[31] Chromatofocusing

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[3 1] C h r o m a t o f o c u s i n g By LALLAN GIRI

The electric charge on the surface of protein molecules is one of the intrinsic properties that have been exploited in designing strategies and methods for protein purification. The separation of proteins on the basis of their charge depends ultimately on their acid-base properties, which are largely determined by their amino acid composition and sequence. The electric charge on protein molecules is influenced by the pH of the solution. At lower or acidic pH values many proteins tend to have a positive net charge, whereas at higher or basic pH they carry a net negative charge. Both positively and negatively charged proteins tend to move in an electric field. However, at a certain pH proteins do not carry a net electric charge and do not move when an electric field is applied. This pH value is called the isoelectric pH or isoelectric point (pI). The charge properties of protein molecules also enable counterions present in the buffer to bind to the protein. Similarly, a charged protein will bind to the surface of a resin or any solid support bearing an opposite charge. The charge properties, the movement of charged protein molecules in an electric field, and the electrostatic interactions between protein molecules or between a resin and a protein molecule have given rise to electrophoresis and ion-exchange chromatography as two of the most powerful methods of protein purification and characterization. The isoelectric pH of proteins has been further exploited to separate proteins by isoelectric focusing and chromatofocusing. Isoelectric focusing by electrophoresis has been described elsewhere in this volume (see [35]). Chromatofocusing is the theme of this chapter. Chromatofocusing or isoelectric focusing by ion-exchange chromatography was first described by Sluyterman et al. Lz They proposed that a pH gradient could be produced in an ion-exchange column packed with an appropriate ion-exchange resin with good buffering capacity. A pH gradient in a column can be created in a manner similar to that of a salt gradient. If a buffer of one pH is mixed gradually with a volume of buffer of another pH in a mixing chamber and effluent from the chamber introduced into the column, a pH gradient is created. Similarly, a pH gradient can be produced internally in the column by taking advantage of the L. A. A. S i u y t e r m a n and O. E l g e r s m a , J. Chromatogr. 150, 17 (1978). 2 L. A. A. S l u y t e r m a n and J. Wijdenes, J. Chromatogr. 150, 31 (1978).

METHODS IN ENZYMOLOGY, VOL. 182

Copyright © 1990by AcademicPress. Inc. All rights of reproduction in any form reserved.

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buffering capacity of the resin. If a buffer of a certain initial pH is passed through an ion-exchange column preequilibrated with a buffer of a different pH, a pH gradient is formed in the column just as if two buffers at different pH were gradually mixed in the mixing chamber of a gradient maker. If such a pH gradient is used to elute proteins bound to the ionexchange resin, the proteins elute in order of their isoelectric points. During this process a focusing effect takes place, resulting in band sharpening, sample concentration, and resolution of peaks corresponding to different pI values of proteins. Mechanism of Chromatofocusing During a chromatofocusing process the individual molecules are continuously changing charged states as the pH gradient develops. Thus, in a descending pH gradient, a single molecular species can change from negative to neutral to positive. When a positively charged column is equilibrated with a starting buffer of high pH, proteins which become negatively charged will initially adsorb to the column. When an elution buffer of low pH is passed through the column, a pH gradient develops and the charge on the protein molecules changes. As the pH gradient moves down the length of the column, proteins are selectively desorbed when the pH is less than or equal to their pl and they are readsorbed when their pH is greater than their pI. Thus, molecules at the rear of the sample zone are the first to be titrated by the low pH buffer and become desorbed as a result of charge repulsion and are carried rapidly to the front of the sample zone due to the high velocity of the moving buffer. In traveling to the front of the sample zone, the proteins encounter an increase in pH which titrates them from their positive form to neutrality and back to their negative form. Once the molecules become negatively charged, they readsorb to the gel matrix and again fall back to the rear of the sample zone. This exchange of molecules between the front and rear of the sample zone results in "focusing" or a continuous narrowing of this zone until it elutes from the column. At this point the pH of the column effluent is approximately the pl of the component of interest. Reagents and Equipment

Ion~Exchange Resins. In principle, any suitable ion exchanger with appropriate buffering capacity can be used. The chosen ion exchanger should be stable in water, salt solutions, organic solvents, and denaturing agents. The resin should be of homogeneous bead size to allow high flow rates, and be rigid enough to prevent fluctuation in bed volume. It should

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also be stable to sterilization by autoclaving at 110-120 °, be free of nonspecific interaction with ampholytes, and be of high chemical and mechanical stability at extreme pH values. Generally, Polybuffer exchangers (PBE) 94 and 118, Sepharose CL, QAE-Sephadex A-25 (all from Pharmacia, Piscataway, N J), DEAE-BioGel A (from Bio-Rad, Richmond, CA), and DEAE-Toyopearl 650 M (from Toyosoda, Tokyo, Japan) have been used as anion exchangers in open conventional columns. For FPLC and HPLC, Mono P (from Pharmacia), and SynChropak AX-300 and AX-500 (from SynChrom, Lafayette, IN), respectively, have been used. For ascending pH gradients (starting with low or acidic pH), one can use any appropriate cation exchanger such as SP-Sephadex C-25 (Pharmacia) or CM-BioGel A (Bio-Rad). 1,3 Although ion-exchange capacity information is provided by manufacturers, it is advisable to verify the capacity experimentally by titration in the desired pH range. Buffers. Chromatofocusing requires two buffer solutions, a starting buffer to equilibrate the ion exchanger and the packed column, and an elution buffer (eluent) to elute bound proteins. It is the elution buffer which generates the pH gradient along the length of the column. The starting buffers are usually 20-30 mM amine buffers. The elution buffers, namely polybuffers 74 and 96 (from Pharmacia), which are a cationic and amphoteric class of buffering species, have been used most commonly, either alone or in combination with ampholytes. The most acidic polybuffer binds to basic polybuffer exchanger (PBE) groups on the resin, increasing H + ions in the vicinity and lowering the pH. This lower pH makes the proteins more positively charged, releasing them from the resin. In descending chromatofocusing the upper limit of the gradient is defined by the pH of the start buffer and the lower limit of the gradient is defined by the pH of the elution buffer. The reverse is true with a cation exchanger in ascending chromatofocusing, which is seldom used. To obtain a linear pH gradient, it is necessary that both buffers have a similar capacity over their working pH range. The pH of the start buffer is normally set 0.4 pH unit above the desired pH to compensate for the fluctuation in pH at the start of the run caused by slight differences in the conductivity of the start buffer and the elution buffer. Table I lists several start buffers and eluents which have been designed exclusively for chromatofocusing in different pH ranges. Columns. A wide range of columns and accessories are available from various suppliers. One can use any open column in the size range of 20 × 1 cm to 60 × 1 cm. Columns of similar sizes, used for other chromato3 A. Murel et al., J. Chrornatogr. 362, 101 (1986).

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graphic procedures such as gel filtration and ion exchange, can also be used for chromatofocusing without any noticeable difference in their resolution. Peristaltic Pump. To maintain a constant flow rate a compact, singlechannel pump without a gradient-forming capability is required. The pump should be able to deliver as low a flow rate as possible to obtain good resolution, and back flow should be minimized. UV Monitor. A sensitive, preferably dual-beam, UV monitor can be used for detecting proteins, nucleic acids, and peptides at 280 and 254 nm. The monitor should be equipped with an appropriate flow cell. Alternatively, the absorbance of individual fractions may be monitored. Recorder. Any reliable chart recorder which can be interfaced with the monitor would be appropriate. Factors Influencing Resolution of Chromatofocusing The resolution in chromatofocusing, as in other column techniques, is determined by the width of the zone as it elutes. This is dependent on many variables which can be optimized in chromatofocusing.4 The most important factors for optimizing a particular protein separation by chromatofocusing are discussed below. Slope o f p H Gradient. A shallow pH gradient gives better resolution. This can be achieved by using low buffer concentrations which give slow and steady pH changes. However, too shallow a gradient can also cause excessive dilution of protein in the eluent. Experimentally, a gradient of 10-15 bed volumes has been found to give good results. 4 Buffers. Most of the separations have been carried out successfully in polybuffers. However, they can also be replaced with appropriate mixtures of conventional buffer components. 1,2 For example, the starting buffer and elution buffer can be identical in their composition, but with two different pH values, representing the upper and lower limits, respectively. Charge on Ion Exchanger. An optimal charge difference between the ion exchanger and the surrounding medium contributes to zone sharpening in chromatofocusing, just as electric field strength contributes to zone sharpening in isoelectric focusing by electrophoresis. Polybuffer exchanger (PBE) resins, which have a high degree of substitution, give good focusing. Column Packing. Any irregularity in column packing can have a 4 Pharmacia Fine Chemicals, Chromatofocusing 17 (1980).

384

P U R I F I C A T I O N PROCEDURES: CHROMATOGRAPHIC METHODS

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marked effect on resolution. The column should be evenly packed and uniformly equilibrated with start buffer. A certain degree of skill is required to achieve a well-packed column. Ions. Monovalent anions other than CI- can be used as the counterions, but it is critical that the anions have a pKa at least two pH units below the lowest point of the gradient chosen. Bicarbonate ions cause fluctuations in the pH gradient. Therefore, all buffers must be degassed before use. Atmospheric COz may cause a plateau in the pH region 5.56.5, depending on the conditions. These effects are most apparent with polybuffer 96 in the pH gradient ending at pH 6, and can be avoided by using acetate as the counterion. On the other hand, acetate is not usually recommended as a counterion with polybuffer 74 because of its higher pKa. Length of Column. Although good results have been obtained with short columns, resolution is superior with longer columns having the same internal diameter. However, very long columns, greater than 50 cm, will result in very long running times. Thus, 20- to 30-cm-long columns have been found to be most appropriate. Flow Rate. Theoretically, the flow rate should not have a major impact on resolution. 1,z However, in practice, a significant difference has been noted since a very low flow rate does give increased resolution (Fig. 1). A higher flow rate can be used until the component of interest is close to elution. Then the flow rate can be decreased about 10-fold, and after the component has been eluted the flow rate can be adjusted back to its original value. Experimental The proper planning of a chromatofocusing experiment is similar to that of other chromatography techniques. The critical stages in designing a chromatofocusing experiment are described below. Choice of Gel and Buffers. One can choose any anion exchanger and buffers or Polybuffer and Polybuffer exchanger resins designed for chromatofocusing. If the isoelectric point of the protein of interest is known, then pH range of the gradient is chosen so that it elutes after one-third to one-half of the pH gradient in order to obtain optimal resolution. If the isoelectric point of the protein is unknown, it can be determined by isoelectric focusing by electrophoresis 5 or by a simple test using ion exchangers. 6,7 When working with an unknown sample, one can select a range, 5 D. Gartin, this volume [33]. 6 "Ion Exchange Chromatography--Principles and Methods." Pharmacia Fine Chemicals, Uppsala, 1987. 7 G. P. Lampson and A. A. Tytell, Anal. Biochem. 11, 374 (1965).

[31] A280

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FIG. 1. Separation of a standard mixture of proteins at two different flow rates. The conditions include Pharmacia column SR 10/50; bed height: 30 cm; sample: 5 ml elution buffer containing cytochrome c (5 mg), ribonuclease (8 mg), and lentil lectin (10 mg); elution: start buffer 0.025 M triethylamine-HCl, pH 11.0, elution buffer 0.0075 mmol/pH unit/ml Pharmalyte pH 8-10.5 equilibrated to pH 8.0. Linear flow rate: (A) 15 cm/hr, (B) 117 cm/hr. (From Pharmacia LKB AB, Uppsala, Sweden.)

such as pH 7-4 for the gradient, since most proteins have pl values in this range. 8 If the desired protein has a pl below 4, it will pass through the column and can be recovered easily. On the other hand, if the pI is above pH 7.0, it will bind to the column and recovery may not be simple. The bound protein has to be eluted with a salt solution, the column reequilibrated with start buffer, and the sample is reequilibrated with a new buffer. Thus, prior information about the pl value of the sample would eliminate these problems. Quantity oflon Exchanger. The amount of gel used will depend on the amount and nature of the sample and contaminants. For most separations, a bed volume of 20-30 ml is sufficient for a protein sample up to 200 mg. It is important to remember that resolution is compromised with excessive amounts of sample. Preparation of Gel. The ion-exchanger gel should be equilibrated with the start buffer. A list of suitable start buffers is given in Table I. The ionexchanger resin can be equilibrated in a sintered glass funnel before pack8 E. Gianazza and P. G. Righetti, J. Chromatogr. 193, 1 (1980).

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ing a column or also in the column after packing. The measured amount of exchanger is poured into the funnel. The start buffer is then passed through the gel at a slow flow rate using aspiration until the pH of the eluent is the same as that of the start buffer. The gel should occasionally be stirred gently to ensure complete equilibration, which usually requires 10-15 bed volumes of start buffer. With Polybuffer exchangers it is not recommended to speed up the equilibration process by using concentrated buffer followed by one to two bed volumes of start buffer since the high capacity of Polybuffer exchangers can make equilibration to a lower ionic strength difficult. After equilibration the resin can be packed into an appropriate column. Column Packing. I t is necessary to have a well-packed column in order to have a successful chromatofocusing experiment. Packing at high flow rates gives the best results. In our laboratories the following method has been used for open columns. 1. The equilibrated gel is dispersed in 150-200 ml of start buffer to make a slurry, and then degassed. 2. The column is mounted vertically. The end of closed outlet tubing is placed approximately 50 cm below the top of the column. 3. Put 3-5 ml of start buffer into the empty column and pour in the gel slurry while mixing it by swirling. If the volume of slurry is greater than that of the column, a packing extension can be used. 4. Open the outlet tubing and allow the gel to settle rapidly. The packing extension can be removed, and the top of the column connected to inlet tubing. 5. Continue to pack the column at a linear flow rate of 100 cm/hr (linear flow rate in centimeters per hour equals milliliters per hour divided by cross-sectional area of the column) until the gel bed has completely settled. Further equilibration can be carried out at a lower flow rate until pH and conductivity of the elueaat match that of the start buffer. Any air b u b b l e s should be removed. 6. The column packing can be checked by passing through a colored marker protein with a very high pI value, such as cytochrome c (pl = 10.5). Use 1 ml of a 2-3 mg/ml solution of cytochrome c in the start buffer. Elute it with the start buffer. This protein should not be adsorbed by the gel and should come through in the void volume. Thus it should come through in an expected elution volume. Sample Preparation and Application. The preparation of a sample depends very much on its nature. Approximately 100 mg of total protein can be applied for every 10 ml of gel bed volume, although this value will vary according to the number of proteins present in the sample. The volume of the sample is not critical, so long as all of the sample is applied

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before the substance of interest is eluted from the column. Nevertheless, it is best if the sample volume does not exceed one-half of the column volume. The sample should not contain salt and should be equilibrated either in start buffer or elution buffer. To ensure even sample application, a layer of 1-2 cm of Sephadex G-25 coarse on the top of the bed is recommended. This serves as a mixing chamber and permits even sample application. The sample should be applied through a syringe or a flow adapter by first running 5 ml of elution buffer, followed by the sample (in elution or start buffer), then switching back to the elution buffer again. In this way sample proteins are never exposed to the extremes of pH. Elution. No special gradient apparatus is required for elution since the gradient is formed in the column automatically. The volume of the gradient is determined by the strength of the elution buffer. The recommendations in Table I are designed to give a gradient volume of approximately 10 column volumes for pH intervals of 3 pH units. Generally, there is a dead volume of 1.5 to 2.5 bed volumes of buffer which passes through the column before the pH in the eluent begins to decrease. Thus, the total amount of buffer required is approximately 12.5 bed volumes. Polybuffer is not suitable for intervals wider than 3 pH units. Flow Rate. From the initial studies L2 it was predicted that in chromatofocusing the separation would be independent of the flow rate. It seems to be true only up to a certain degree. A large variation in flow rate does change the resolution. A linear flow rate of 30-40 cm/hr has been found to give consistently good results in our laboratories. Monitoring. The eluent can be monitored for protein at 280 or 254 nm. Monitoring at 254 nm should be avoided when using Polybuffers because they absorb slightly at this wavelength. The pH of the eluent should also be monitored either by using a pH flow cell electrode or by measuring the pH of the individual fractions soon after they have been collected. Both the UV absorbance and the pH measurement can be recorded with a twochannel chart recorder. Regeneration. A column can be regenerated and used several times without repacking. The gel should be washed with two or three bed volumes of a 1 M NaCl solution to remove any bound substances. Strongly bound proteins can be removed by washing with 0.1 M HCI. If HCI is used, the gel must be reequilibrated to a higher pH as soon as possible after washing. The column should then be reequilibrated with the start buffer until the eluent pH is the same as that of start buffer.

Separation of Polybuffer or Ampholytes from Protein Although Polybuffer and ampholytes generally do not interfere with enzyme assays, amino acid analysis, or the Coomassie Blue protein as-

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say, they may form a complex with copper ions, thus interfering with other protein determination procedures (see [6] in this volume). Several methods can be used to remove Polybuffer or ampholytes from proteins. Precipitation. The simplest method is precipitation with ammonium sulfate. Solid ammonium sulfate is added to the relevant fractions to a suitable concentration (80% saturation) and the sample is allowed to stand for 1-2 hr or until the protein precipitates. Since the protein is at its pI, it should be fairly simple to precipitate. The precipitate is collected by centrifugation and 'washed several times with saturated ammonium sulfate. An alternate method would be to place the fractions of interest in dialysis tubing and dialyze against saturated ammonium sulfate. Gel Filtration. Polybuffer or ampholytes can also be removed from most proteins by gel filtration on Sephadex G-75. If the fractions are small enough, prepacked disposable Sephadex desalting columns can also be used. Other chromatography techniques such as hydrophobic interaction chromatography (HIC) and affinity chromatography can also be used to remove Polybuffer and ampholytes. The methods for these techniques are described elsewhere in this volume. 9,J°

Chromatofocusing as a One-Step Separation Technique Chromatofocusing has been used as an adjunct method to other chromatographic methods, generally as a final purification step. However, a number of papers have described a purification in which chromatofocusing was used as the sole chromatographic method (e.g., Ref. 11).

Chromatofocusing in Denaturing Agents Separation and purification of proteins by chromatofocusing have also been carried out in the presence of dissociating agents such as urea, DMSO (dimethyl sulfoxide), formamide, ethylene glycol, and nonionic detergents such as Nonidet P-40 (NP-40) and Triton X-100 (e.g., Ref. 12). The success of chromatofocusing with any of these dissociating agents depends to a large extent on their interaction with the molecules in the sample. For example, when detergents are used to solubilize proteins, at a concentration approaching the critical micellar concentration (cmc), there may be association of protein molecules. For very hydrophobic protein molecules, dissociation and solubilization may not be as successful as one 9 R. K e n n e d y , this v o l u m e [27]. ~0 S. Ostrove, this volume [29]. 11 I. Kaivaria et al., Thromb. Res. 29, 459 (1983). iz H. Bloemendal and G. G r o e n e w o u d , Anal. Biochem. 117, 327 (1981).

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might expect. This may result in a range of molecular complexes which may perturb the focusing. Furthermore, some molecules may be so heavily coated with detergent that the charge-dependent interactions necessary for chromatofocusing are not efficient. Thus, a certain degree of caution would have to be exercised about the concentration of detergents versus the nature of proteins in the sample. High-Performance Chromatofocusing Although chromatofocusing had proved to be a powerful technique for protein separation, it still requires several hours to complete a single successful experiment. Thus, the demand and needs for protein purification in a short amount of time forced researchers to apply the principles and techniques of chromatofocusing to high-performance liquid chromatography (HPLC) and fast protein liquid chromatography (FPLC). The chromatofocusing principles were successfully integrated into the FPLC concept by Pharmacia. 13 Approximately at the same time it was also applied to HPLC by Wagner and Regnier, j4 who demonstrated that compared with other HPLC procedures, chromatofocusing was superior based on the number of protein components it resolved in a single run. FPLC chromatofocusing has found widespread application in research as the method of choice for resolving isoenzymes and molecular species with very similar charge characteristics and has proved promising for both analytical and preparative separation of proteins. Chromatofocusing on FPLC does not require any special modification of the regular FPLC system.13,~5 Concluding Remarks Chromatofocusing has become very popular as a simple and rapid method for protein purification to attain reasonable purity. The resolution of this method enables one to separate almost identical molecules differing in pI by as little as 0.05 pH unit. This method has advantages of separating and concentrating proteins during the same run. In several instances the resolution of components inseparable by other chromatographic methods has been obtained by chromatofocusing. Although generally a pH gradient range of 10-4 has been suggested, some separation has been achieved even)at a much lower pH range. t3 R. M. Mullerand L. Soderberg, Int. Syrnp. Proteins, Pept. Polynucleotides, Baltimore, Md. Abtsr. No. 714 (1982). 14G. Wagnerand F. Regnier,Anal. Biochem. 126, 37 (1982). ~5"FPLC Ion Exchangeand Chromatofocusing--Principlesand Methods."PharmaciaAB, Uppsala, 1985.

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High-performance chromatofocusing has demonstrated advantages over conventional methods particularly with regard to reproducibility of the experimental conditions, speed, and resolution at both the analytical and preparative scales. A wealth of published literature on conventional and FPLC chromatofocusing exists. A compiled reference list can be obtained from Pharmacia LKB Biotechnology, Inc. Acknowledgments I wish to thank my colleagues Drs. David Englert and Bengt Osterlund for reading the manuscript, and Maggie Gustin and John Kubowitz for their help in figure preparation. I would also like to thank Dr. D. A. Hart, Faculty of Medicine, Universityof Calgary, Canada for sharing his unpublished results with us.

[32] H i g h - P e r f o r m a n c e L i q u i d C h r o m a t o g r a p h y : E f f e c t i v e Protein Purification by Various Chromatographic Modes

By ROMAN M. CaIcz and FRED E. REGNmR Introduction Chromatographic resolution of biological macromolecules in all cases except size-exclusion chromatography is a surface-mediated process, i.e., there is differential adsorption of solutes at the surface of the chromatographic packing material. Optimization of a chromatographic separation is nothing more than a solute adsorption. Structural characterization tells us that biological macromolecules differ physically in their size and shape, charge, hydrophobicity, and arrangement of functional groups within their three-dimensional structure. It is not surprising that the major chromatographic modes by which biopolymers can be fractionated are by size-exclusion chromatography (size and shape discrimination), ion-exchange chromatography (charge discrimination), hydrophobic interaction chromatography (surface hydrophobicity), reversed-phase chromatography (general hydrophobicity), immobilized metal affinity chromatography (surface-available histidines), and bioaffinity chromatography (distribution of specific amino acids at the surface of proteins). It is unlikely that this repertoire of chromatographic fractionation modes will increase to any extent during the next decade. METHODS IN ENZYMOLOGY, VOL. 182

Copyright © 1990 by Academic Press, Inc. All fights of reproduction in any form reserved.