rates depend on the strength of the elution buffer as related to the affinity of the sample. The goal is to use a buffer that will easily strip the desired protein from the ligand without damaging it, the ligand, or the matrix. The elution flow rate can be as high as the wash flow, but is always lower than the flow rate used for packing the matrix. The flow rate during reequilibration can also be very rapid. At this point, only the coupled ligand should be left on the matrix. Therefore, flow rates up to the packing rate can be used to save time. Flow rates should not exceed about 80% of the flow rate used to pack the resin. This avoids compression during the chromatography run. Another factor that determines the maximum flow rate is the stability of the matrix. In order to avoid gel compression and deformation of the beads do not exceed the maximum flow rate recommended for the matrix. Also, try to avoid turbulence and high shear rates due to rapid buffer flow in the matrix when loading or eluting. The manufacturer can generally provide information on the best flow rates for all steps. Nonspecific Interactions Nonspecific interactions, if a problem, can usually be avoided by using a salt concentration between 0.1 to 0.5 M since in this range nonspecific ionic attractions are greatly reduced. These salt concentrations are usually not so high as to make hydrophobic interactions between the protein and the matrix or ligand a problem. As always, the manufacturer of the matrix usually can supply the specific information needed to prevent nonspecific interactions. Other methods that may be used to decrease nonspecific interactions include the addition of agents such as glycerol up to a concentration of about 10% (no higher due to increased viscosity of the buffer, resulting in
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higher back pressures and lower flow rates). Low levels of detergent (below the critical micelle concentration) are also useful in reducing nonspecific interactions. Detergents, however, can interfere with both ligand and protein binding, and not every ligand can be safely used with all detergents. For example, a ligand may dissociate or denature in the presence of detergents and some may interact with the active site of the ligand, lowering its affinity for the protein. Specific vs. Nonspecific Elution Specific elution of the protein of interest is always the best method to use in affinity chromatography (see Table IV). This type of elution is the result of a competitive action of the eluent for the ligand. An eluent is chosen that has a greater affinity for the ligand than the protein so that it will displace the protein from the ligand. The eluent can then be removed by a more stringent cleaning of the matrix. An example of a specific eluent is the use of c~-methylglucoside to elute samples from concanavalin A (ConA)-Sepharose. If there are no known specific eluents for the protein of interest then nonspecific elution may be used (e.g., elution using a salt gradient, changing the pH or temperature). Design conditions so that the protein of interest is eluted separately from the majority of contaminating proteins. One procedure is to raise the eluting buffer concentration to a level just below that at which the desired protein starts to be eluted, followed by an
TABLE IV ELUT1ON CONDITIONS Ligand
Eluent
Protein A
Acetic acid Glycine c~-D-Methylmannoside Borate buffer C~-D-Methylglucoside Temperature Salt Salt Urea Arginine pH NAD ÷, NADP ÷ Salt
ConA
Lysine Blue dye Gelatin 5'-AMP
Specific
Nonspecific X
X X X X X X X X X X X
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increasing shallow gradient. This results in sharper peaks and greater purity. If there are any proteins remaining bound to the matrix after the protein of interest has been completely eluted, the elution buffer strength can be increased rapidly to remove this remaining material. Elution is most often done in the "forward" direction, i.e., the same direction as sample application. Ideally, the sample should bind to the upper third to half of the column. Molecules with the highest affinity for the ligand will bind near the top of the column, while the remainder will bind further down the column as the affinity decreases. If the protein of interest is bound near the top of the column then the rest of the proteins can be more easily eluted in the forward direction, leaving the protein of interest on the column. Even if this protein moves down during the preliminary elution it will not come off the column. At this point if the eluent flow can be reversed and a strong eluent used, the sample can also be eluted off the top of the column in a sharper peak and in a shorter time, thus limiting exposure of the protein to potentially harsh conditions. Such a flow reversal can be accomplished by turning the column upside down, or using valves to allow the eluent to flow from the bottom of the column to the top. When reversing the direction of flow in a column always make sure that flow adaptor are used to prevent the loss of the matrix through the top of the column. The use of reversed elution to yield a more concentrated sample is valid only in certain situations. These occur when the desired protein binds more strongly than the other proteins and when it is bound to the upper half of the matrix. In all other cases it is still best to elute the sample in the forward direction. Measurement of the elution profile is usually done by monitoring parameters such as the Aaso or fluorescence. Specific assays for the protein of interest, such as enzyme activity, can yield information on the concentration and the condition of the separated material, and should be used whenever possible. Detection of any ligand that has leached off the matrix is usually difficult and requires specific assays. Radioimmunoassays (RIA) for ligand or matrix material are often useful in these situations. Electrophoresis (i.e., S D S - P A G E and immunoelectrophoresis) may also be used to determine protein purity, activity, and the extent of leaching. Regeneration Thoroughly clean resins prior to their reuse. Regeneration means that any material that remains on the resin must be removed and the gel reequilibrated with starting buffer. If all the material is not removed, and the ligand not properly prepared, the efficiency of the gel will be impaired.
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This will result in less material binding in successive runs and a concomitant loss in resolution. With proper care an affinity resin can be used multiple times. The actual number of uses depends on the sample, the ligand, and the elution conditions. Clean the gel with either a higher concentration of the specific eluent or by using a high concentration of a nonspecific agent such as sodium chloride (e.g., 0.5-1 M). Increasing the salt concentration is usually effective in removing nonspecifically bound material as well as some of the specifically bound sample that may be left on the resin. Take care not to damage the bound ligand or to alter its activity. In some cases high salt levels cause proteins to change their conformation. 5 If the ligand is a protein, its active site may be altered, causing it to lose some or all of its binding capacity or affinity for the sample. Some procedures are gentle enough for almost all gels. A general regeneration scheme (recommended by Pharmacia LKB Biotechnology, Piscataway, NJ) 6 follows. However, if the manufacturer provides a specific method for regeneration, then follow their advice: 1. Wash with 10 column volumes of Tris-C1 (0.1 M, pH 8.5) containing 0.5 M NaCI. 2. Follow with 10 column volumes of sodium acetate (0.1 M, pH 4.5) containing 0.5 M NaCI. 3. Reequilibrate with 10 column volumes of starting buffer. Any regeneration procedure requires buffer volumes up to 10 times the column volume. This assures that all areas of the resin have been reached and cleaned. Be sure those cations or anions needed for ligand stability are added to the regeneration buffer. These ions are usually present in the start and elution buffers, but are often overlooked in the regeneration buffer. For example, ConA requires Ca 2÷ and Mg 2÷ or Mn 2÷ at concentrations of 110 mM to maintain its tertiary structure. Resins are usually stable to most regeneration buffers, but if in doubt check with the resin manufacturer. Sterilization If the sample is to be kept sterile, the affinity column and gel must also be sterilized. Take special care throughout the entire process to assure the maintenance of this sterility. The gel can often be sterilized by autoclav5 p. H. von Hippel and T. Schleich, in "Structure and Stability of Biological Macromolecules" (S. N. Timasheff and G. D. Fasman, eds.). Dekker, New York, 1969. 6 Pharmacia LKB, "Affinity Chromatography: Principles and Methods," p. 88. Piscataway, New Jersey, 1983.
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ing, and is more easily accomplished before ligand attachment. The ligand can be filter sterilized before coupling to the resin. Coupling can be done under aseptic conditions such as with sterile buffers, air, and vessels. Depyrogenation of the matrix with agents such as sodium hydroxide is also best done prior to ligand attachment. If sterilization is necessary after the ligand is attached, take care to avoid altering the ligand or the linkage to the matrix. Autoclaving is not usually feasible at this stage since proteins and most other biological material are denatured under these conditions. Possible solutions to the sterilization of sensitive gels include radiation treatment or chemical sterilization. One gentle method for the sterilization of a sensitive gel-ligand system follows7: 1. Equilibrate the column with 2% chlorhexidine diacetate and 0.2% benzoyl alcohol. 2. Let stand for 4 days. 3. Wash with sterile buffer; a neutral phosphate buffer or the start buffer can be used. 4. Reequilibrate with the chlorhexidine diacetate (2%) and benzoyl alcohol (0.2%). 5. Rewash with the sterile buffer. 6. Store in 0.5% chlorhexidine diacetate and 0.05% benzoyl alcohol. Gel Storage Storage of the gel after preparation is usually quite easy. The actual conditions used for proper storage are dependent on the ligand that is bound to the matrix. In general, 4° is the preferred temperature. This lowers the possibility of bacterial growth and does not harm either the matrix or the ligand. Avoid freezing since this may rupture the matrix. It is best not to store a gel in the middle of the coupling process. This is especially true with CNBr gels, since they will lose activity rapidly at the pH used for activation. Clean the gel before storage by removing all residual material that is known to adhere to the column. This will allow for easier reuse of the matrix. In general, store the gel at temperatures below 8 ° but not frozen. Store all affinity resins in the presence of antibacterial agents such as chlorhexidine digluconate (or acetate), sodium azide, 20% ethanol, and thimerosal (do not use this with SH-active ligands, e.g., thiolpropyl-Sepharose). Base the choice of the antibacterial agent on the stability of the ligand to 7 S. S. Block, ed., "Disinfection, Sterilization, and Preservation." Lea & Fabiger, Philadelphia, Pennsylvania, 1977.
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that agent and the charge characteristics of the gel-ligand combination. Is it anionic, cationic, or neutral? Is it temperature sensitive? Is it subject to degradation by enzymatic action? To maintain the same level of activity after storage as existed previously, choose a bacteriostatic agent that will not bind to the gel matrix or ligand, and one that is easily washed out when the gels are reused (e.g., ethanol). Carefully remove all of the storage solution prior to reuse to prevent denaturation of the sample. Do not freeze the gel at any time. This will disrupt the matrix and can lead to fine particles that can interfere with the buffer flow. Again, follow the manufacturer's advice for proper storage.
[30] A f f i n i t y C h r o m a t o g r a p h y : S p e c i a l i z e d T e c h n i q u e s
By STEVEN OSTROVE and SHELLY WEISS This chapter discusses some specialized affinity chromatography techniques: cell affinity chromatography, metal chelate affinity chromatography, covalent affinity chromatography, and other binding techniques and the scaling up of affinity chromatography. It will be a guide in the use of these techniques and give a start in understanding the reasons behind their use. In addition, some of the possible problems and danger areas associated with these techniques are described. Not all of the specific methodologies available for separation by affinity chromatography will be reviewed in this chapter, nor will it provide an exhaustive list of examples for each technique. As you read this chapter, and try to use the techniques, however, you will find new and different ways to accomplish your separation task. Certain assumptions need to be made before we begin: First, that you are aware of general affinity chromatography procedures; second, that you know how some parameters such as temperature, pH, ionic strength, and flow rates affect affinity separations (see [29] in this volume). Cell Affinity Chromatography Isolating cells by affinity chromatography requires some special considerations due to the size and sensitivities of the living cell. Cells can be separated by affinity chromatography in two ways: either by binding the cell directly to the matrix as one binds a protein, or by binding a protein or METHODS IN ENZYMOLOGY, VOL. 182
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
