Chromatography Affinity

8/6/2019 Chromatography Affinity

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580 CHITIN AND CHITOSAN Vol. 1

CHROMATOGRAPHY, AFFINITY

Introduction

One of the most important developments in enzymology has been the introduction

of a rapid and highly efficient means of protein purification utilizing the highly

selective ability of enzymes to recognize certain biological compounds or their

analogues. This technique, termed affinity chromatography, involves the immobi-

lization 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 contam-inating proteins have no such recognition. Virtually hundreds of proteins have

been purified in this way, using a wide variety of bioligands immobilized in a ma-

trix; enzyme inhibitors, coenzymes, antibodies, and even other enzymes may be

useful bioligands in the purification of a particular protein (1). A classification of

the types of affinity chromatography is as follows (2):

Bioselective adsorption is the process where the affinity is based on biologi-

cally relevant binding. It includes group-specific ligands, eg, lectins and nucleotide

cofactors (NAD, AMP), and specific ligands, eg, certain less common cofactors (vi-

tamin B12), receptor proteins, and antibodies as used in immunosorbents.Chemiselective adsorption is the process where the affinity is based on chem-

ically defined nonbiological interactions. It includes hydrophobic chromatography,

ion-exchange chromatography, covalent chromatography (active thiols, Hg2+, etc),

and borate complexes.The basic application of affinity chromatography involves several steps, as

illustrated in Figure 1. First, the necessary affinity-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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Vol. 1 CHROMATOGRAPHY, AFFINITY 581

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

enzyme for the adsorbent. This is normally achieved by dialysis or enzyme precip-

itation. The crude extract is applied to the column, and contaminating proteins,

which ideally have no affinity for the column under the conditions employed, are

removed by washing. The protein to be purified remains bound to the column

because of its affinity for the immobilized ligand. It is subsequently removed by

eluting the column with a solution of the free ligand. Alternatively the enzymemay 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 affinity

for the immobilized ligand.

Efficient purification by affinity chromatography depends on the nature of

the ligand, the methods used in the preparation of the chromatographic packing,


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582 CHROMATOGRAPHY, AFFINITY Vol. 1

the type of inert matrix, and the methods for both attaching the ligand to

the matrix and eluting the enzyme from the bioselective adsorbent. Nonspe-

cific adsorption of proteins, which can be defined as the retention of proteins

by some general factor, eg, ion exchange, hydrophobic interactions, and charge-

transfer complexation, may cause a large variety of proteins to be nonspecifically

retained.

Finally, it must be decided whether affinity chromatography is employed

primarily for purification or primarily as a method for studying enzymesubstrate

interaction. The choice of the final 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 highpressure, require beads with high mechanical stability. Affinity purification as

a final step following a long series of conventional procedures requires a small

column. When affinity chromatography is employed in the last stages of purifi-

cation as a polishing step, high yields of the valuable partially purified enzyme

are especially important. Even a small percentage of nonspecific 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 nonspecific

adsorption is essential.

Nonspecific adsorption must not occur in the derivatized matrix, even

though the untreated matrix might have considerable nonspecific 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 affinity chromatography (hplac), a rapidly grow-

ing technique, the matrix must be mechanically stable and capable of sustain-

ing 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 chiefly because of the hydrophilicity of the under-

ivatized matrix (5). It might be thought that derivatized agarose would not have

more nonspecific adsorption than its untreated counterpart. However, this is not

the case, and this observation confirms 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 nonspecific adsorption, the chemicalstability and ease of derivatization of the matrix must be considered. Agarose

provides the least nonspecific 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 nonspecific adsorption.


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Other matrices have certain advantages and disadvantages for specific

applications. Regenerated cellulose, polyacrylamide, and cross-linked dextrans

generally yield high capacity adsorbents as defined 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 ac-

cessible 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 affinitychromatography. It is a linear polysaccharide containing alternating residues ofD-galactose and 3,6-anhydro-L-galactose (6).

Agarose is prepared by mixing a hot aqueous solution of specially purified

agar (26%) with an organic solvent, eg mineral oil, and a small amount of de-

tergent. 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 de-creases porosity.

The agar base, and thus agarose, is a naturally occurring polysaccharide

with many ionic residues, chiefly 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 significant 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 100C

or in 4 M sodium iodide, whereas agarose cross-linked with epichlorohydrin is

essentially unaffected under the same condition (


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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 exam-

ined 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 purification based on bioselective adsorption, or

affinity chromatography, was in reality a specifically tailored ion-exchange chro-

matography, which may or may not have given better results than classical ion-

exchange methods (12).

Even though there is some question about the mode of action of some bioselec-

tive adsorbents based on cyanogen bromide activation, enzymes can be purified

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(2,3-

epoxypropoxy)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 Is-rael (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 beperformed on columns of porous glass with excellent results (2).

Controlled-pore glass was developed when it was discovered that borosili-

cate glass heated to 700800C 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 additionalalkali 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 large-

pore-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 finely 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 nonspecific

adsorption

Early synthetic procedures for preparing glass for affinity chromatography orenzyme immobilization involved refluxing -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 75C (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 nonspecific adsorption in cer-

tain enzyme preparations, others do not (18). Nonspecific adsorption is minimized

by coating the glass with glycidoxypropyltrimethoxysilane, followed by appropri-

ate 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 deriva-

tized in a manner similar to that used for agarose, although the same reservationexpressed above for cyanogen bromide activation of agarose, namely its ionogenic

nature, applies to glycidoxy controlled-pore glass.

-Galactosidase has been purified from cell-free extracts ofAspergillus 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

purification of-galactosidase (20). The aniline-modified material has a capacity

75% of that containing the bound inhibitor and produces -galactosidase with

about 50% of the specific activity of that produced with the affinity packing con-

sisting 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 in-teractions are not known.

Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) polymerases have

been separated and purified on DNA-glass columns, effecting more rapid purifica-

tion than affinity 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 affinity-chromatography procedure (21) de-pends upon the physical trapping of DNA fibers in a mesh of cellulose fibers. The

DNAcellulose is prepared by drying DNA and cellulose together in an appropri-

ate 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 affinity-chromatographic matrix; its principal advan-

tage is its low cost. This is rarely an overriding consideration since the enzymes

to be purified are usually expensive. Cellulose was employed in 1953 in one of

the earliest affinity-chromatography separations, where tyrosinase was purified

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 adsor-

bent including DNAcellulose for the purification of DNA-binding proteins.Polyacrylamide. Polyacrylamide is familiar to most biochemists, since itserves 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 affinity-chromatographic support. In fact, it

is not often used because the beads are not sufficiently 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 deriva-

tization. Trisacryl, a product of LKBIBF, is the polyamide of Tris,

tris(hydroxymethyl)aminomethane, and acrylic acid. It is a promising matrix ma-

terial 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

affinity electrophoresis, where enzymes are purified on a support medium con-

taining an immobilized bioselective adsorbent (31). This technique has two ad-

vantages over affinity chromatography: A highly porous polyacrylamide can be

employed because the material does not assume a spherical form but is castin a glass or plastic supporting-gel tube; and the separation of two or more

substances with similar affinity 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 purified by elec-

trophoresis on acrylamide-derived gels prepared from alkenyl-O-glycosides and a


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bisacrylamide (32). The phytohemagglutinins have also been separated by biose-

lective adsorption chromatography with a similar glycoside-containing polyacry-

lamide gel derivative (32).Other Matrices. A large number of other matrices have been employed, in-

cluding 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 poten-

tial for nonspecific 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 nonspecific adsorption. Proper derivatization can eliminate

nonspecific adsorption or, as with cyanogen bromide activation, create nonspecific

adsorption in matrices having no prior nonspecific adsorption properties.

Ligands

The ligand to be immobilized can be an inhibitor, a substrate, substrate ana-

logue, coenzyme, or any other biomolecule with an affinity for a specific site (ac-

tive site, allosteric site, membrane-binding site, etc) on the protein to be purified.

Such biomolecules are usually small (mol wt 500), although macromolecules have

been successfully employed, eg, with the purification of soybean trypsin inhibitor

on immobilized trypsin as a bioselective adsorbent. The precise type of ligand

for any purification is dictated by the nature of the enzyme and the aim of the

investigation.

The ligand must have sufficient affinity for the enzyme being purified to re-

tain it on the ligandmatrix complex (44). In addition, the bioselective adsorption

of the enzyme to the ligand must exceed the nonspecific adsorption of the proteinsto the matrix under the conditions of the preparation. Even under optimum con-

ditions, some nonspecific adsorption is certain to occur. Enzyme adsorption is due

to a bioselective component and heterologous, nonspecific factors (45).

By altering the chromatographic conditions, eg, ionic strength, pH, and di-

electric constant, the ability of the enzyme to be specifically adsorbed is enhanced

and/or the nonspecific adsorption is decreased. For example, the bioselective affin-

ity of lipoamide dehydrogenase on propyllipoamide glass has been shown to in-

crease 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 nonspecific binding is the purification of lactate dehydrogenase on N6-

(6-aminohexyl)-AMP-agarose (46). When lactate dehydrogenase is purified on this

bioselective adsorbent, yields of only 60% are obtained by elution with the reduced

form (NADH) of nicotinamide adenine dinucleotide (NAD+). Addition of ethyleneglycol, and thus further alteration of the dielectric constant of the elution buffer,

was shown to change the conformation of the enzyme, thus lowering its affinity

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 sufficiently large ligandenzyme at-

traction or binding energy for enzyme retention is that the enzymeligand bind-

ing must be loose enough to permit dissociation of the enzyme from the column

under appropriate conditions. Such binding can be so tight that enzyme dissocia-

tion is extremely difficult. 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 ad-

sorbent by an appropriate method, eg, elution with free estradiol, changes in pH or

ionic strength, or even the use of mild denaturants. Normal affinity chromatogra-

phy is precluded by the inability to elute the purified enzyme-immobilized enzyme

complex. Purification 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 hydrosulfite 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 armsto 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 bedirectly 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 com-

plexes were used for the purification of insulin-receptor protein, the derivative

with the longest spacer arm was the most effective (53). Similarly, guanine deam-

inase 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

affinity packing contains a OCH2CH2 spacer arm between the ligand and thematrix (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). Occasion-

ally 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) Ligandattachment through a spacer molecule, true ligandenzyme interaction; and (c) Spacerarm too long, the effective distance of ligand from matrix is too short to permit adequateligandenzyme interaction.

Spacers are not always needed, and if possible, should not be incorporated in

the packing since they can contribute to nonspecific adsorption. Early investiga-

tors often ignored spacerenzyme interactions, and many results are obscured

by the ion-exchange effect of spacers. Ion-exchange has been observed when

neuraminidase is purified on a column of p-aminophenyloxamic acid coupled to

agarose. Neuraminidase was initially reported to be retained (55) and specificallyeluted from such a column at pH 5.5, but it has since been shown that these prepa-

rations yield neuraminidase contaminated with hemagglutinin, hemolysin, and

phospholipase C (56). If the purification is performed at pH 7.5, only the siali-

dase is retained by the column. This suggests strongly that chiefly 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 nonspecific 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 con-

trols are required to demonstrate that the chromatographic separation is solely a

result of bioselective adsorption.Hydrophobic Chromatography. The observation that affinity chro-

matography separations were attributed chiefly to hydrophobic interaction be-tween the spacers and one or more enzymes (57) stimulated the development of

so-called hydrophobic chromatography (58). Similar to ion exchange, hydropho-

bic chromatography depends upon chemiselective adsorption, in this case hy-

drophobic interaction between the enzyme and an alkyl or aromatic arm bound

to an agarose matrix. Affinity chromatography on such materials frequently


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yields active enzyme preparations in cases where conventional ion-exchange chro-

matography results in substantial, if not total, enzyme denaturation. Thus, -

isopropylmalate isomerase was purified in the presence of high concentrations

of ammonium sulfate and glycerol, both of which stabilize the enzyme (59). The

glycerol serves to weaken the affinity of the enzyme for the matrix, whereas am-

monium sulfate increases binding.

Applications

Applications of affinity chromatography include (60)

(1) Protein purification

a. Enzymes

b. Antibodies and antigens

c. Binding or receptor proteinsd. Complementary proteins

e. Repressor proteins

(2) Separative procedures

a. Cells and viruses

b. Denatured and chemically modified proteins from native proteins

c. Nucleic acids and nucleotides

(3) Concentration of dilute protein solutions

(4) Storage of otherwise unstable proteins in immobilized form

(5) Investigation of kinetic sequences and mechanisms

(6) Medical applications

a. Extracorporeal adsorbents

b. Immunoassays

The most spectacular applications of affinity chromatography are in ther-

apeutic and clinical uses. For example, immunoadsorption chromatography has

been used to treat familial hypercholesterolemia (61) by passing the hypercholes-

terolemic blood through an extracorporeal shunt consisting of a blood cell separa-

tor and, on the plasma side, an immunosorbent column containing antibodies to

plasma low density lipoprotein (LDL), and finally back to the blood supply.

Because elevated blood cholesterol concentrations, which cause heart at-

tacks in the victims, are directly related to high LDL levels, the removal of LDL-

cholesterol in the extracorporeal shunt effects considerable relief from hyperc-

holesterolemia. After saturation, the immunosorbent anti-LDL column is removedfrom 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, preg-

nancy 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 continuous-flow blood-cell-separator centrifuge and immunoadsorbent column (b) in the arteriovenousshunt. Samples were derived from theflow 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 affinity chromatography is the purification

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-affinity chromatography. For example, thermophilicDNA-ligase can be purified (63) from Thermus thermophilus, using DNAcellulose

chromatography as the key step. This enzyme, unlike many classical DNA ligases,

is extremely heat stable and withstands 37C for 1 week and 65C for 2 days. This

is only one of hundreds, if not thousands, of enzymes prepared by means of affinity

chromatography that have been commercialized or have commercial potential.


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An area of growing importance is the purification of fusion proteins produced

by fusing the coding sequence for a desired protein with a second sequence, or

tag, engineered for the future purification 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 chelat-

ing column or IMAC (64,65).

Excellent examples are the purification of recombinant chloramphenicol

acetyltransferase, dihydrofloate reductase, and green fluorescent 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 purified

on a Co2+-carboxymethylaspartate agarose Superflow. The latter is synthesized

from Superflow agarose, which is one of the new generation highly cross-linked

matrices produced for commercial high flow rates and high dynamic capacities.

Purities greater than 95% could be obtained with yields greater than 75%.

The enzyme -galactosidase was purified directly from crude cell extractsusing expanded bed chromatography on another second generation matrix, Ni 2+

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 purifica-

tion from a crude unclarified cell extract (67).

-Galactosidase, the enzyme missing in Gauchers disease, has been the

subject of numerous affinity-chromatographic preparations, both because of its

clinical importance and its potential use in the food industry. Reports on -

galactosidase purification by affinity chromatography have been obscured because

a weak inhibitor, p-aminophenyl--D-thiogalactose, was used as ligand in many

of the earlier studies. These purifications of this enzyme using this inhibitor

occur through a combination of ion-exchange and hydrophobic chromatography

(12,57). For example, -galactosidase was purified on alkyl amine-glass coupled to

p-aminophenyl--D-thiogalactose via malonic and azelaic spacer arms (20). With acontrol column containing aniline coupled to the controlled-pore glass in the same

way as the inhibitor, significant purification of the enzyme was obtained. Similar

results were found with aniline, or the inhibitorp-aminophenyl--D-thiogalactose,

coupled to agarose or glass by means of an anilide or azo linkage.

A growing tendency in affinity chromatography is to utilize combinato-

rial libraries and phage display techniques to identify new ligands for protein

purification.

For example, the recombinant human insulin precursor protein, M13, has

been purified by using a biomimetic ligand produced using a combinatorial sys-

tem (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 (trichlorotri-azine) bound through one of its reactive chlorine residues to amino agarose. The

amines reacted sequentially with the remaining reactive chlorine residues, yield-

ing completely substituted triazines bound to agarose. When these were screened

for their M13 protein binding ability, the result was an affinity 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 random-

ized Protein A receptor domain using phage display selection techniques from

about a 40-million-member library (69). The selected peptides, termed affibodies,

were so stable that the peptides, immobilized on HiTrap NHS Sepharose, could

be repetitively sanitized by treatment with 0.5 M sodium hydroxide.

Lastly, affinity 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 throm-

bospondin was first purified from outdated blood platelets using successive affin-

ity chromatography on heparinagarose and gelatinagarose. The purified en-

zyme was then partially proteolytically digested with thermolysin or plasmin. The

lysate was chromatographed on heparinagarose to yield purified 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 affinity chro-matography closely correlated with the value determined by traditional tech-

niques. An affinity 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 confirms the previous hypoth-

esis 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 cellu-

lose has been employed to purify and study sugars and nucleic acids. Aminopropyl

glass and itsp-phenylene diisocyanate derivative have been employed as a support

for solid-phase Edman degradation of peptides (73). Enormous advances includ-

ing the synthesis of an active enzyme, ribonuclease A (74), have been made in the

synthesis of peptides by the Merrifield solid-phase method.

A mild reducing agent, the dihydrolipoyl derivative of aminopropyl glass, has

been employed for the reduction of various protein and peptide disulfides (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 oxida-

tion of methionine. When lysozyme, for example, is photo-oxidized in the presenceof 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 sul-

foxide, 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 an-

ticipated in the near future.

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W. H. Scouten, Baylor University.

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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 Syn-

thetic 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 Affinity Chromatography, Elsevier, Amsterdam, 1979.

WILLIAM H. SCOUTEN

University of Texas

Chromatography Affinity

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