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Introduction
One of the most important developments in enzymology has been the introduction of a rapid and highly efcient means of protein purication utilizing the highly selective ability of enzymes to recognize certain biological compounds or their analogues. This technique, termed afnity chromatography, involves the immobilization of an appropriate ligand in such a way that the enzyme is still capable of recognizing and binding to the immobilized form of the ligand, whereas contaminating proteins have no such recognition. Virtually hundreds of proteins have been puried in this way, using a wide variety of bioligands immobilized in a matrix; enzyme inhibitors, coenzymes, antibodies, and even other enzymes may be useful bioligands in the purication of a particular protein (1). A classication of the types of afnity chromatography is as follows (2): Bioselective adsorption is the process where the afnity is based on biologically relevant binding. It includes group-specic ligands, eg, lectins and nucleotide cofactors (NAD, AMP), and specic ligands, eg, certain less common cofactors (vitamin B12 ), receptor proteins, and antibodies as used in immunosorbents. Chemiselective adsorption is the process where the afnity is based on chemically dened nonbiological interactions. It includes hydrophobic chromatography, ion-exchange chromatography, covalent chromatography (active thiols, Hg2+ , etc), and borate complexes. The basic application of afnity chromatography involves several steps, as illustrated in Figure 1. First, the necessary afnity-chromatographic packing, or bioselective adsorbent, as it may be more appropriately termed, is synthesized. Next, a cell-free extract containing the desired enzyme is prepared. This extract is freed of any endogenous substrate or biomolecule that might compete with the
Encyclopedia of Polymer Science and Technology. Copyright John Wiley & Sons, Inc. All rights reserved.

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Fig. 1. The steps of afnity chromatography: (a) A bioligand is immobilized; (b) A crude extract is prepared and freed from endogenous substrate; (c) The substrate-free extract is applied to the chromatographic packing of immobilized bioligand from step (a); (d) Unwanted protein is removed by washing; and (e) The desired protein is eluted, possibly with a soluble bioligand (2).

enzyme for the adsorbent. This is normally achieved by dialysis or enzyme precipitation. The crude extract is applied to the column, and contaminating proteins, which ideally have no afnity for the column under the conditions employed, are removed by washing. The protein to be puried remains bound to the column because of its afnity for the immobilized ligand. It is subsequently removed by eluting the column with a solution of the free ligand. Alternatively the enzyme may be eluted by altering the chromatographic conditions, ie, pH, ionic strength, dielectric constant, etc, in such a way that the enzyme no longer retains its afnity for the immobilized ligand. Efcient purication by afnity chromatography depends on the nature of the ligand, the methods used in the preparation of the chromatographic packing,

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the type of inert matrix, and the methods for both attaching the ligand to the matrix and eluting the enzyme from the bioselective adsorbent. Nonspecic adsorption of proteins, which can be dened as the retention of proteins by some general factor, eg, ion exchange, hydrophobic interactions, and chargetransfer complexation, may cause a large variety of proteins to be nonspecically retained. Finally, it must be decided whether afnity chromatography is employed primarily for purication or primarily as a method for studying enzymesubstrate interaction. The choice of the nal system to be used is governed by the nature of the application.

Matrices
The selection of an appropriate inert matrix is of great importance and depends on the type of separation desired. Large columns, or those employed under high pressure, require beads with high mechanical stability. Afnity purication as a nal step following a long series of conventional procedures requires a small column. When afnity chromatography is employed in the last stages of purication as a polishing step, high yields of the valuable partially puried enzyme are especially important. Even a small percentage of nonspecic adsorption or protein denaturation on the column would limit the usefulness of the procedure. For this reason a hydrophilic, nonionic matrix with little capacity for nonspecic adsorption is essential. Nonspecic adsorption must not occur in the derivatized matrix, even though the untreated matrix might have considerable nonspecic adsorption. For example, virgin glass adsorbs enzymes in an active form, yet dextran-coated glass or glass treated with hydrophilic silanes exhibits little or no adsorption (4) of ribonuclease and other proteins. In high performance liquid afnity chromatography (hplac), a rapidly growing technique, the matrix must be mechanically stable and capable of sustaining very high pressures. High pressure chromatography resins of the Trisacryl or TSK-type may supplement the silica-based hplac matrices. Several activated resin forms are commercially available (Pierce Chemical Co., Rockford, Ill.). The most widely used matrix is beaded agarose, a common gel-permeation chromatography packing used chiey because of the hydrophilicity of the underivatized matrix (5). It might be thought that derivatized agarose would not have more nonspecic adsorption than its untreated counterpart. However, this is not the case, and this observation conrms that it is the derivatized chromatographic packing, and not the matrix, that must possess the appropriate properties for a bioselective adsorbent. In addition to mechanical stability and nonspecic adsorption, the chemical stability and ease of derivatization of the matrix must be considered. Agarose provides the least nonspecic adsorption, fair chemical stability, and a rather poor and often limiting mechanical stability whereas controlled-pore glass provides the greatest mechanical stability and ease of derivatization, with acceptable capacity but often with unacceptable amounts pf nonspecic adsorption.

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Other matrices have certain advantages and disadvantages for specic applications. Regenerated cellulose, polyacrylamide, and cross-linked dextrans generally yield high capacity adsorbents as dened chemically, ie, the amount of ligand bound per gram, but often much of their surface is not available to large macromolecules; thus the effective capacity, ie, the amount of ligand accessible to a particular enzyme, is generally low. In other words, many of the ligands are buried in the matrix in such a way so as to be inaccessible to the enzyme. Agarose. This material is among the most useful matrices for afnity chromatography. It is a linear polysaccharide containing alternating residues of D-galactose and 3,6-anhydro-L-galactose (6).

Agarose is prepared by mixing a hot aqueous solution of specially puried agar (26%) with an organic solvent, eg mineral oil, and a small amount of detergent. The aqueous solution is mixed rapidly with the organic phase to form droplets that, upon cooling, form agarose beads (7). These beads are fragile even to the touch. Cross-linking with epichlorohydrin increases their strength but decreases porosity. The agar base, and thus agarose, is a naturally occurring polysaccharide with many ionic residues, chiey carboxylate and sulfate, which are removed by hydrolysis and reduction. Commercial agarose beads contain up to 0.37% sulfur (8), indicating the presence of a signicant number of ionic groups in commercial agarose. Agarose also presents other problems: It lacks thermal stability, cannot be dried or frozen readily, and shrinks and swells upon changes of ionic strength or dielectric constant of the medium, especially in the presence of organic solvents. Thus, commercial agarose is completely soluble in dimethyl sulfoxide at 100 C or in 4 M sodium iodide, whereas agarose cross-linked with epichlorohydrin is essentially unaffected under the same condition (<1% soluble) (8). Agarose is frequently used as the cyanogen bromide derivative. This activated matrix reacts with proteins and other molecules with amino groups (5,911) and covalently couples them to the agarose surface. The most serious problem encountered with agarose is the formation of an unstable isourea function from the reaction of cyanogen bromide-activated agarose with primary amines. This isourea forms a positively charged derivative:

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Until 1973 the ionogenic nature of cyanogen bromide-activated agarose was not known, and many early investigations using this method must be examined closely. Furthermore, it should be noted that cyanogen bromide is toxic and presents an explosion hazard when impure. Derivatized agarose often possesses ion-exchange properties in addition to, if not in place of, bioselective adsorption. In many instances it is suspected that what was hitherto thought to be a purication based on bioselective adsorption, or afnity chromatography, was in reality a specically tailored ion-exchange chromatography, which may or may not have given better results than classical ionexchange methods (12). Even though there is some question about the mode of action of some bioselective adsorbents based on cyanogen bromide activation, enzymes can be puried by ligands coupled to cyanogen bromide-activated agarose. However, there are many methods for derivatizing agarose that are not ionogenic. Coupling can be employed by means of bifunctional reagents, including bisoxiranes, eg, 1,4-bis(2,3epoxypropoxy)butane, and diphenyl sulfone (13). The reactions of bisoxiranes are shown below:

where R is the ligand to be attached to the agarose. Other activation methods are performed with carbonyldiimidazole or tresyl chloride (14); both yield uncharged materials. Coupling with carbonyldiimidazole (CDI) is very rapid, but the bond formed, a substituted carbamate, is not as stable as the secondary amine formed with the slower tresyl chloride.

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A group of activation reagents similar to CDI have been developed in Israel (11). They resemble phosgene and produce immobilized enzymes with bonds identical to those produced by CDI. One of their advantages is that p-nitrophenyl chloroformate can be used to prepare an activated matrix that has the property of releasing the yellow p-nitrophenol anion as coupling occurs, thereby permitting one to easily monitor the reaction visually:

Controlled-Pore Glass. Many bioselective adsorption separations can be performed on columns of porous glass with excellent results (2). Controlled-pore glass was developed when it was discovered that borosilicate glass heated to 700800 C separates into borate and silicate phases (15,16). The borate phase is removed by an acid etching process, leaving a porous 96% silica network. These pores, ca 4.5 nm in diameter, can be enlarged by additional alkali etching to produce porous silica beads with nominal pore diameters of 4.52500 nm. The pore distribution (10%) is the most uniform of any porous chromatographic support. Controlled-pore glass with pore diameters above 250 nm are generally too friable for chromatographic application, although largepore-diameter glass, up to about 400 nm, has been employed.

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Another type of inorganic matrix that is similar to controlled-pore glass but considerably less expensive can be prepared by fusing inorganic compounds such as nely pulverized silica and zirconium oxide to form a porous body. Although the pore distribution is less uniform, the surface composition is almost uniform; in the case of silica, the surface is nearly 100% silica. Controlled-pore

glass is 96+% silica, but alkali leeching facilitates the migration of boron to the surface, which may in fact contain as much as 30% boron (15). The numerous boron Lewis acid sites that result may adsorb ammonia or nucleophiles such as protein amines to yield positively charged centers, both of which lead to nonspecic adsorption Early synthetic procedures for preparing glass for afnity chromatography or enzyme immobilization involved reuxing -aminopropyltriethoxysilane with the beads (17) under various conditions; the beads were coated with multiple layers of silane, which were not as stable as desired. A more stable layer is obtained by employing aqueous silanization at pH 3.75 and 75 C (17). Nonetheless, aminoalkyl glass, or any other silanized glass material, is unstable in aqueous solution at pH values much above 7. Similar materials prepared from zirconium oxides are, conversely, stable in base and labile in acid. Some derivatives of aminopropyl glass exhibit nonspecic adsorption in certain enzyme preparations, others do not (18). Nonspecic adsorption is minimized by coating the glass with glycidoxypropyltrimethoxysilane, followed by appropriate treatments to yield a highly stable glass with a hydrophilic surface because of the presence of hydroxyl groups in the coating. This surface can be further derivatized in a manner similar to that used for agarose, although the same reservation expressed above for cyanogen bromide activation of agarose, namely its ionogenic nature, applies to glycidoxy controlled-pore glass. -Galactosidase has been puried from cell-free extracts of Aspergillus niger (19) by using a bioselective adsorbent consisting of the inhibitor -aminophenyl-D-thiogalactose coupled to controlled-pore glass by means of an amide linkage using a soluble carbodiimide as the coupling reagent. However, the same matrix prepared with aniline as a ligand in place of the inhibitor results in a substantial purication of -galactosidase (20). The aniline-modied material has a capacity 75% of that containing the bound inhibitor and produces -galactosidase with about 50% of the specic activity of that produced with the afnity packing consisting of inhibitor-controlled-pore glass. A combination of ionic and hydrophobic forces results in a reasonably selective adsorbent whose function is not totally based upon enzyme-active siteinhibitor interaction. The exact nature of the interactions are not known. Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) polymerases have been separated and puried on DNA-glass columns, effecting more rapid purication than afnity chromatography on DNAcellulose (21,22). The DNA is coupled

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to aminopropyl glass beads, using the method for covalent coupling of DNA to cellulose with a soluble carbodiimmide as the coupling reagent (23). Silica and porous glass can be used as matrices for hplac (2426). In this technique, where mechanical stability under pressure is critical and pH changes are transient, porous silica matrices are useful. Cellulose. A widely used DNA afnity-chromatography procedure (21) depends upon the physical trapping of DNA bers in a mesh of cellulose bers. The DNAcellulose is prepared by drying DNA and cellulose together in an appropriate buffer, followed by washing the untrapped DNA. However, as one might expect from a method involving physical trapping, DNA slowly elutes. Bulk cellulose is a poor afnity-chromatographic matrix; its principal advantage is its low cost. This is rarely an overriding consideration since the enzymes to be puried are usually expensive. Cellulose was employed in 1953 in one of the earliest afnity-chromatography separations, where tyrosinase was puried on a column of aromatic cellulose ethers (27). Currently, cellulose (28) or related polysaccharides (29) are used in a number of applications as a bioselective adsorbent including DNAcellulose for the purication of DNA-binding proteins. Polyacrylamide. Polyacrylamide is familiar to most biochemists, since it serves as the support for polyacrylamide gel electrophoresis and a medium for gel-permeation chromatography (see also ACRYLAMIDE POLYMERS). For the latter purpose, preformed spherical beads, Biogel-P, are available from Bio-Rad Laboratories, Richmond, California. These beads are hydrophilic and chemically stable and have a uniform pore diameter and mesh size. These desired traits, and the fact that the polyacrylamide beads are readily derivatized, suggest that it should be an excellent afnity-chromatographic support. In fact, it is not often used because the beads are not sufciently porous to permit proteins to have access to ligands bound within the beads. The lack of normal porosity for polyacrylamide beads is aggravated by the shrinkage of the gel occurring upon derivatization. As a result, the effective capacity of polyacrylamide gel is very low for all except the smallest enzymes. Several derivatives of polyacrylamide have been formulated that have the capacity for much larger pore sizes and less shrinkage upon derivatization. Trisacryl, a product of LKBIBF, is the polyamide of Tris, tris(hydroxymethyl)aminomethane, and acrylic acid. It is a promising matrix material that can be derivatized by all of the methods available for agarose but with far greater mechanical stability. Czechoslovakian investigators have prepared other hydrophilic gels based on hydroxyalkylmethacrylates (30). These supports exhibit increased porosity, with exclusion limits of up to a molecular weight of 108 . An interesting application of polyacrylamide has been in the technique of afnity electrophoresis, where enzymes are puried on a support medium containing an immobilized bioselective adsorbent (31). This technique has two advantages over afnity chromatography: A highly porous polyacrylamide can be employed because the material does not assume a spherical form but is cast in a glass or plastic supporting-gel tube; and the separation of two or more substances with similar afnity for the same ligand is greatly increased since charge and gel-permeation effects, as well as bioselective adsorption, contribute to the migration of proteins. Phytohemagglutinins have been puried by electrophoresis on acrylamide-derived gels prepared from alkenyl-O-glycosides and a

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bisacrylamide (32). The phytohemagglutinins have also been separated by bioselective adsorption chromatography with a similar glycoside-containing polyacrylamide gel derivative (32). Other Matrices. A large number of other matrices have been employed, including starch (33), cross-linked dextrins (34,35), a vinyl maleimide polymer (36), chitin (37), mannan (38), and insolubilized proteins (39). The number of potential matrices is almost limitless, and most of the matrices used for immobilization of enzymes have potential application in bioselective adsorption. Among these are nylon (40), metal oxides (41), maleic anhydrideethylene co-polymers (42), and polystyrene derivatives (43). Few of these have been used because of their potential for nonspecic adsorption either by charge, as with the metal oxides, or by hydrophobic interactions (as would be the case with polystyrene). The derivatized matrix must be free of nonspecic adsorption. Proper derivatization can eliminate nonspecic adsorption or, as with cyanogen bromide activation, create nonspecic adsorption in matrices having no prior nonspecic adsorption properties.

Ligands
The ligand to be immobilized can be an inhibitor, a substrate, substrate analogue, coenzyme, or any other biomolecule with an afnity for a specic site (active site, allosteric site, membrane-binding site, etc) on the protein to be puried. Such biomolecules are usually small (mol wt 500), although macromolecules have been successfully employed, eg, with the purication of soybean trypsin inhibitor on immobilized trypsin as a bioselective adsorbent. The precise type of ligand for any purication is dictated by the nature of the enzyme and the aim of the investigation. The ligand must have sufcient afnity for the enzyme being puried to retain it on the ligandmatrix complex (44). In addition, the bioselective adsorption of the enzyme to the ligand must exceed the nonspecic adsorption of the proteins to the matrix under the conditions of the preparation. Even under optimum conditions, some nonspecic adsorption is certain to occur. Enzyme adsorption is due to a bioselective component and heterologous, nonspecic factors (45). By altering the chromatographic conditions, eg, ionic strength, pH, and dielectric constant, the ability of the enzyme to be specically adsorbed is enhanced and/or the nonspecic adsorption is decreased. For example, the bioselective afnity of lipoamide dehydrogenase on propyllipoamide glass has been shown to increase with the addition of 10% acetone to the aqueous enzyme solution. A good example of the use of an organic solvent in the elution buffers to eliminate nonspecic binding is the purication of lactate dehydrogenase on N 6 (6-aminohexyl)-AMP-agarose (46). When lactate dehydrogenase is puried on this bioselective adsorbent, yields of only 60% are obtained by elution with the reduced form (NADH) of nicotinamide adenine dinucleotide (NAD+ ). Addition of ethylene glycol, and thus further alteration of the dielectric constant of the elution buffer, was shown to change the conformation of the enzyme, thus lowering its afnity for the immobilized (NAD+ ) analogue. The enzyme was eluted from the column with buffers containing about 43% ethylene glycol, with a recovery of about 45% of the activity.

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The corollary to the requirement of a sufciently large ligandenzyme attraction or binding energy for enzyme retention is that the enzymeligand binding must be loose enough to permit dissociation of the enzyme from the column under appropriate conditions. Such binding can be so tight that enzyme dissociation is extremely difcult. For example, uterine estradiol-receptor proteins can be removed from serum or uterine cellular extracts by exposure to estradiol coupled to various matrices, eg, cellulose, glass, agarose, or a vinyl maleimide polymer (36,44,47,48), but estradiol-receptor protein is not eluted from the bioselective adsorbent by an appropriate method, eg, elution with free estradiol, changes in pH or ionic strength, or even the use of mild denaturants. Normal afnity chromatography is precluded by the inability to elute the puried enzyme-immobilized enzyme complex. Purication can be successful using estradiol immobilized on agarose by an azo linkage. Uterine cell extracts are applied to this column and unbound proteins are removed. The estradiol-receptor proteinadsorbent complex is then treated with hydrosulte to cleave the azo linkage between the agarose and the coupled estradiol, and the resulting estradiol-receptor complex is freed of estradiol by extensive dialysis. Attachment. Numerous methods of attachment of ligands or spacer arms to the appropriate matrix are given in References 44 and 4952. Attachment must be performed in such a way that few charged, ionogenic, or hydrophobic residues remain after derivatization. This fact has only recently been appreciated, and therefore must be kept in mind when reviewing the earlier literature. Cyanogen bromide or bisoxiranes can be employed in reactions similar to those shown earlier for matrix activation. Other methods include azo coupling (51), ester formation (51,52), and amide formation (52).

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Spacer Arms between Matrix and Ligand. Although a ligand may be directly attached to an activated matrix, an intervening organic group usually serves as the point of attachment and/or as a spacer or arm to separate the ligand from the matrix. For example, when a series of eight agaroseinsulin complexes were used for the purication of insulin-receptor protein, the derivative with the longest spacer arm was the most effective (53). Similarly, guanine deaminase was not retained by the inhibitor, 9-phenyl-guanine, when it was bound directly to cyanogen bromide-activated agarose, whereas it is retained when the afnity packing contains a OCH2 CH2 spacer arm between the ligand and the matrix (54). The spacer arm positions the ligand a certain distance from the matrix in order to reduce steric interference from the matrix (see Figs. 2a and 2b). Occasionally a spacer arm may interfere with the bioselective adsorption process because hydrophobic ligands fold back (Fig. 2c).

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Fig. 2. (a) Direct ligand attachment, little or no ligandenzyme interaction; (b) Ligand attachment through a spacer molecule, true ligandenzyme interaction; and (c) Spacer arm too long, the effective distance of ligand from matrix is too short to permit adequate ligandenzyme interaction.

Spacers are not always needed, and if possible, should not be incorporated in the packing since they can contribute to nonspecic adsorption. Early investigators often ignored spacerenzyme interactions, and many results are obscured by the ion-exchange effect of spacers. Ion-exchange has been observed when neuraminidase is puried on a column of p-aminophenyloxamic acid coupled to agarose. Neuraminidase was initially reported to be retained (55) and specically eluted from such a column at pH 5.5, but it has since been shown that these preparations yield neuraminidase contaminated with hemagglutinin, hemolysin, and phospholipase C (56). If the purication is performed at pH 7.5, only the sialidase is retained by the column. This suggests strongly that chiey ion exchange is operating at pH 5.5; at pH 7.5 bioselective adsorption predominates. On the other hand, the use of long hydrophobic side chains as spacers may lead to nonspecic hydrophobic proteinspacer interactions. These observations show that the spacer, as all other components of the system, must have minimal interaction with protein molecules. They also demonstrate that appropriate controls are required to demonstrate that the chromatographic separation is solely a result of bioselective adsorption. Hydrophobic Chromatography. The observation that afnity chromatography separations were attributed chiey to hydrophobic interaction between the spacers and one or more enzymes (57) stimulated the development of so-called hydrophobic chromatography (58). Similar to ion exchange, hydrophobic chromatography depends upon chemiselective adsorption, in this case hydrophobic interaction between the enzyme and an alkyl or aromatic arm bound to an agarose matrix. Afnity chromatography on such materials frequently

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yields active enzyme preparations in cases where conventional ion-exchange chromatography results in substantial, if not total, enzyme denaturation. Thus, isopropylmalate isomerase was puried in the presence of high concentrations of ammonium sulfate and glycerol, both of which stabilize the enzyme (59). The glycerol serves to weaken the afnity of the enzyme for the matrix, whereas ammonium sulfate increases binding.

Applications
Applications of afnity chromatography include (60) (1) Protein purication a. b. c. d. e. Enzymes Antibodies and antigens Binding or receptor proteins Complementary proteins Repressor proteins

(2) Separative procedures a. Cells and viruses b. Denatured and chemically modied proteins from native proteins c. Nucleic acids and nucleotides (3) (4) (5) (6) Concentration of dilute protein solutions Storage of otherwise unstable proteins in immobilized form Investigation of kinetic sequences and mechanisms Medical applications a. Extracorporeal adsorbents b. Immunoassays The most spectacular applications of afnity chromatography are in therapeutic and clinical uses. For example, immunoadsorption chromatography has been used to treat familial hypercholesterolemia (61) by passing the hypercholesterolemic blood through an extracorporeal shunt consisting of a blood cell separator and, on the plasma side, an immunosorbent column containing antibodies to plasma low density lipoprotein (LDL), and nally back to the blood supply. Because elevated blood cholesterol concentrations, which cause heart attacks in the victims, are directly related to high LDL levels, the removal of LDLcholesterol in the extracorporeal shunt effects considerable relief from hypercholesterolemia. After saturation, the immunosorbent anti-LDL column is removed from the extracorporeal shunt and regenerated by elution of the LDL with glycine HCl buffer at pH 3.0 (see Fig. 3). Other important clinical applications include solid-phase (heterogeneous) immunoassays (62). These are widely used in therapeutic drug monitoring, pregnancy testing, and allergy testing, among others, as well as in research.

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Fig. 3. Plasma-separator-membrane system and LDL adsorbent (a) and the continuousow blood-cell-separator centrifuge and immunoadsorbent column (b) in the arteriovenous shunt. Samples were derived from the ow system at A, B, and C in time intervals of 40 min. Pumps of the blood-cell-separator centrifuge (b): 1, citrate anticoagulant (13 mL/min); 2, heparin (1 mL/min, 80,000 units/L of saline); 3, leukocytes; 4, erythrocytes (1040 mL/min); 5, plasma (1040 mL/min); 6, lubrication (1 mL/min, 2000 units of heparin/L of saline) (51).

The largest single application of afnity chromatography is the purication of enzymes and other proteins. As the biotechnological revolution has proceeded, the demand for new DNA-related enzymes has increased. Many of these are only available by means of DNA-afnity chromatography. For example, thermophilic DNA-ligase can be puried (63) from Thermus thermophilus, using DNAcellulose chromatography as the key step. This enzyme, unlike many classical DNA ligases, is extremely heat stable and withstands 37 C for 1 week and 65 C for 2 days. This is only one of hundreds, if not thousands, of enzymes prepared by means of afnity chromatography that have been commercialized or have commercial potential.

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An area of growing importance is the purication of fusion proteins produced by fusing the coding sequence for a desired protein with a second sequence, or tag, engineered for the future purication of the fusion product. Usually a unique sequence is inserted between these two such that the desired protein can readily be cleaved from the tag using a very selective protease. While many such tag sequences exist, the most popular is a poly-6-histidine that readily binds via a chemiselective process to an immobilized metal ion chelating column or IMAC (64,65). Excellent examples are the purication of recombinant chloramphenicol acetyltransferase, dihydrooate reductase, and green uorescent protein, each one of which was fused to a natural polyhistidine tag consisting of a 19 amino acid sequence from lactate dehydrogenase (66). All three fusion proteins were puried on a Co2+ -carboxymethylaspartate agarose Superow. The latter is synthesized from Superow agarose, which is one of the new generation highly cross-linked matrices produced for commercial high ow rates and high dynamic capacities. Purities greater than 95% could be obtained with yields greater than 75%. The enzyme -galactosidase was puried directly from crude cell extracts using expanded bed chromatography on another second generation matrix, Ni2+ loaded streamline chelating resin. The natural histidine content of the enzyme permitted the enzyme to be recovered in almost 90% yield and 5.95-fold purication from a crude unclaried cell extract (67). -Galactosidase, the enzyme missing in Gauchers disease, has been the subject of numerous afnity-chromatographic preparations, both because of its clinical importance and its potential use in the food industry. Reports on galactosidase purication by afnity chromatography have been obscured because a weak inhibitor, p-aminophenyl--D-thiogalactose, was used as ligand in many of the earlier studies. These purications of this enzyme using this inhibitor occur through a combination of ion-exchange and hydrophobic chromatography (12,57). For example, -galactosidase was puried on alkyl amine-glass coupled to p-aminophenyl--D-thiogalactose via malonic and azelaic spacer arms (20). With a control column containing aniline coupled to the controlled-pore glass in the same way as the inhibitor, signicant purication of the enzyme was obtained. Similar results were found with aniline, or the inhibitor p-aminophenyl--D-thiogalactose, coupled to agarose or glass by means of an anilide or azo linkage. A growing tendency in afnity chromatography is to utilize combinatorial libraries and phage display techniques to identify new ligands for protein purication. For example, the recombinant human insulin precursor protein, M13, has been puried by using a biomimetic ligand produced using a combinatorial system (68). Initially the x-ray structure of the protein was used to design a ligand that putatively would bind to a known portion of the protein sequence. This was followed by combinatorial synthesis of a group of ligands similar to the designed ligand based on the reaction of various amines with cyanuric acid (trichlorotriazine) bound through one of its reactive chlorine residues to amino agarose. The amines reacted sequentially with the remaining reactive chlorine residues, yielding completely substituted triazines bound to agarose. When these were screened for their M13 protein binding ability, the result was an afnity matrix used to purify the protein to over 95% purity with a recovery above 90%.

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Similarly small protein ligands have been created by selection from randomized Protein A receptor domain using phage display selection techniques from about a 40-million-member library (69). The selected peptides, termed afbodies, were so stable that the peptides, immobilized on HiTrap NHS Sepharose, could be repetitively sanitized by treatment with 0.5 M sodium hydroxide. Lastly, afnity chromatography has been utilized as a basic research tool to study proteins and enzymes. In one such study heparinagarose was used to purify the heparinbinding domain of platelet thrombospondin (70). The thrombospondin was rst puried from outdated blood platelets using successive afnity chromatography on heparinagarose and gelatinagarose. The puried enzyme was then partially proteolytically digested with thermolysin or plasmin. The lysate was chromatographed on heparinagarose to yield puried heparin-binding fragments. Similarly, the dissociation constant of staphlococcal nuclease was determined for free and matrix-bound thymidine-3 -(p-aminophenylphosphate)- 5 -phosphate, a competitive inhibitor of staphlococcal nuclease (71). The dissociation constant for the soluble ligand that was determined by using quantitative afnity chromatography closely correlated with the value determined by traditional techniques. An afnity chromatographic method was used for kinetic studies of lactate dehydrogenase (72). The enzyme was applied to a bioselective matrix consisting of oxamate covalently coupled to agarose in the presence or absence of the reduced form (NADH) of NAD. With NADH present at concentrations of 102 M or higher, the enzyme was retained, but when NADH was removed from the elution buffer, the enzyme was eluted. This conrms the previous hypothesis of a compulsory-ordered mechanism for lactate dehydrogenase, that is, a mechanism in which NADH binding must precede substrate binding. Further studies using the same procedure demonstrated that the nicotinamide portion, but not the adenosine portion, of NADH was needed for the substrateenzyme interaction. Immobilized Biochemicals. Immobilized biomolecules, or bioreagents, are being synthesized in increasing numbers. For example, dihydroxyboryl cellulose has been employed to purify and study sugars and nucleic acids. Aminopropyl glass and its p-phenylene diisocyanate derivative have been employed as a support for solid-phase Edman degradation of peptides (73). Enormous advances including the synthesis of an active enzyme, ribonuclease A (74), have been made in the synthesis of peptides by the Merrield solid-phase method. A mild reducing agent, the dihydrolipoyl derivative of aminopropyl glass, has been employed for the reduction of various protein and peptide disuldes (18). An immobilized mild oxidizing reagent has also been prepared by the azo coupling of methylene blue to porous glass (75). This reagent photolytically catalyzes the formation of singlet oxygen, which in turn oxidizes protein methionyl, tyrosyl, and tryptophenyl residues. Under certain conditions this can be limited to the oxidation of methionine. When lysozyme, for example, is photo-oxidized in the presence of methylene blue in 84% acetic acid, only 5% of the activity is retained (76). This reduction results from the oxidation of one protein methionine to methionine sulfoxide, a process that is reversed by reducing the enzyme methionine sulfoxide with thiols, thus restoring 85% of the original activity.

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Numerous other examples of both bioselective adsorption and the use of immobilized biomolecules have been reported. Many new applications can be anticipated in the near future.

BIBLIOGRAPHY
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GENERAL REFERENCES
Refs. 1, 3, 4, 8, 4654, and 63 are also good general references. Ref. 1 has an excellent detailed discussion of techniques A. H. Nishikawa, in W. H. Scouten, ed., Solid Phase Biochemistry: Analytical and Synthetic Aspects (Chemical Analysis: A Series of Monographs on Analytical Chemistry and its Applications), WileyInterscience, New York, 1983, p. 17. W. H. Scouten, Am. Lab. 6, 23 (1974). C. R. Lowe, Introduction to Afnity Chromatography, Elsevier, Amsterdam, 1979.

WILLIAM H. SCOUTEN University of Texas

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