580 CHITIN AND CHITOSAN Vol.

CHROMATOGRAPHY, AFFINITY

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 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 puried 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 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 biologi-
cally relevant binding. It includes group-specic ligands, eg, lectins and nucleotide
cofactors (NAD, AMP), and specic ligands, eg, certain less common cofactors (vi-
tamin B12 ), receptor proteins, and antibodies as used in immunosorbents.
Chemiselective adsorption is the process where the afnity is based on chem-
ically 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.
Vol. 1 CHROMATOGRAPHY, AFFINITY 581

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) Un-
wanted 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 precip-
itation. 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,
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-
cic adsorption of proteins, which can be dened 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 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 puri-
cation 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 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 chiey because of the hydrophilicity of the under-
ivatized 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.
Vol. 1 CHROMATOGRAPHY, AFFINITY 583

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 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 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 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, 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 acti-
vated 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:
584 CHROMATOGRAPHY, AFFINITY Vol. 1

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 purication based on bioselective adsorption, or
afnity chromatography, was in reality a specically 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 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(2,3-
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.
Vol. 1 CHROMATOGRAPHY, AFFINITY 585

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 be

performed on columns of porous glass with excellent results (2).
Controlled-pore glass was developed when it was discovered that borosili-
cate 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 large-
pore-diameter glass, up to about 400 nm, has been employed.
586 CHROMATOGRAPHY, AFFINITY Vol. 1

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 cer-
tain enzyme preparations, others do not (18). Nonspecic 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 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 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 puried on DNA-glass columns, effecting more rapid purica-
tion than afnity chromatography on DNAcellulose (21,22). The DNA is coupled
Vol. 1 CHROMATOGRAPHY, AFFINITY 587

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) de-
pends 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 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 afnity-chromatographic matrix; its principal advan-
tage 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 adsor-
bent 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 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
afnity electrophoresis, where enzymes are puried on a support medium con-
taining an immobilized bioselective adsorbent (31). This technique has two ad-
vantages 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 elec-
trophoresis on acrylamide-derived gels prepared from alkenyl-O-glycosides and a
588 CHROMATOGRAPHY, AFFINITY Vol. 1

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 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 ana-

logue, coenzyme, or any other biomolecule with an afnity for a specic site (ac-
tive 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 re-
tain 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 con-
ditions, 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 di-
electric constant, the ability of the enzyme to be specically adsorbed is enhanced
and/or the nonspecic adsorption is decreased. For example, the bioselective afn-
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 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.
Vol. 1 CHROMATOGRAPHY, AFFINITY 589

The corollary to the requirement of a sufciently 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 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 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 afnity chromatogra-
phy 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).
590 CHROMATOGRAPHY, AFFINITY Vol. 1

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 com-
plexes were used for the purication 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
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). Occasion-
ally a spacer arm may interfere with the bioselective adsorption process because
hydrophobic ligands fold back (Fig. 2c).
Vol. 1 CHROMATOGRAPHY, AFFINITY 591

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 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 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 prepa-
rations yield neuraminidase contaminated with hemagglutinin, hemolysin, and
phospholipase C (56). If the purication is performed at pH 7.5, only the siali-
dase 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 con-
trols are required to demonstrate that the chromatographic separation is solely a
result of bioselective adsorption.
Hydrophobic Chromatography. The observation that afnity chro-
matography separations were attributed chiey 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. Afnity chromatography on such materials frequently
592 CHROMATOGRAPHY, AFFINITY Vol. 1

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

matography 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 am-
monium sulfate increases binding.

Applications

Applications of afnity chromatography include (60)

(1) Protein purication

a. Enzymes
b. Antibodies and antigens
c. Binding or receptor proteins
d. Complementary proteins
e. Repressor proteins
(2) Separative procedures
a. Cells and viruses
b. Denatured and chemically modied 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 afnity 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 nally 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 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, preg-
nancy testing, and allergy testing, among others, as well as in research.
Vol. 1 CHROMATOGRAPHY, AFFINITY 593

Fig. 3. Plasma-separator-membrane system and LDL adsorbent (a) and the continuous-
ow 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.
594 CHROMATOGRAPHY, AFFINITY Vol. 1

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 chelat-
ing 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 purica-
tion 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 combinato-
rial 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 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 afnity matrix used to
purify the protein to over 95% purity with a recovery above 90%.
Vol. 1 CHROMATOGRAPHY, AFFINITY 595

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 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 throm-
bospondin was rst puried from outdated blood platelets using successive afn-
ity chromatography on heparinagarose and gelatinagarose. The puried en-
zyme 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 chro-
matography closely correlated with the value determined by traditional tech-
niques. 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 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 its p-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 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 oxida-
tion 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 sul-
foxide, a process that is reversed by reducing the enzyme methionine sulfoxide
with thiols, thus restoring 85% of the original activity.
596 CHROMATOGRAPHY, AFFINITY Vol. 1

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.

BIBLIOGRAPHY

Afnity Chromatography in EPST 1st ed., Suppl. Vol. 2, pp. 1938, by W. H. Scouten, Sr.,
Bucknell University; Chromatography, Afnity, in EPSE 2nd ed., Vol. 3, pp. 531548; by
W. H. Scouten, Baylor University.
1. J. Turkova, Bioafnity Chromatography, 2nd ed., Elsevier, Amsterdam, 1993.
2. W. H. Scouten, ed., Solid Phase Biochemistry, Wiley-Interscience, New York, 1983, p.
1.
3. W. H. Scouten, Afnity Chromatography, Wiley-Interscience, New York, 1981, includes
detailed discussion of techniques.
4. Pierce Reviews, Pierce Chemical Co., Rockford, Ill., Jan. 1975.
5. J. Porath and co-workers, J. Chromatogr. 86, 53 (1973).
6. T. La as,
J. Chromatogr. 111, 373 (1975).
7. C. Araki, Bull. Chem. Soc. Jpn. 29, 543 (1966).
8. J. Porath, Biochimie 55, 943 (1973).
9. R. Axen, J. Porath, and S. Ernback, Nature (London) 214, 1302 (1967).
10. J. Kohn and M. Wilchek, in Ref. 1, p. 599.
11. J. Kohn and M. Wilchek, Anal. Biochem. 115, 375 (1981).
12. A. H. Nishikawa and P. Bailon, Arch. Biochem. Biophys. 168, 576 (1975).
13. L. Sundberg and J. Porath, J. Chromatogr. 90, 87 (1974).
14. K. Nilsson and K. Mosbach, Biochem. Biophys. Res. Commun. 102, 449 (1981).
15. W. J. Haller, Chem. Phys. 42, 686 (1961).
16. H. H. Weetall and A. M. Filbert, in W. B. Jakoby and M. Wilchek, eds., Methods in
Enzymology, Vol. 34, Academic Press, Inc., New York, 1974, p. 59.
17. W. Haller, Nature 206, 693 (1965).
18. W. H. Scouten, Chem. Phys. 42, 288 (1961).
19. J. H. Woychik and M. V. Wondolowski, Biochim. Biophys. Acta 289, 347 (1972).
20. G. Baum, J. Chromatogr. 104, 105 (1975).
21. B. M. Alberts and co-workers, Cold Spring Harbor Symp. Quant. Biol. 30, 289
(1968).
22. R. M. Litman, J. Biol. Chem. 243, 6222 (1968).
23. M. S. Poonian, A. J. Schlabach, and A. Weissbach, Biochemistry 10, 424 (1971).
24. S. Ohlson and co-workers, FEBS Lett. 93, 5 (1978).
25. P. O. Larsson and co-workers, in C. Giddings and co-workers, eds., Advances in Chro-
matography, Vol. 21, Marcel Dekker, Inc., New York, 1983, p. 41.
26. S. Ohlson and M. Glad, in I. Chaiken, M. Wilchek, and I. Parikh, eds., Afnity Chro-
matography and Biological Recognition, Academic Press, Inc., New York, 1983, p. 241.
27. L. S. Lerman, Proc. Natl. Acad. Sci. U.S.A. 39, 232 (1953).
28. J. R. Uren, Biochim. Biophys. Acta 236, 67 (1971); G. W. Jameson and D. T. Elmore,
Biochem. J. 124, 66 (1974).
29. D. Rickwood, Biochim. Biophys. Acta 269, 47 (1972).
30. J. Turkova, J. Chromatogr. 91, 267 (1974).
31. V. Horejsi and J. Kocourek, Biochim. Biophys. Acta 336, 338 (1974).
32. V. Horejsi and J. Kocourek, Biochim. Biophys. Acta 297, 346 (1973).
33. P. Cuatrecasas, Nature 228, 1327 (1970).
34. L. Eldjarn and E. Jellum, Acta Chem. Scand. 17, 2610 (1963).
Vol. 1 CHROMATOGRAPHY, AFFINITY 597

35. R. Axen and J. Porath, Nature 210, 367 (1966).

36. B. Vonderharar and G. C. Mueller, Biochim. Biophys. Acta 176, 626 (1969).
37. T. Imoto and K. Yagishita, Agric. Biol. Chem. 37, 1191 (1973).
38. J. Sandula and L. Kuniak, J. Chromatogr. 91, 293 (1975).
39. S. Tomino and K. Paigen, The Lactose Operon, Cold Spring Harbor Symposium, Cold
Spring Harbor, New York, 1970, p. 233.
40. W. E. Hornby and H. Filipusson, Biochim. Biophys. Acta 220, 343 (1970).
41. H. H. Weetall and L. S. Hersh, Biochim. Biophys. Acta 206, 54 (1970).
42. H. Fritz and co-workers, Angew. Chem. 78, 775 (1966).
43. W. Ledingham and W. Hornby, FEBS Lett. 5, 118 (1969).
44. P. Cuatrecasas and C. B. Annsen, Annu. Rev. Biochem. 40, 259 (1971).
45. Ref. 2, p. 44.
46. C. R. Lowe and K. Mosbach, Eur. J. Biochem. 52, 99 (1975).
47. E. V. Jensen, E. P. De Sombro, and P. W. Jungblut, in L. Martini, F. Frascini, and M.
Motta, eds., Hormonal Steroids, Excerpta Median Foundation, Amsterdam, 1967, p.
492.
48. H. Truong and co-workers, FEBS Lett. 35, 289 (1973).
49. P. Cuatrecasas, in G. R. Stark, ed., Biochemical Aspects of Reactions on Solid Supports,
Academic Press, Inc., New York, 1971, p. 79.
50. P. Cuatrecasas, Agric. Food Chem. 19, 600 (1971).
51. H. H. Weetall, Sep. Purif. Methods 2, 199 (1973).
52. W. B. Jakoby and M. Wilchek, Methods in Enzymology, Vol. 34, Academic Press, Inc.,
New York, 1974.
53. P. Cuatrecasas, Proc. Natl. Acad. Sci. U.S.A. 69, 1277 (1972).
54. B. R. Baker and H. U. Siebeneick, J. Med. Chem. 14, 799 (1974).
55. P. Cuatrecasas and G. Illiano, Biochem. Biophys. Res. Commun. 44, 178 (1971).
56. J. I. Rood and R. G. Wilkinson, Biochim. Biophys. Acta 334, 168 (1974).
57. B. H. J. Hofstee, Biochem. Biophys. Res. Commun. 50, 751 (1973).
58. B. H. J. Hofstee, Enzyme Technol. Dig. 2, 90 (1974); S. Shaltiel and Z. Er-el, Proc. Natl.
Acad. Sci. U.S.A. 70, 778 (1973); Z. Er-el, Y. Zaidenzaig, and S. Shaltiel, Biochem.
Biophys. Res. Commun. 49, 383 (1972).
59. R. Bigelis and H. E. Umbarger, J. Biol. Chem. 250, 4315 (1975).
60. C. R. Lowe and P. D. G. Dean, Afnity Chromatography, John Wiley & Sons, Inc.,
London, 1974.
61. W. Stoffel and T. Demant, Proc. Natl. Acad. Sci. U.S.A. 78, 611 (1981).
62. M. Oellich, in H. U. Bergmeyer, ed., Methods of Enzymates Analyses, 3rd ed., Vol. I,
Verlog Chemie, Weinheim, Germany, 1983, p. 233.
63. M. Takahashi, E. Yamagushi, and T. Uchida, J. Biol. Chem. 259, 10041 (1984).
64. L. Kagedal, in J.-C. Janson and L. Ryden, eds., Protein Purication, 2nd ed., Wiley-Liss,
New York, 1998, p. 311.
65. J. Portah and co-workers, Nature (London) 258, 598 (1975).
66. S. F. Teng and co-workers, J. Chromatogr. B 740, 1 (2000)
67. R. H. Clemmitt and H. A. Chase, J. Chromatogr., A 874, 27 (2000)
68. K. Sproule and co-workers, J. Chromatogr., B 740, 17 (2000)
69. K. Nord and co-workers, J. Biotechnol. 80, 45 (2000)
70. V. V. Dixit and co-workers, J. Biol. Chem. 259, 10100 (1984).
71. B. M. Dunn and I. M. Chaiken, Biochemistry 14, 2343 (1975).
72. P. OCarra and S. Barry, FEBS Lett. 21, 281 (1972).
73. E. Wachter and co-workers, FEBS Lett. 35, 97 (1973).
74. R. B. Merrield, J. Am. Chem. Soc. 85, 2149 (1963).
75. C. Lewis and W. H. Scouten, J. Chem. Educ. 53, 395 (1976).
76. G. Jori, G. Galiazzo, and E. Scoffone, Biochemistry 8, 2868 (1969).
598 CHROMATOGRAPHY, AFFINITY Vol. 1

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 Afnity Chromatography, Elsevier, Amsterdam, 1979.

WILLIAM H. SCOUTEN
University of Texas