[311               AFFINITY CHROMATOGRAPHY            345

[ 3 l] Affinity Chromatography

By PEDRO CUATRECASAS AND CHRISTIAN B. ANFINSEN

General Considerations

Conventional procedures of protein purification are generally based
on relatively small differences in the physicochemical properties of the
proteins in the mixture. They are frequently laborious and incomplete,
and the yields are often low. The selective isolation and purification of
enzymes and other biologically important macromolecules by "affinity
chromatography" exploits the unique biological property of these pro-
teins or polypeptides to bind ligands specifically and reversibly.1-7 The
method is related in principle to the use of "immunoadsorbents" intro-
duced as chemically defined materials for chromatographic separation
of antibodies by Campbell and his colleagues in 195 1s and subsequently
employed widely as a standard immunological procedure.s Affinity chro-
matography, as summarized in the present article, exploits the phenom-
enon of specific biological interaction in a large variety of protein-
ligand systems. A solution containing the macromolecule to be purified
is passed through a column containing an insoluble polymer or gel to
which a specific competitive inhibitor or other ligand has been covalently
attached. Proteins not exhibiting appreciable affinity for the ligand will
pass unretarded through the column, whereas those which recognize
the inhibitor will be retarded in proportion to the affinity existing under
the experimental conditions. The specifically adsorbed protein can be
eluted by altering the composition of the solvent so that dissociation oc-
curs.

In principle, specific adsorbents can be used to purify enzymes, anti-
bodies, nucleic acids, cofactor or vitamin-binding proteins, repressor
proteins, transport proteins, drug or hormone receptor structures,
sulfhydryl group containing proteins, and peptides formed by organic

`P. Cuatrecasas. M. Wilchek, and C. B. Anfinsen, Proc. Nat. Acnd. Sci. U. S.61,636 (1968).
2P. Cuatrecasas,J. Biol. C/tern. 245,3059 (1970).
3P. Cuatrecasas, in Biochemical Aspects of Solid State Chemistry, ed. G. R. Stark. h'ew York,
Academic Press. In press.
`P. Cuatrecasas,J. Agr. Food Chem.. In press (I 971).
"I. Kato, and C. B. Anfinsen.]. Biol. Chem. 244, 1004 ( 1969).
eP. Cuatrecasas and C. B. Anfinsen, Annu. Rev. B&hem., 40, In press (1971).
`P. Cuatrecasas, Nature, 228, 1327 (1970).
sD. H. Campbell, E. Leuscher, and L. S. Lermann, Proc. Nat. Acad. Sci. U.S. 37,575 (1951).
sl. Silman and E. Katchalski, Annu. Rev. B&hem. 35,873 (1966).


346         SEPARATION BASED ON SPECIFIC AFFINITY      PII
synthesis. Affinity chromatography may also be useful in concentrating
dilute solutions of proteins, in removing denatured forms of a purified
protein, and in the separation and resolution of protein components
resulting from specific chemical modifications of purified proteins. In-
herent advantages of this method of purification are the rapidity and
ease of a potentially single-step procedure, the rapid separation of the
protein to be purified from inhibitors and destructive contaminants, e.g.,
proteases, and protection from denaturation during purification by
active site ligand-stabilization of protein tertiary structure.
Successful application of the methods will depend in large part on
how closely the particular experimental conditions chosen permit the
ligand interaction to simulate that observed when the components are
free in solution. Careful consideration must therefore be given to the
nature of the solid matrix, the dependence of the interaction on the
structure of the ligand, the means of covalent attachment, and the con-
ditions selected for adsorption and elution. The specific conditions for
purification of a particular protein must be highly individualized since
they will reflect the uniquely specific biological property of selective
interaction with a given ligand. The following general considerations
should serve as guidelines in the preparation of adsorbents for affinity
chromatography.
Solid Matrix Support. An ideal insoluble support should possess the
following properties: (1) It must interact very weakly with proteins in
general, to minimize the nonspecific adsorption of proteins. (2) It should
exhibit good flow properties which are retained after coupling. (3) It
must possess chemical groups which can be activated or modified, under
conditions innocuous to the structure of the matrix, to allow the chem-
ical linkage of a variety of ligands. (4) These chemical groups should be
abundant in order to allow attainment of a high effective concentration
of coupled inhibitor (capacity), so that satisfactory adsorption can be
obtained even with protein-inhibitor systems of low affinity. (5) It must
be mechanically and chemically stable to the conditions of coupling and
to the varying conditions of pH, ionic strength, temperature, and pres-
ence of denaturants (e.g., urea, guanidine hydrochloride) which may be
needed for adsorption or elution. Such properties also permit repeated
use of the specific adsorbent. (6) It should form a very loose, porous net-
work which permits uniform and unimpaired entry and exit of large
macromolecules throughout the entire matrix; the gel particles should
preferably be uniform, spherical, and rigid. A high degree of porosity
is an important consideration for ligand-protein systems of relatively
weak affinity (dissociation constant of 10e5 M or greater),`since the con-
centration of ligand freely available to the protein must be quite high to


[311                AFFINITY CHROMATOGRAPHY           347

permit interactions strong enough to physically retard the downward
migration of the protein through the column. This probably explains
why certain solid supports, such as cellulose, have been used so success-
fully as immunoadsorbents but have been of only minimal utility in the
purification of enzymes.

The use of hydrophilic cellulose derivatives in the purification of anti-
bodies is described in detail elsewhere in this volume and need not be
discussed further here. In some cases cellulose derivatives have been
used in -the purification of enzymes: ( 1) aminophenol diazotized to cellu-
lose containing resorcinol residues in ether linkage has been used to
purify tyrosinase'O; (2) flavokinase has been specifically adsorbed to car-
boxymethyl cellulose and to cellulose containing flavin derivatives"*i2;
(3) avidin has been partially purified with the use of cellulose reacted
with biotinyl chloride. l3 Derivatives of cellulose also have been used to
purify nucleotides,`* complementary strands of nucleic acids,15 and cer-
tain species of transfer RNA. l6 Although cellulose derivatives may be
advantageous in specific instances, they are generally much less useful
than the agarose derivatives because their fibrous and nonuniform
character impedes proper penetration of large protein molecules.
Highly hydrophobic polymers, such as polystyrene, display poor com-
munication between the aqueous and solid phases. A technique for the
immobilization of organic substances on glass surfaces, described recent-
ly by Weetall and Hersh," may be a promising tool for the preparation
of specific adsorbents.

Various polysaccharide hydrophilic polymers are very useful solid
supports. Commercially available,  cross-linked dextran derivatives
(Sephadex, Pharmacia) possess most of the desirable features listed
above except for their low degree of porosity. For this reason they are
relatively ineffective as adsorbents for purification of enzymes of even
low molecular weight. However, beaded derivatives of agarose,i* another
polysaccharide polymer, have nearly all the properties of an ideal ad-
sorbent'+ and are commercially available (Pharmacia; Bio-Rad Labora-
tories). The beaded agarose derivatives have a very loose structure
which allows molecules with a molecular weight in the millions to diffuse

`OL. S. Lerman, Proc. Nat. Acad. Sci. U. S. 39,232 (1953).
"C. Arsenis and D. B. McCormick,J. Biol. Chem. 239,3093 (1964).
`*C. Arsenis and D. B. McCormick,]. Biol. Chen. 241,330 (1966).
`sD. B. McCormick, Anal. Biochem. 13, 194 (1965).
"E. G. Sander, D. B. McCormick, and L. D. Wright,]. Chromatogr. 21.4 19 (1966).
`sE. K. F. Bautz and B. D. Holt, Proc. Nat. Acad. Sci. U. S. 48,400 ( 1962).
`% L. Erhan, L. G. Northrup, and F. R. Leach, Proc. Nat. Acad. Sci. U. S. 53, 646 (1965).
"H. H. Weetall and L. S. Hersh, Eiochim. Biophys. Ada 185,464 (1969).
`sS. HjertPn, Arch. Eiochem. Biophys. 93,446 (1962).


348          SEPARATION BASED ON SPECIFIC AFFlNlTY      1311

readily through the matrix. These polysaccharides can readily undergo
substitution reactions by activation with cyanogen halides,1g*20 are very
stable, and have a moderately high capacity for substitution.*s3*' Syn-
thetic polyacrylamide gels also possess many desirable features and are
available commercially in beaded, spherical form, in various pregraded
sizes and porosities (Bio-Gel, Bio-Rad Laboratories). Recently described
derivatization procedures permit attachment of a variety of ligands and
proteins to polyacrylamide beads. **`**r These beads exhibit uniform
physical properties and porosity, and the polyethylene backbone endows
them with physical and chemical stability. Preformed beads are available
which permit penetration of proteins with molecular weights of about
one half million (Bio-Gel P-300). Their porosity, however, is diminished
during the chemical modifications required for attachment of ligands,
and in this respect the polyacrylamide beads are inferior to those of
agarose. **** The principal advantage of polyacrylamide is that it possesses
a very large number of modifiable groups (carboxamide). Thus, highly
substituted derivatives may be prepared for use in the purification of
enzymes, which exhibit poor affinity for the attached ligand.*p**

Comidmutions in Selecting a Ligund. The small molecule to be cova-
lently linked to the solid support must be one that displays special and
unique affinity for the macromolecule to be purified. It can be a substrate
analog inhibitor, effector, cofactor, and, in special cases, substrate. En-
zymes requiring two substrates for reaction may be approached by im-
mobilizing one of the substrates, provided sufficiently strong binding is
displayed toward that substrate in the absence of the other. Also, a sub-
strate may be used if it binds to the enzyme under some conditions that
do not favor catalysis, i.e., in the absence of metal ion, if the pH de-
pendence of Km and of kat are different, or at lower temperatures.
The small molecule to be insolubilized must possess chemical groups
that can be modified for linkage to the solid support without abolishing
or seriously impairing interaction with the complimentary protein. If
the strength of interaction of the free complex in solution is very strong,
i.e., a K{ of about 1 mh4, a decrease in affinity of 3 orders of magnitude
upon preparation of the insoluble derivative may still leave an effective
and selective adsorbent. The important parameter is the effective experi-
mental affinity- that displayed between the protein in solution and the
insolubilized ligand under the experimental conditions chosen. In prac-
tice it has been very difficult to prepare such adsorbents for enzyme-

`"J. Porath, R. Ax&, and S. Ernblck, Nature (London) 215,1491(1967).
*`JR. Ax&n, J. Porath, and S. ErnbZck, Nature (London) 2 l&l302 (1967).
*J. K. Inman and H. M. Din&, Biochemistry 8.4074 (1969).
"E. Steers, P. Cuatrecasas, and H. Pollard,J.Btil. Chrm.246, 196 (1971).


r311               AFFINITY CHROMATOGRAPHY            349

inhibitor systems whose dissociation constants under optimal conditions
in solution are greater than 5 mM. ** It is possible in theory, however, to
prepare adequate adsorbents for such systems if a sufficiently large
amount of inhibitor can be coupled to the solid support.
It is becoming increasingly clear that, for successful purification by
affinity chromatography, the inhibitor groups critical in the interaction
with the macromolecule to be purified must be sufficiently distant from
the solid matrix to minimize steric interference with the binding pro-
cess.****** Steric considerations appear to be most important with proteins
of high molecular weight. The problem may be approached by prepar-
ing an inhibitor with a long hydrocarbon chain, an "arm," attached to
it, which can in turn be coupled to the insoluble support. Alternatively,
such a hydrocarbon extension "arm" can first be attached to the solid
support.*

General Considerations in the Preparation oj`SpeciJic Adsorbents. The ligand
must be coupled to the solid support under mild conditions that are
tolerated well by both ligand and matrix. The coupled gel must be washed
exhaustively to ensure total removal of the material not covalently bound.
In some cases, such as with highly aromatic compounds, e.g., estradiol,
complete removal of adsorbed material is very difficult and may require
many days of continuous washing. Organic solvents may occasionally
be required for this purpose. In rare instances, i.e., Congo red, effective
washing can be achieved only by washing the column with large quanti-
ties of the protein to be purified, i.e., amyloid, followed by elution to
regenerate the column. Whenever possible, radioactivity or other sensi-
tive indicators should be present on the ligand to assist in monitoring
the washing procedure. Aqueous dimethyl formamide (50%, v:v) has
been useful in accelerating the washing of agarose and polyacrylamide
derivatives; both tolerate this solvent well.

An accurate method for determining the amount of material attached
to the solid support should be available. This is preferably done by de-
termining the amount of ligand (by radioactivity, absorbance, amino acid
analysis, etc.) released from the substituted matrix by acid or alkaline
hydrolysis. Exhaustive digestion with pronase or carboxypeptidase, fol-
lowed by amino acid analysis, has been used in some cases in which oligo-
peptides are attached to agarose. It is also possible to determine the
radioactive content of the unhydrolyzed solid support by assay in suspen-
sion with Cab-0-Sil (Beckman): estimates made in this way are generally
low even if appropriate internal standards are used for correcting effi-
ciencies of counting. An alternative means of quantitation is to estimate
the amount of ligand not recovered in the final washings. This is less
accurate when a very large excess of ligand is added during the coupling


350         SEPARATION BASED ON SPECIFIC AFFINITY      [311

procedure or when appreciable noncovalent adsorption to the solid
matrix occurs which demands large volumes of solvent for thorough
washing. It is operationally more useful to express the degree of ligand
substitution on the solid matrix in terms of concentration, such as micro-
moles of ligand per milliliter of packed gel, rather than on the basis of
dry weight.
Conditions for Adsorption and Elution of Proteins. The specific conditions
for adsorption are dictated by the specific properties of the protein to be
purified. Affinity purification need not be restricted to column pro-
cedures. In certain circumstances it may be advisable to use batch purifi-
cation methods. This is the case, for example, when small amounts of
protein are to be extracted from very crude or particulate protein mix-
tures with an adsorbent of very high affinity. Column flow rates in such
cases may be severely compromised, and purification can be more expe-
ditiously carried out by adding a slurry of the specific adsorbent to the
crude mixture, followed by batchwise washing and elution. In some
cases involving very high affinity complexes, such as observed with cer-
tain antibody-antigen systems, it is preferable to adsorb the protein to the
solid support and to wash extensively while the gel is packed in a column,
elute the protein by removing the matrix and incubating in suspension
in an appropriate buffer. For reasons of thoroughness of mixing, higher
dilution of the insoluble lig-and, and easier control of time and tempera-
ture, this means of elution requires less drastic conditions and the yields
may be higher.
Protein specifically adsorbed to a solid carrier matrix will eventually
emerge from the column without altering the properties of the buffer if
the affinity for the l&and is not too great. With this type of elution,
however, the protein is generally obtained in dilute form. In most cases
elution of the protein will require changing the pH, ionic strength, or
temperature of the buffer. Dissociation of proteins from very high-
affinity adsorbents may require protein denaturants, such as guanidine
hydrochloride or urea. Ideal elution of a tightly bound protein should
utilize a solvent which causes sufficient alteration in the conformation of
the protein to decrease appreciably the affinity of the protein for the
ligand but which is not sufficiently severe to completely denature or un-
fold the protein. The eluted protein should be neutralized, diluted, or
dialyzed at once to permit prompt reconstitution of the native protein
structure. This may be tested by rechromatographing the purified pro-
tein to determine changes in adsorption to the affinity column. Elution
of the specifically adsorbed protein can also be achieved with a solution
containing a specific inhibitor or substrate. The inhibitor can either be
the one that is covalently linked to the matrix (and must be used at higher
concentrations), or preferably another, stronger competitive inhibitor.


[3Il                AFFINITY CHROMATOGRAPHY            351

An alternative method of eluting the specifically bound protein is to
selectively cleave the matrix-ligand bond, thus removing the intact
ligand-protein complex. Excess ligand can then be removed by dialysis
or by gel filtration on Sephadex. This approach can be applied to ligands
attached to Sepharose by azo linkage, and by thiol or alcohol ester bonds.
These will be discussed under the appropriate sections.

Preparation and Use of Agarose Bead Adsorbents

Of the beaded agarose derivatives commercially available, Sepharose
4B (Pharmacia) is the most useful for affinity chromatography. It is more
porous than the 6B derivative, and has considerably greater capacity
than the 2B gels. Chemical compounds containing primary aliphatic or
aromatic amines can be coupled directly to agarose beads after activa-
tion of the latter with cyanogen bromide at alkaline pH (Scheme I). The
chemical nature of the intermediate formed by cyanogen halide treat-
ment of polysaccharide derivatives is not known, but the products
formed upon coupling with amino compounds appear to be principally
derivatives of amino carbonic acid and isourea.23 Notably, both of these
postulated linkage groups retain the basicity of the amino group of the
coupled ligand. It must be borne in mind, therefore, that even if the
charge on the ligand amino group is known to contribute to the binding
interaction, the resultant agarose ligand gel may nevertheless demon-
strate considerable binding effectiveness.

(a)    u-CH,NH,       t NHCH*-R

(b)

(cl    HINKH,LNHI      t NHV3fzLNH,

               SCHEMEI

Beaded agarose gels, unlike the cross-linked dextrans, cannot be dried
or frozen, since they will shrink severely and essentially irreversibly.
Similarly, they will not tolerate many organic solvents. Dimethyl form-
amide (50%, v:v) and ethylene glycol (50%, v:v) do not adversely affect
the structure of these beads. These solvents are quite useful in situations
where the compound to be coupled is relatively insoluble in water
(e.g., steroids, thyroxine, tryptophan derivatives) since the coupling step

"J. Porath, ~Valure (London) 2 18,834 (1968).


352          SEPARATION BASED ON SPECIFIC AFFINITY      1311

can be carried out in these solvents. Similarly, the final, coupled deriva-
tive can be washed with these solvents to remove strongly adsorbed or
relatively water-insoluble material.
Agarose beads, both before and after activation and coupling, ex-
hibit very little nonspecific adsorption of proteins provided the ionic
strength of the buffer is 0.05 M or greater. The coupled, substituted
adsorbents of agarose can be stored at 4" in aqueous suspensions with
an antibacterial preservative (sodium azide, toluene), for periods of time
limited only by the stability of the bound inhibitor or ligand; they should,
as mentioned above, not be frozen or dried for storage. Agarose beads
tolerate 0.1 M NaOH and 1 N HCl for at least 2-3 hours at room tem-
perature without detectable adverse alteration of their physical proper-
ties, and without cleavage of the covalently linked ligand, if the latter
itself does not possess labile bonds. The specific adsorbents formed from
these beads can therefore be used repeatedly even after exposure to
relatively extreme conditions; the limitation in this respect is more likely
determined by the stability of the ligand than of the matrix. Agarose
beads are not destroyed by exposure to temperatures as high as 45" for
many minutes. They tolerate quite well 6 M guanidine*HCl or 7 M urea
solutions for prolonged periods; a very small amount of dissolution may
occur after exposure to 6 M guanidine.HCl for 2-3 days at room tem-
perature. These protein denaturants may therefore be used to aid in
the elution of specifically bound proteins, for thorough washing of col-
umns in preparation for re-use, and in washing off protein that is tightly
adsorbed during the coupling procedure.
Procedure.2 A given volume of decanted agarose, containing a mag-
netic stirring bar, is mixed with an equal volume of water and the elec-
trodes of a pH meter are placed in this suspension. The procedure is
performed in a well ventilated hood. Finely divided solid cyanogen bro-
mide (50-300 mg per milliliter of packed agarose) is added at once to
the stirred suspension, and the pH is immediately raised to 11 with
NaOH. The molarity of the NaOH solution will depend on the amounts
of agarose and cyanogen bromide added; it should vary between 2 M
(for 5-10 ml agarose and l-3 g of cyanogen bromide) and 8 M (for
100-200 ml of agarose and 20-30 g of cyanogen bromide). The pH is
maintained at 11 by constant manual titration. Temperature is main-
tained at about 20" by adding pieces of ice as needed. The reaction is
complete in 8- 12 minutes, as indicated by the cessation of base uptake;
no solid CNBr should remain. A large amount of ice is then rapidly
added to the suspension, which is transferred quickly to a Biich?er fun-
nel (coarse disk) and washed under suction with cold buffer. The buffer
should be the same as that which is to be used in the coupling stage, and


[311                AFFINITY CHROMATOGRAPHY            353

the volume of wash should be IO-15 times that of the packed agarose,
larger volumes (20-30 times) should be used if a protein is to be coupled.
The Biichner funnel, containing the moist, washed agarose is removed
from the filtering flask and its outlet is covered tightly with Parafilm.
The co'mpound to be coupled, in a volume of cold buffer equal to the
volume of packed agarose, is added to the agarose and the suspen-
sion is immediately mixed (in the Biichner funnel) with a glass stirring
rod. The entire procedure of washing, adding the ligand solution, and
mixing should consume less than 90 seconds. It is important that these
procedures be performed rapidly and that the temperature be lowered,
since the "activated" agarose is unstable. The suspension is transfer-
red from the Biichner funnel to a beaker containing a magnetic mixing
bar and is gently stirred at 4" for 16-20 hours. Care must be taken to
avoid vigorous stirring since Sepharose can be physically dirupted,
resulting in material with poor flow rates. The substituted agarose is
then washed with large volumes of water and appropriate buffers until
it is established with certainty that ligand is no longer being removed.

The quantity of ligand coupled to agarose can be controlled by
varying several parameters .2 Most important is the amount of ligand
added to the activated agarose (Table I). When highly substituted
derivatives are desired, the amount of inhibitor added should, if possible,
be 20-30 times greater than that which is desired in the final product.
For ordinary procedures, loo-150 mg of cyanogen bromide is used per
milliliter of packed agarose, but much higher coupling yields can be
obtained if this amount is increased to 250-300 mg (Table II). The
larger amounts of CNBr should be used only if highly substituted deriv-
atives are desired since greater heat is evolved and longer reaction times

TABLE I

EFFICIENCY OF COUPLING OF 3'-(4-AMINOPHENYLPHOSPHORYL)DEOXYTHYMIDINE 5'-
PHOSPHATE TO AGAROSE, AND CAPACITY OF THE RESULTING; ADSORBENT FOR
               STAPHYLOCOCCAL NUCI.EASE"

       phfoles of inhibitor/ml agarose      Mg of nucelaseiml agarose
Expt.           Added   Coupled           TheoreticaP   Found

A               4.1       2.3                 44        -
B              2.5        1.5                 28       8
C              I.5        I.0                 19        -
D              0.5        0.3                  6       1.2
"One hundred milligrams of cyanogen bromide was added per milliliter of packed agarose,
and coupling was performed in 0.1 M NaHCO,,, pH Y.O. Data from P. Cuatrecasas,
M. Wilchek. and C. B. Anfinsen, Proc. Nat. Acad. Sci. U.S. 61, 636 (1968).
*Assuming equimolar binding.


354          SEPARATION BASED ON SPECIFIC AFFINITY       r311

TABLE II

EFFECT OF pH ON THE COUPLING OF ["Cl-ALANINE TO ACTIVATED AGAROSE~

Conditions for coupling reaction                 Alanine coupled

   Buffer       PH                @moles per ml agarose)

Sodium Citrate, 0.1 M     6.0                           4.2
Sodium phosphate, 0.1 M   7.5                           8.0
Sodium borate, 0.1 M     8.5                            1 I.0
Sodium borate, 0. I M     9.5                          12.5
Sodium carbonate, 0.1 M   10.5                           10.5
Sodium carbonate, 0.1 M   11.5                           0.2

"Sixty milliliters of packed Sepharose 4B was mixed with 60 ml of water and treated with
15 mg of cyanogen bromide as described in the text. To the cold, washed activated Sepha-
rose was added 2.2 mmoles of ["C]alanine (0.1 &i//lmole) in 60 ml of cold distilled water,
and 20-ml aliquots of the mixed suspension were added rapidly to beakers containing 5
ml of cold 0.5 M buffer of the composition described in the table. The final concentration
of alanine was 15 mM. After 24 hours, the suspensions were thoroughly washed, aliquots
were hydrolyzed by heating at 110" in 6 N HCI for 24 hours, and the amount of released
radioactivity was determined. Data from P. Cuatrecasas, J. Viol. Chem. 245, 3059 (1970).

are required. The pH at which the coupling stage is performed will also
determine the degree of coupling since it is the unprotonated form of
the amino group which is reactive. Compounds containing an a-amino
group (pK about 8) will couple optimally at a pH of about 9.5-10.0
(Table II). Higher pH values should not be used, since the stability of
the activated Sepharose decreases sharply above pH 10.5 (Table II).
Compounds with primary aliphatic aminoalkyl groups, such as the E-
amino group of lysine or ethylamine (pK about lo), should be coupled
at pH values of about 10, and a large excess of ligand should be added.
The most facile coupling occurs with compounds bearing aromatic
amines, due to the low pK (about 5) of the amino group; very high coup-
ling efficiencies are obtained at pH values between 8 and 9 (Table I).

It is possible, in some cases, to increase the amount of ligand coupled
to agarose by repeating the activation and coupling procedures of the
already substituted material, provided the inhibitor is stable for about
10 minutes at pH 11.

Quantitation of the amount of amino acid or protein coupled to
agarose is best determined by hydrolyzing a lyophilized aliquot of ma-
terial in 6 N HCI, 1 lo", for 24 hours. After hydrolysis, the brown-black
suspension is filtered twice by passing through a Pasteur pipette con-
taining glass wool, or through a Millipore filter; the solution is dried and
its composition is determined with an amino acid analyzer.   -
The operational capacity for specific adsorption of the agarose
adsorbents' (Table I) can be tested by passing slowly an amount of en-


[311              AFFINITY CHROMATOGRAPHY            355
zyme or protein in excess of the theoretical capacity through a sample of
adsorbent packed in a column, washing with buffer until negligible pro-
tein emerges in the effluent, and eluting (Fig. 1). Alternatively, small
samples pf preferably pure protein can be added successively to the
column until detectable protein or enzymatic activity emerges in the
effluent.' The total amount added, or that eluted, is considered the
operational capacity.

Examples 1: Purijication of Staphylococcal Nuclease with a Competitive In-
hibitor.' The inhibitor of staphylococcal nuclease, 3'-(4-aminophenyl-
phosphoryl)deoxythymidine 5'-phosphate (pdTp-aminophenyl) is an
ideal ligand for preparation of specific agarose adsorbents because it
is a strong competitive inhibitor (K,, 10e6 M), its 3'-phosphodiester
bond is not cleaved by the enzyme, it is stable at pH values of 5-10, the

t-O.1 M tris pH 7.5 -507  Acetx acld

0     IO     20       IO     20

           Froctlon number

FIG. 1. Determination of the operational capacity of a column of agarose-RNase-S-
protein (0.8 X 19cm) for RNase-S-peptide. The column having a bed volume of 9.6 ml and
containing 27 mg of bound S-protein on the basis of amino acid analysis, was washed with
200 ml of 0.1 M Tris.HCI buffer, pH 7.5. It was then perfused with 3.8 ml of an aqueous
solution containing 4.2 mg of S-peptide. After thorough washing with buffer (0.1 M
TriseHCI, pH 7.5) bound S-peptide (2.3 mg) was eluted with 50% acetic acid. Since 27 mg
of S-protein should combine, theoretically, with approximately 5 mg of S-peptide, it may
be calculated that the binding efficiency of the immobilized S-protein was about 45%. Data
from 1. Kato and C. B. Anfinsen,J. Biol. Chem. 244. 1004 (1969).


356         SEPARATION BASED ON SPECIFIC AFFINITY      [3Il

pK of the aromatic amino group is low, and the amino group is relatively
distant from the basic structural unit (pTp-X) recognized by the enzy-
matic binding site .24 This inhibitor couples to agarose with high effi-
ciency, and the resulting adsorbent has a high capacity for the enzyme
(Table I). Columns containing this specific adsorbent completely and
strongly adsorb samples of pure and crude nuclease (Fig. 2). Virtually
no enzyme escapes from such columns with washes exceeding 50 times
the column bed volume if the amount of protein applied does not exceed
30% of the operational capacity. Elution is accomplished by washing
with solutions of low pH (acetic acid, pH 3) or high pH (NH,OH, pH 11).
The eluted protein emerges in a very small volume, and the material can
be lyophilized directly.

If very crude enzyme solutions with total protein concentration greater
than 20 mg/ml are passed through such columns, some of the enzyme
escapes, probably because of interaction with the major protein fraction.
Rechromatography usually will allow complete removal of the residual

0
.

- 2.5

- 0.5

Effluent (ml)

Frc. 2.  Purification of staphylococcal nudease by affinity adsorption chromatography
on a nuclease-specific agarose column (0.8 X 5 cm) (sample B, Table 1.). The column
was equilibrated with 50 mM borate buffer, pH 8.0, containing 10 mM CaCI,. Approxi-
mately 40 mg of partially purified material containing about 8 mg of nuclease was applied
in 3.2 ml of the same buffer. After 50 ml of buffer had passed through the column, 0.1 M
acetic acid was added to elute the enzyme. Nuclease, 8.2 mg, and all the original activity
was recovered. The flow rate was about 70 ml per hour. Data from P. CuatrecasaS, M. Wil-
chek, and C. B. Anfinsen, Proc. Nat. Acad. Sci. U.S. 61, 636 (1968).

%P. Cuatrecasas, M. Wilchek, and C. B. Anfinsen, Biochemistry 8.2277 (1969).


[311                AFFINITY CHROMATOGRAPHY           357

enzyme. Very dilute solutions of enzyme should be passed through these
columns at moderately slow flow rates. The columns can be used re-
peatedly, and over protracted periods, without detectable loss of effec-
tiveness. These affinity adsorbents can also be used in the separation of
active and inactive nuclease derivatives from samples subjected to chemi-
cal modification25'27 (as will be described), and they are effective in
stopping enzymatic reactions.*
Example 2: Purification of a-Chymotrypsin with an Inhibitor Containing an
((Arm."1 An agarose adsorbent containing a relatively weak competitive
inhibitor, n-tryptophan methyl ester, (K,, 10e4 M) is relatively ineffective
in adsorbing cY-chymotrypsin even though 10 pmoles of inhibitor are
present per milliliter of agarose (Fig. 3B). Only a slight retardation
of the enzyme is observed. However, by interposing a G-carbon chain
between the same inhibitor and the agarose matrix, dramatically
stronger adsorption of the enzyme occurs (Fig. 3C). This illustrates the
marked steric interference that results when the ligand is attached too
closely to the supporting gel. DFP-treated a-chymotrypsin (Fig. 3D),
chymotrypsinogen, pancreatic ribonuclease, subtilisin, and trypsin are
not adsorbed by this specific affinity adsorbent. Greater than 90% recov-
ery is obtained on elution with distilled water titrated to pH 3 with acetic
acid. It is also possible to elute the protein with a 0.0 18 M solution of the
competitive inhibitor, &phenylpropionamide (K,, 7 mM).

Example 3: Purijication of DAHP Synthetase with an EffectorFa An agarose
adsorbent containing L-tyrosine (3.2 ~moles/ml), an effector of 3-
deoxy-o-arabinoheptulosonate 7-phosphate (DAHP) synthetase (Ki,
5 x lo-" M), was capable of separating this enzyme from a crude protein
mixture (Fig. 4). As with cu-chymotrypsin, the enzyme is merely retarded,
and it is not necessary to change the nature of the buffer to obtain
elution. Nevertheless, a very useful derivative is obtained which sepa-
rates the weakly adsorbed enzyme from the bulk of the protein and from
a related enzyme.

Example 4: Batch PuriJication of a Vitamin-Binding ProteinFg An early
example of affinity chromatography was the purification of avidin on
biotincellulose.*3 A much more effective adsorbent for avidin can be
prepared by coupling a derivative of biotin containing a 6-carbon chain,
biocytin (e-N-biotinyl+lysine), to Sepharose. The lysyl portion serves
as an "arm" to hold the essential structural portion of the vitamin, the

25P. Cuatrecasas, M. Wilchek, and C. B. Anfinsen,J. Biol. Chem. 244,4316 (1969).
*sP. Cuatrecasas,J. Biol. Chem. 245,574 (I 970).
*`H. Taniuchi and C. B. Anfinsen,j. Biol. Chem. 244.3864 (1969).
*SW. C. Chan and M. Takahashi, Biochem. Biophvs. Res. Commun. 37.272 (1969).
*OP. Cuatrecasas and M. Wilchek, Biochem. Biophys. Res. Commun. 33,235 (1968).


358        SEPARATION BASED ON SPECIFIC AFFINITY      [311

2.0 -



1.5 -

I 1 I r , 1     II IV 11 I I, I, I, 1,
     (a)                (cl             (3.2)
  Unsubstituted       Sepharose coupled with
   sephorose      o -aminocoproyl-D-tryptophon   !
                   methyl ester

  1.0 -
2
g D.5-    I           I
N
i
:  !I s-L,_,_._* I A&&&&+&&,   ,
x            (b)                        (d)
&
L.7 2.0   Sepharose coupled with         DFP treated chymotrypsln
9 t D-tryptophan methyl ester t       [sephorose OS I" (c )]

Effluent (ml)

FIG. 3. Affinity chromatography of ochymotrypsin on inhibitor-agarose columns.
The columns (0.5 X 5 cm) were equilibrated and run with 50 mM Tris.HCl buffer, pH 8.0.
Each sample (2.5 mg) was applied in 0.5 ml of the same buffer. Fractions of 1 ml were col-
lected at a flow rate of about 40 ml per hour at room temperature. cx-Chymotrypsin was
eluted with 0.1 M acetic acid, pH 3.0 (arrows). Peaks preceding the arrows in B, C, and D
were devoid of enzyme activity. In preparing the agarose adsorbent, 65 pmoles of in-
hibitor was added per milliliter of Sepharose, 10 pmoles of which was found to be covalently
attached, yielding an effective concentration of inhibitor, in the column, of about 10 mM.
Data from P. Cuatrecasas, M. Wilchek, and C. B. Anfinsen, Proc. Nat. Acud. Sci. U.S. 61,636
(1968).

ureido and thiophan rings, at a good distance from the matrix. Adsor-
bents containing very small amounts (0.02 pmole per milliliter of
agarose) of this compound which binds very tightly to avidin (&, 1 O-l5 M)
can be used to purify, in a batchwise manner, avidin from crude egg
white in a single step (Table III). The binding of avidin is so strong that
elution must be performed with 6 M guanidine*HCl, pH 1.5.

Example 5: Purijication of Thyroxine-Binding Protein from Serum. Columns
containing agarose with N-(6-aminocaproyl)thyroxine,"O or thyroxine30*31
itself, remove most of the thyroxine-binding globulin From serum. This
                                                   -

sOR. A. Pages, H. J. Cahnmann, and J. Robbins, personal communication (1969).
"`J. Pensky and J. S. Marshall, Arch. Eiochm. Biophys. 135,304 (1969).


r311              AFFINITY CHROMATOGRAPHY            359

2 4 6 8 IO I2 I4 I6 I8

-I 6

Froctlon number

FIG. 4.  Purification of tyrosine-sensitive 3-deoxy-o-arabinoheptulosonate 7-phosphate
synthetase by affinity chromatography. Chromatography of crude yeast extract on tyrosine-
Sepharose column (0.6 X 15.5 cm), equilibrated with 0.2 M sodium phosphate buffer. pH
6.5 containing &SO, (0.1 mM) and PMSF (0.1 mM). The sample consisted of 2 ml of
crude extract, and I-ml fractions were collected. C+O. Tyrosine-sensitive DAHP syn-
thetase; a-+, phenylalanine-sensitive DAHP synthetase;  - , protein concentration.
Pooled fractions are indicated (I and II). Data from W. W. C. Chan and M. Takahashi,
Biochem. Eiophys. Res. Commun. 37, 272 (1969).

protein, which has a dissociation constant of about lo-`OM for thyroxine
and which is present at very low concentrations in serum (lo-20 pglml),
remains strongly adsorbed to the agarose after the column is washed
with 0.1 M NaHC03 buffer, pH 9.0, it is eluted with 4 mM NaOH, pH
1 1.4.30 Although not pure, the eluted protein is enriched considerably
by this single-step procedure. Sepharose-4B, activated with 100 mg of
cyanogen bromide per milliliter of Sepharose by the previously described
procedures, is coupled with thyroxine or aminocaproyl-thyroxine (3
pmoles per milliliter of Sepharose) in 50% (v:v) ethylene glycol (to en-
hance thyroxine solubility), 50 mM NaHCO,, pH 9.6; about 1.8 pmoles
of thyroxine derivative is coupled per milliliter of agarose. Adsorbed
[rz51]thyroxine is completely removed by washing with 20 volumes of the
ethylene glycol buffer followed by 20 volumes of 0.1 M NaHCO,, pH


360         SEPARATION BASED ON SPECIFIC AFFINITY       f311

TABLE III

PURIFICATION OF AVIDIN FROM EGG WHITE BY BIOCYTIN-AGAROSE CHROMATOGRAPHY

Sample

Protein      Avidin
(mg)      (mg)

Specific activity
(pg biotin
per mg protein)

Purification
  factor

Egg white
Washings of
Sepharose
Elution from
Sepharose

8000        2.2          0.0037


8000        0.2          0.0003


1.5"        1.6         14.6             4000

"Based on .?$`,z of 1.57 (M. D. Melamed and N. M. Green, Bi0chem.J. 89, 591 (1963).
Two milliliters of biocytin-Sepharose were added to 25 ml of 0.2 izf NaHCOs, pH 8.7,
containing 8 g of crude, dried egg white. After a few minutes the solution was centrifuged
at 20,000 g for IO minutes. The Sepharose was suspended in bicarbonate buffer and
poured onto a I X 5 cm column. The column was washed with about 25 ml of 3 M guani-
dine.HCI. pH 4.5, and the avidin was eluted with 6 M guanidine.HCl at pH 1.5. Avidin
concentration was determined by addition of biotin "C followed by dialysis for 24 hours
                                      ,
at room temperature against large volumes of 0.1 iI4 NaHCO, at pH 8.2: it was assumed
that 13.8 pg of biotin are bound per milligram of avidin. Data from P. Cuatrecasas and M.
Wilchek. Biochem. Biophys. Res. Commun. 33, 235 (1968).

9.0. These studies indicated that ethylene glycol (50%, v:v), but not
dimethyl sulfoxide, which was also investigated, is well tolerated by
agarose.

Example 6: Resolution of Chemically Modzjied Enzyme Mixtures.25-27 Studies
of the functional effects of chemical modifications of purified enzymes
frequently reveal incomplete loss of enzymatic activity. It is often diffi-
cult to determine whether this results from an altered protein possessing
diminished catalytic properties or to residual native enzyme. In the lat-
ter case, separation of the active native and the catalytically inert proteins
may not be possible. In certain cases, both problems may be resolved by
using affinity adsorbents. Modification of staphylococcal nuclease by
attachment of a single molecule of an affinity labeling reagent through
an azo linkage to an active site tyrosyl residue, resulted in loss of 83 % of
the enzyme activity. 26 Chromatography of this enzyme solution on a
nuclease-specific agarose column revealed that the residual activity
was due entirely to a 20% contamination of native enzyme; complete
resolution of the two components was possible by affinity chromatography
(Fig. 5).2s Similar separation was obtained with partially active prepara-
tions of staphylococcal nuclease modified with bromoacetamidophenyl
affinity labeling reagents,"_ and residual native nuclease could be sep-
arated from an inactive peptide fragment obtained by specific tryptic
cleavage.27


1311                AFFINITY CHROMATOGRAPHY            361

r

20

Effluent [ml)

FIG. 5. Affinity chromatography on specific agarose column (Table I, Fig. 2) of
staphylococcal nuclease treated with a 1.7-fold molar excess a diazonium labeling reagent
derived from pdTp-aminophenyl. About 3 mg of chemically modified nuclease, contain-
ing 17% of the DNase activity of the native enzyme, were applied to a 0.5 X 7 cm column
which contained Sepharose 4B conjugated with the inhibitor, pdTp-aminophenyl (0.8
pmole per milliliter of Sepharose). The column was equilibrated and developed with 50
mM borate buffer, pH 8.0, and IO mM CaC&. The bound enzyme was eluted with NH,OH.
pH 11 (arrow). The small amount of enzymatic activity present in the early, unretarded
peak could be removed by rechromatography of this peak through the same column. The
specific activity of the small amount of protein adsorbing strongly to the column was iden-
tical with that of native nuclease. Data from P. Cuatrecasas,J. Biol. Chem. 245,574 (1970).

Example 7: Other Enzymes. Papain has been purified with an agarose
adsorbent to which glycylgiycyl-(o-benzyl)-L-tyrosine-L-arginine had been
coupled by the cyanogen bromide procedure described earlier.32 Bovine
pancreatic ribonuclease can be purified with an agarose adsorbent

3ZS. Blumberg. I. Schechter, and A. Berger, fsrdJ. Chem. 7, 125 p (I 969).


362          SEPARATION BASED ON SPECIFIC AFFINITY      [311

containing 5'-(4-aminophenylphosphoryl)uridine-2'(3')-phosphate, pre-
pared by the cyanogen bromide method.=

Preparation of and Coupling to Modified Agarose Beads

A number of stable chemical derivatives of agarose beads, which can
be rapidly and easily prepared under mild aqueous conditions,2-3*4*7 will
be presented in order to provide greater versatility to the general
method. Since selective adsorbents must be tailored to the special char-
acteristics of the individual protein, and to the equally stringent ligand
requirements of that protein, it is useful to have alternative methods of
attaching ligands to agarose. In many cases it is much easier to modify
the matrix support than the ligand. More specifically, these derivatiza-
tions provide procedures especially applicable to cases in which (1) an
amino group on the ligand is not available or its synthesis is difficult; (2)
hydrocarbon chains of varying length need to be interposed between the
matrix and the ligand; and (3) only the mildest eluting conditions, i.e.,
neutral pH, absence of protein denaturants, are tolerated by the ad-
sorbed enzyme. In the last case, the protein ligand complex is removed
intact by specific cleavage of the ligand matrix bond. Some of the
derivatives described below are now available commercially (Affitron
Corp.).

w-Aminoalkyl Derivatives of Agarose Beads.2,7

Aliphatic diamine compounds, such as ethylenediamine, may be sub-
stituted directly to agarose by the cyanogen bromide procedure de-
scribed earlier (Scheme I, C). Although on theoretical grounds cross-
linking might be expected to occur, in practice very little, if any, can be
detected. This is probably due to the large excess of diamine added
during the coupling stage, so that the amino groups on the agarose
must compete with a very large excess of amino groups free in solution.
It is possible, therefore, to prepare agarose derivatives having free amino
groups which extend a good distance from the solid matrix, depending
on the nature of the group, -(CH,),. The w-aminoalkyl groups can be
used, in turn, for attachment of other functional groups, ligands, or
proteins.

Procedure-Preparation of Aminoethyl Agarose. To an equal volume of
a Sepharose4B water suspension is added 250 mg of cyanogen bromide
per milliliter of packed gel, and the reaction is performed as described
above. The washed, activated Sepharose is suspended in an equal volume
of cold distilled water containing 2 mmoles of ethylenediamine per

"M. Wilchek and M. Gorecki, Eur. J.Biochem. 11,491 (1969).


[311                AFFINITY CHROMATOGRAPHY            363

milliliter of Sepharose; the pH of this solution is adjusted to 10.0 with
6 N HCl before it is added to the Sepharose. After reaction for 16 hours
at 4", the gel is washed with large volumes of distilled water. This
treatment r'esults in a derivative having about 12 pmoles of aminoethyl
group per milliliter of Sepharose. Different w-aminoalkyl derivatives
can be prepared in a similar way by using other diamines. 3,3'-Diamino-
dipropyl amine has been one of the most useful for a variety of pur-
poses.2*3,22
Color Test with Sodium 2,4,6-Trinitrobenzenesuljkaate (TNBS)*
This simple color test, modified2 from that described by Inman and
Dintzis,*l is very useful in following the course of the various agarose
and polyacrylamide derivatizations to be described. One milliliter of
saturated sodium borate is added to a slurry (0.2-0.5 ml, in distilled
water) of the agarose or polyacrylamide gel. Three drops of a 3%
aqueous solution of sodium 2, 4,6-trinitrobenzenesulfonate are added;
the stock solution may be stored, in a brown glass bottle, for at least
2 weeks. At room temperature the color reaction of the gel beads is
complete within 2 hours. The following color products are formed with
various derivatives: unsubstituted agarose or polyacrylamide beads,
yellow; derivatives containing primary aliphatic amines, orange;
derivatives containing primary aromatic amines, red-orange; un-
substituted hydrazide derivatives, deep red; carboxylic acid and bromo-
acetyl derivatives, yellow. The degree of substitution of amino gel
derivatives by carboxylic acid ligands, and of hydrazide gels by amino
group containing ligands, can be conveniently estimated from the
relative color intensity of the washed, coupled gel.
Coupling Carboxylic Acid Ligands to Aminoethyl Agarose.2v7
Ligands containing free carboxyl groups may be coupled directly
to the o-aminoalkyl-Sepharose derivatives described above with a
water-soluble carbodiimide (Scheme II, A). Two examples of such
reactions are presented below.
Procedure a: Preparation of Estradiol-Sepharose,34 Three hundred
milligrams of 3-U-succinyl-[3H]estradiol (prepared by reacting [3H]-
estradiol with succinic anhydride in 50% dimethyl formamide-pyridine;
specific activity, 0.3 #Z/mole) is added in 40 ml of dimethyl formamide
to 40 ml of packed aminoethyl Sepharose-4B (2 pmoles of aminoethyl
groups per milliliter). The dimethyl formamide is required to solubilize
estradiol. The pH of this suspension is brought to 4.7 with 1 N HCI.
Five hundred milligrams (2.6 mmoles) of I-ethyl-3-(3-dimethylamino-

s'P. Cuatrecasas and G. A. Puca, unpublished observations (1970).


364         SEPARATION BASED ON SPECIFIC AFFINITY      I311

  AgarW                      Water

(a)  t                      soluble        R
     NH(CH2),NH2 + R-COOH               NH(CHI),NHC~
                         Carbodiimide
                        0
  AgaKW
(b) j       ii                          0
                                               II
    NH(CHI),NH2 + BYCHICO-N 3 - t NH(CH1),NHCCH,Br
                                        R-NH1
                        0

                                              R u
                             / \   OH
                                                  -
                             R-l-1

   1  NvNH

Alkylated derivative

AtWO%
                              NH(CH, )x NHCCH,CH,COH

NH(CH,),NHCCH&HJNH-R

  AgaPX

Cd) t NH(CHx),NH2 + 02N --u
             , \ fl  DMF N,%O.      '
                     cm,---+  NH(CH,),NHC        NH2
                     -     Hz0
                                        HNO,

OH

AZ0 Derivative

         +=o                   CH,

Agaro=
t      H*Y
(e)    NHW&.NHz +  H,C-CH      pH 9.1          I '
         IkOCH, 4" YEgf~~Dooode
                                Thiol ether derivatives   Thiol ester derivatives

                       SCHEMEII

propyl) carbodiimide, dissolved in 3 ml of water, is added over a 5-
minute period and the reaction is allowed to proceed at room tempera-
ture for 20 hours. The substituted agarose is then washed Continuously,
while packed in a column or on a Biichler funnel with 50% aqueous
dimethyl formamide until radioactivity is absent from the effluent. The


r311               AFFINITY CHROMATOGRAPHY            365

completeness of washing is checked by collecting 200 ml of effluent wash,
lyophilizing, adding 2 ml of dimethyl formamide, and determining
radiosctivity. Before use, it is essential to incubate the agarose in an
albumin buffer (3%, v/v) to determine if there is release of substances
into the medium which are capable of inhibiting the binding of 3H
estradiol to the uterine binding protein, this is an excellent way of
detecting non-covalently bound estradiol. About 0.5 pmole of [3H]-
estradiol are covalently bound to the agarose matrix. This is determined
by counting the Sepharose in Cab-0-Sil, or by hydrolyzing the ester
bond by exposure to 0.1 N NaOH for 60 minutes at room temperature,
followed by centrifugation and estimating radioactivity in the super-
natant fluid. With such derivatives it is possible to elute strongly ad-
sorbed serum estradiol binding protein by cleaving the estradiol agarose
bond with mild base, thus avoiding the use of protein denaturants.
This estradiol-agarose derivative is very effective in extracting estradiol
binding proteins from serum and from calf uterine extracts.34 Estradiol
has been attached to polyvinyl and cellulose polymers by different pro-
cedures.35

Procedure b: Preparation of Organomercurial-agarose for Separation of
SH-proteins?*' Packed aminoethyl-Sepharose, 35 ml (capacity, 10
pmoles/ml) is suspended in a total volume of 60 ml of 40% dimethyl
formamide; 2 mmoles of sodium p-chloromercuribenzoate is added;
the pH is adjusted to 4.8; and 5 mmoles of 1-ethyl-3-(3-dimethyl-
aminopropyl) carbodiimide are added. The pH is maintained at 4.8
by continuous titration with 0.1 N NaOH for one hour. After reaction at
room temperature for another 18 hours, the substituted agarose is
washed with 4 liters of 0.1 M NaHC03, pH 8.8, over an 8-hour period.
Complete substitution of the agarose amino groups is confirmed by the
TNBS color test, which yields a yellow gel. The derivative can bind 40-50
mg of horse hemoglobin per milliliter of packed gel. The effectiveness
of the binding, compared to that of a derivative prepared by attaching
a bifunctional organomercurial to SH-Sephadex G25 by much more
complicated procedures,36 is reflected in the fact that solutions of low
pH (2.7, acetic acid) or complexing agents, such as EDTA (0.1 M),
remove the protein only very slowly. The most effective elution is
achieved by passing a 50 mM cysteine or dithiothreitol solution into the
column and stopping the flow for 1 or 2 hours before collecting the
effluent. Advantages of the present procedures include the ease of

%B. Vonderhaar and G. C. Mueller, Biochim. Biophys. Acta 176,626 (1969).
"L. Eldjarn and E. Jellum, A& Chem. Sand. 17.2610 (1963).


366          SEPARATION BASED ON SPECIFIC AFFINITY      [311

preparation, the use of agarose with its inherently large capacity to bind
large proteins by permitting their diffusion throughout the mesh, the
circumvention of conditions which irreversibly destroy the agarose beads,
i.e., drying and dioxane, and the stability of the final derivative.

Preparation of Bromoacetyl Agarose Derivative.s.2*7

Bromoacetamidoethyl-Agarose can be prepared under mild aque-
ous conditions by treating aminoethyl-Sepharose-4B with O-bromo-
acetyl-N-hydroxysuccinimide (Scheme II, B). This derivative of agarose
can react with primary aliphatic or aromatic amines as well as with
imidazole and phenolic compounds. Additionally, proteins readily
couple to bromoacetamidoethyl-Sepharose, forming insoluble de-
rivatives in which the protein is located at some distance from the solid
support.

Procedure. In 8 ml of dioxane are dissolved 1 .O mmole of bromoacetic
acid and 1.2 mmole of N-hydroxysuccinimide. To this solution, 1 .l
mmole of dicyclohexylcarbodiimide is added. After 70 minutes, di-
cyclohexylurea is removed by filtration, and the entire filtrate, or
crystalline bromoacetyl-N-hydroxysuccinimide ester, is added, without
further purification, to a suspension, at 4", which contains 20 ml of
packed aminoethyl-Sepharose (2 pmoles of amino groups per milliliter)
in a total volume of 50 ml at pH 7.5 in 0.1 M sodium phosphate. After
30 minutes, the product is washed with 2 liters of cold 0.1 N NaCl.
Quantitative reaction of the amino groups occurs as shown by the loss
of orange color with the TNBS test. The bromoacetamidoethyl-Sepha-
rose gel can be stored at 4" as a suspension in distilled water (pH about
5.5), or can be treated with a primary amine, R-NH2 (Scheme II, B).
Reaction with 10 mM pdTp-aminophenyl as a 50% (v:v) suspension
in 0.1 M NaHCO,, pH 8.5, for 3 days at room temperature, followed
by reaction for 24 hours at room temperature with 0.2 M P-amino-
ethanol to mask unreacted bromoacetyl groups, results in attachment
of 0.8 pmole of inhibitor per milliliter of packed agarose. A similar
reaction with [3H]5'-GMP resulted in the covalent attachment of 0.2
pmolesiml. Such bromoacetyl-Sepharose derivatives have also been
used to insolubilize [3H]estradiol for use in the purification of serum
and uterine estradiol binding proteins.34 Proteins can be similarly
attached by reacting in 0.1 M NaHC03, pH 9.0, for 2 days at room
temperature, or for longer periods at lower temperatures.

Preparation of Succinylaminoethyl-Agaros2*7                   d
This derivative is prepared by treating aminoethyl-Sepharose with
succinic anhydride in aqueous media at pH 6.0 (Scheme II, C). One


[311                AFFINITY CHROMATOGRAPHY           367

millimole of succinic anhydride is added per milliliter of packed amino-
ethyl-Sepharose (capacity, 8 @moles/ml), in an equal volume of distilled
water a't 4", and the pH is raised to and maintained at 6.0 by titrating
with 20% NaOH. When no further change in pH occurs, the suspension
is left for 5 more hours at 4". Complete reaction of the amino group
occurs as shown by the TNBS color reaction. Compounds containing
primary amino groups can be coupled at pH 5 to such carboxyl con-
taining Sepharose derivatives in the presence of the water-soluble
carbodiimide,  l-ethyl-3-(3-dimethylaminopropyl) carbodiimide, by
the same procedures described above.

Example: Purification of Bacterial /3-Galactosidase.22 The very "long"
diamine derivative, 3, 3' diaminodipropylamine, was coupled to
Sepharose (about 10 pmoles/ml of gel) by the procedure described for
the preparation of aminoethylagarose. The succinyl derivative was then
prepared as described above. One millimole of p-aminophenyl-p-n-
thiogalactopyranoside is added to 30 ml of the succinyl gel suspended
in 50 ml of distilled water. The pH is adjusted to 4.7, and 7.5 mmoles
of l-ethyl-3-(3dimethylaminopropyl) carbodiimide in 3 ml of water,
are added dropwise over a 5-minute period. The pH is maintained at
4.7 for 1 hour by titrating with 0.1 N NaOH. The suspension is stirred
at room temperature for another 16 hours, then washed with about
12 liters 0.1 N NaCl over an S-hour period. The adsorbent, prepared
in this way, binds &-alactosidase from several bacterial sources very
strongly; elution of the protein from such a column is achieved by
passage of a buffer having a pH of 10, or with a substrate-containing
buffer. It is notable that gel derivatives prepared by attaching the same
ligand directly to agarose, or by the diazotization procedure (described
below), were ineffective in binding the enzyme. This compound, which
is a very weak competitive inhibitor with a Ki of about 5 mM, must be
attached to the solid matrix backbone by a rather long "arm."

Preparation of p-Aminobenzamidoethyl-Agarose for Coupling Cornpour&
via Azo Linkage.2*7

Diazonium derivatives of agarose, which are capable of reacting with
phenolic and histidyl compounds, can be prepared under mild aqueous
conditions from p-aminobenzamidoethyl-Sepharose (Scheme II, D).
The azo-substituted agarose gels retain the properties of good flow
rate, porosity, capacity, and stability, all of which are necessary for
effective affinity chromatography.

Procedure. Aminoethyl-Sepharose, in 0.2 M sodium borate, pH 9.3,
and 40% dimethyl formamide (v:v), is treated for 4 hours at room
temperature with 70 mM p-nitrobenzoyl azide (Eastman). The sub-


368          SEPARATION BASED ON SPECIFIC AFFINITY      ISJI

stitution is complete, as judged by the loss of color reaction with TNBS.
The p-nitrobenzamidoethyl-Sepharose is washed extensively with 50%
dimethyl formamide and is reduced by reaction for 60 minutes at
40" with 0.2 M sodium dithionite in 0.5 M NaHC03 at pH 8.5. The
p-aminobenzamidoethyl-Sepharose derivative, in 0.5 N HCI, can be
diazotized by treating for 7 minutes at 4" with 0.1 M sodium nitrite.
This diazoniumSepharose derivative may be utilized at once, without
further washing, by adding the phenolic, histidyl, or protein component
to be coupled in a strong buffer such as saturated sodium borate, and
adjusting the pH with NaOH to 8 (histidyl) or 9.2 (phenolic); the reaction
is allowed to proceed for about 16 hours at 4". Strong tertiary amines,
such as triethylamine, should not be used as buffers during the azoti-
zation reaction since they can react with the diazonium intermediate.
Agarose derivatives containing estradiol in azo linkage at C-2 and C-4
of the A ring remove estradiol binding proteins from human serum
and from calf uterine extracts.34
An important advantage of the azo-linked ligand derivatives of
agarose or of polyacrylamide beads is that passage of a solution of
0.1 M sodium dithionite in 0.2 M sodium borate at pH 9, through such
columns causes rapid and complete release of the bound inhibitor by
reducing the azo bond. This allows easy and accurate estimation of the
quantity of inhibitor bound to the gel. Of greater importance, the pro-
cedure permits elution of the intact protein-inhibitor complex under
relatively mild conditions. For example, serum estradiol binding protein
is denatured irreversibly by exposure to pH 3 or 11.5, and by low
concentrations of guanidine hydrochloride (3 M) or urea (4 M). The
protein, which binds estradiol very tightly (K, about 10mg M), can be
removed in active form from the agarose-estradiol gel by reductive
cleavage of the azo link with dithionite, but not with buffers of low
pH or with guanidine-HC1.34
Preparation of Tyrosyl-Agarose for Coupling Diazotized Compound?*'
A tripeptide containing a COOH-terminal tyrosine residue can be
attached to agarose by the cyanogen bromide procedure described
earlier. Ligands containing an aromatic amine, which can be diazotized,
can then be coupled in high yield through azo linkages to the tyrosyl
moiety (Scheme III). The ligand thus extends considerably from the
matrix backbone. An example of such a coupling procedure which has
been used to insolubilize deoxythymidine 5'-phosphate 3'-p-amino-
phenylphosphate (for staphylococcal nuclease) and p-aminophenyl-
&n-thiogalactoside (for &galactosidase), and which is Zpplicable to
many other compounds is the following: 100 pmoles of inhibitor is


r311               AFFINITY CHROMATOGRAPHY            369

Sepharore  + HINCH,CONHCH,CONHCHCII~




                CNBr

NHCH2CONHCH2CONHCHCH~         OH

NHCtI,CONHCH2CONHCHCH2  / \
   -Q- OH
                    -

                       N=N         R


           SCHEME III

dissolved in 1.5 ml of 1.5 N HCI, and placed in an ice bath; 700 pmoles
of NaN02, in 0.5 ml of water, is added over a l-minute period to the
stirred ligand solution. After 7-8 minutes the entire mixture, without
further purification, is added to a rapidly mixing suspension, in an ice
bath, of tyrosyl-Sepharose containing 0.2 M NapCOB. The pH is rapidly
adjusted to 9.4. After 3 hours the agarose suspension is transferred
to a Biichner funnel and washed. If dimethyl formamide is to be used
in the reaction mixture, the buffer should be sodium borate in order to
prevent precipitation of the salt. Staphylococcal nuclease is readily
purified in one step by passage through a column of such a derivative
containing the competitive inhibitor, pdTp-aminophenyl, in azo
linkage. Congo red dye has also been attached to Sepharose in this
manner; small amounts of amyloid protein are agarose adsorbed
to this matrix after passage of crude material dissolved in 4 M guanidine.
HCl at pH 6; elution is achieved with 6 M guanidine*HCl at pH 3. These
l&-and-protein complexes can be removed by treating the gel with
sodium dithionite, as described earlier for other gels containing azo-
linked ligands.

Preparation of Sulfhydryl Agarose Derivative?
Thiol groups can be introduced into w-aminoalkyl agarose derivatives


370         SEPARATION BASED ON SPECIFIC AFFINITY      [311

by reaction with homocysteine thiolactones by using procedures similar
to those used for thiolation of proteins (Scheme II, E).~`s~~ Five grams
of N-acetylhomocysteine thiolactone is added to a cold (4") suspension
consisting of an equal volume (50 ml) of aminoethyl Sepharose 4-B and
of 1 .O M NaHC03, pH 9.7. The suspension is stirred gently for 24 hours
at 4", then washed with 8 liters of 0.1 N NaCI. The agarose derivative is
then incubated in 0.05 M dthiothreitol, 0.5 M TrisHCl buffer, pH 8.0,
for 30 minutes at room temperature. The suspension is washed with 2
liters of 0.1 M sodium acetate buffer, pH 5.0. At this stage a strong
red-brown color is obtained with the sodium trinitrobenzenesulfonate
color test. The color produced with this reagent is completely lost
after treating a sample of the thiol agarose with 50 mM iodoacet-
amide in 0.5 M NaHC03, pH 8.0, for 15 minutes at room temperature,
followed by washing with distilled water. This confirms that complete
substitution of the amino-agarose groups has occurred. The derivative
can be stored for many months as an aqueous suspension at 4" provided
that it is treated, before use, with dithiothreitol as described above.

The degree of sulfhydryl substitution can be conveniently determined
by reacting the thiol-agarose with 5,5'-dithio-bis-(2-nitrobenzoic acid)3g
at pH 8, followed by centrifugation and determination of the absorb-
ancy at 412 rnk in the supernatant. By this procedure, the derivative
described above contains 0.58 pmole of sulfhydryl per milliliter of
packed agarose. Reaction with 10 mM [`4C]iodoacetamide in 0.1 M
NaHCO,, pH 8.0, for 15 minutes at room temperature, results in up-
take of 0.65 pmole of radioactivity per milliliter of agarose.

Ligands or proteins that contain alkyl halides can be readily coupled
to sulfhydryl agarose derivatives through stable thioether bonds. The
reactivity of the thiol gels with heavy metals may be of special value in
affinity chromatography. Furthermore, agarose derivatives containing
free sulfhydryl groups can be used to couple ligands by thiol ester
linkage, as described below.

Coupling Ligands to Agarose by Thiol Ester Bonds.2 Ligands containing a
free carboxyl group can be coupled to sulfhydryl-agarose with water
soluble carbodiimides. Although the thiol ester bonds thus formed are
stable at neutral pH, cleavage is readily achieved by exposure for a
few minutes to pH 11.5, or by treatment with 1 N hydroxylamine for
about 30 minutes. Thus, it is possible by specific chemical cleavage to
remove the intact ligand-protein complex from an adsorbent containing
the specifically bound protein.

s7R. Benesch and R. E. Benesch, J. Amer. Chem. Sot. 78,1597 (1956).
"R. Benesch and R. E. Benesch, Proc. Nat. Acad. Sci. U. S. 44,848 (1958).
s9G. L. Ellman, Arch. Biochem. Biophys. 82,70 (1959).


[311               AFFINITY CHROMATOGRAPHY            371

N-Acetylglycine (0.2 M) is added to 40 ml of packed sulfhydryl-
agarose (containing 0.7 pmole of thiol per milliliter of packed agarose)
adjusted to 80 ml with distilled water. The pH is adjusted to 4.7, and
1.5 g of l-ethyl-3-(3-dimethylaminopropyl) carbodiimide, dissolved
in 3 ml of water, is added. The pH is maintained at 4.7 for 1 hour by
continuous titration. The suspension is allowed to stir gently for 8-12
hours at room temperature. The agarose is then washed with 2 liters
of 0.1 N NaCl. At this stage virtually no color is obtained with the sodium
trinitrobenzene sulfonate color test, and the sulfhydryl content, de-
termined with 5,5'-dithiobis-(2-nitrobenzoic acid),3s is about 0.03
pmole per milliliter of Sepharose. All the sulfhydryl groups remaining
free on the Sepharose are masked by treatment with 0.02 M iodoacet-
amide for 20 minutes at room temperature in 0.2 M NaHC03, pH
8.0. 3-0-Succinylestradiol has been coupled to sulfhydryl-Sepharose
by these procedures. The resulting adsorbent is very effective in binding
estradiol binding proteins from serum and uterine extracts.34

Preparation of Derivatives of Polyacrylamide Beads

As discussed earlier, the principal advantage of polyacrylamide beads
over agarose is that a much higher degree of substitution is possible
with the former. However, the considerably lower porosity of currently
available beads may limit their use in the affinity chromatography of
very large proteins. Another consideration is that some shrinkage of
the gels occurs during formation of the acyl azide intermediate2; shrink-
age would be expected to lead to decreased porosity. Thus, it may be
possible to prepare an adsorbent containing a very high concentration
of ligand, much of which is inaccessible to the protein to be purified.**
Only the most porous beads, Bio-Gel P-300 (Bio-Rad Labs), have been
found effective for the purification of staphylococcal nuclease, a protein
with a molecular weight of 17,000. The procedures, based on those
described by Inman and Dintzis, *l described below, involve conversion
of the carboxamide side groups to hydrazide groups which in turn
are converted with nitrous acid into azyl azide derivatives, thereby allow-
ing ready reaction with aliphatic and aromatic primary amines.

Preparation of Hydrazide Derivatives (Scheme IV, 1).2*21 Swollen poly-
acrylamide beads, in a volume of water equal to 1.5 times the value of
the hydrated gel and containing 3-6 M hydrazine hydrate are heated in
a constant temperature bath to between 45" and 50". The procedure is
carried out under constant stirring in a stoppered vessel in a hood.
The time required for reaction is a function of the degree of derivati-
zation desired; the amount of substitution varies linearly for at least
an 8-hour period (Fig. 6). Thereafter, the hydrazide-acrylamide


372

SEPARATION BASED ON SPECIFIC AFFINITY


   Polyacrylamide
   1 + $N,,,,,
            H,NNH,

[311

2 FR, ; +INu-R
NaNO>
  HCI         R-NH2

SCHEME IV

derivative is washed in a hood with large volumes of 0.1 M NaCl until
the washes are free of hydrazine as determined by the TNBS color
reaction. Bio-Gel P-300 derivatives are conveniently washed by re-
peated decantation using large volumes of saline, rather than on a
Biichner funnel with its slower flow rate. Approximately 30 pmoles of
hydrazide per milliliter of packed resin (Bio-Gel P-300) are formed
after treatment for 3 hours at 47" in 3 A4 hydrazine. Greater substi-

4.0- , ( , ( , ( , ,

012345678
     Time of heotlng (hours)

1

FIG. 6. Time course of the reaction between polyacrylamide Bio-Gel P-60, 100-200
Mesh) and aqueous hydrazine. Molar concentrations of hydrazine and temperature are
indicated. Curve C is the average time course of carboxyl group formation during the runs
for curves A and B. Specific capacity, C', refers to mmoles of hydrazide group found on the
amount of derivative produced from 1 g of the original dry polyacrylamide. Similar curves
are to be expected for Bio-Gel beads other than P-60. Data from J. K. kiman and H. M.
Dintzis, Biockemictry 8, 4074 (1969).


[311               AFFINITY CHROMATOGRAPHY           373

                                                      -
.

tution on Bio-Gel P-300 is not recommended because the physical
properties of the beads are adversely affected. The polyacrylamide-
hydrazide derivatives may be stored in aqueous media in the presence
of a preservative, such as sodium azide or toluene, for at least 5 months.

Coupling of Primary Amines via the Azyl Azide Procedure (Scheme IV,
2 3).3*1s Primary aliphatic or aromatic amines can be coupled via the
a;yl azide derivative without intermediate washings or transfers. The
hydrazide polymer (packed volume, 50 ml) is washed, and suspended
in twice its volume of 0.3N HCl and the entire suspension is placed in
an ice bath. Ten milliliter of 1.0 M NaNOz are added rapidly under
vigorous magnetic stirring. After 90 seconds, the amine is added
rapidly in about 20 ml of cold 0.2 M Na2C03, and the pH is raised to
9.4 with 4 M NaOH. After reaction for 2 hours at 4", the suspension is
washed with distilled water and suspended for 5 hours in 2 M NH4CI-1
M NH,OH, pH 8.8, in order to convert unreacted hydrazide to the
carboxamide form. If a high degree of substitution is desired, the
amine should be added in amounts lo- to 20-fold greater than that of
the hydrazide content of the gel.

Other Polyacrylamide Derivatization.s.2 In general, the specific poly-
acrylamide adsorbents are more useful when the ligand is attached at
some distance from the matrix. For this purpose o-aminoalkyl, bromo-
acetamidoethyl, Gly-Gly-Tyr, and p-aminobenzamido ethyl derivatives
can be prepared by procedures identical to those described earlier.
Very satisfactory affinity adsorbents have been prepared for the puri-
fication of staphylococcal nuclease by many of these procedures. A
derivative obtained by coupling pdTp-aminophenyl directly to Bio-Gel
P-300 via the acyl azide step, which contained 12 pmoles of inhibitor
per milliliter of gel, effectively adsorbed 22 mg of nuclease per milli-
liter of resin from a crude enzyme preparation. Enzyme was recovered
by elution with NH40H at pH 11. Other ligands which have been
coupled to polyacrylamide beads include Congo red, p-aminophenyl-
/3-n-thiogalactopyranoside, 5'-GMP, estradiol, adenosine 3', 5'-cyclic
monophosphate, and several amino acids.

Coupling of Proteins and Peptides to Agarose for Ajfinity Chromatography

The previously described desirable features of agarose, particularly
its porosity, permit the preparation of insoluble protein derivatives
which can be used to exploit reversible macromolecular interactions.
In addition to attaching proteins or peptides to agarose directly by the
cyanogen bromide procedure, it is possible to use the bromoacetyl or
the diazonium derivatives described earlier. Proteins coupled to such gels
extend some distance from the matrix backbone by an "arm." This may


374         SEPARATION BASED ON SPECIFIC AFFINITY      [311

be important in decreasing steric difficulties when interactions with
other macromolecules are being studied. For example, purification or
isolation of intact cells or particulate cell structures by insolubilized
proteins may best be achieved with such derivatives.3
An important consideration in the covalent attachment of a bio-
logically active protein to an insoluble support is that the protein
should be attached to the matrix by the fewest possible bonds. This will
enhance the probability that the attached macromolecule will retain its
native tertiary structure. Proteins react with cyanogen bromide-activated
agarose through the unprotonated form of their free amino groups.
Since most proteins are richly endowed with lysyl residues, which are
exposed to the solvent, it is likely that such molecules will have multiple
points of attachment to the resin when the coupling reaction is per-
formed at pH 9 or higher. The number of linkage points and the re-
sultant biologic activity of the insolubilized protein can be controlled by
manipulating the pH of the coupling reaction. Proteins having few
amino groups, e.g., insulin, can be preferentially attached to agarose
through the a-amino group by coupling at a relatively low pH (5.0) and
high ionic strength. 4o Proteins with a large number of lysyl residues,
and any protein of high molecular weight, yield much more efficient
adsorbents when coupled to agarose at pH values in the neighborhood
of 6.0-6.5 (see example 2, Fig. 7). *s3 Only a small fraction of the lysyl
residues, and the a-amino groups, will be significantly nonionized
and thus capable of reaction with the cyanogen bromide-activated
agarose.

In spite of the low pH, adsorbents containing a large amount of pro-
tein can still be obtained by the procedures described above by (1)
using agarose activated with relatively large amounts of cyanogen
bromide (250 mg per milliliter of packed Sepharose), (2) performing
the coupling procedure at pH 6, and (3) using high protein concen-
trations during the coupling reaction.2'3

Because of its greater porosity, Sepharose 2B should be used for
studies involving the isolation of particulate structures, such as cells,
cell membranes, or viruses. Protein-agarose derivatives are quite
stable when stored in suspension at 4", and it is generally possible to
use them repeatedly.

It should be stressed that when the biological activity of insoluble
ligands or proteins is to be studied, special care must be taken to ensure
complete removal of adsorbed, noncovalently bound material. This may
require long and tedious washing procedures. It is recommended that,
whenever possible, insolubilized protein derivatives be washed ex-

`OP. Cuatrecasas, Proc. Nat. Ad. Sci. LT. S. 63,450 (1969).


[311                AFFINITY CHROMATOGRAPHY           375

!O -





15 -'





IO -





5,





OL

\      (al

I

n-nll-d-

-


         (b)





              t
  I
i!dL
1   3  5   7   05  87  89

                         Effluent (ml)
FIG. 7. Affinity chromatography patterns of [sH]insulin on columns containing purified
antiporcine insulin sheep immunoglobulin which had been coupled by the cyanogen bro-
mide procedure to agarose at pH 9 (A) and at pH 6.5 (B). Forty milligrams of purified
y-globulin, in 10 ml of 0.2 M sodium citrate, pH 6.5, or 8 ml of 0.2 M sodium bicarbonate,
pH 9.0, were added to 5 ml of Sepharose 48 activated with 300 mg of cyanogen bromide
per milliliter of gel. Based on the recovery of absorbancy (280 mp) in the washes, about
90-95% of the protein was coupled in both cases. The specific adsorbent was washed with
20 times its volume of 6 M guanidine.HCl before use. [`H]Acetyl insulin, 40 pg, in 1 ml of
50 mM borate buffer at pH 8.5 containing 0.1 N NaCI, and 0.5% bovine albumin, was ap-
plied to a 12 X 0.6 cm column. The column was then washed with 85 ml of the above buffer,
and elution of bound insulin was accomplished with 6 M guanidine.HCl (arrow). The theo-
retical capacity for insulin binding, based on the amount of antibody covalently linked and
on the capacity of the antibody to bind insulin in solution, was about 30 pg for both adsor-
bents. Column A adsorbed 2 pg of insulin, or 7% of its theoretical capacity, whereas col-
umn B adsorbed 23 pg. or 77% its theoretical capacity.

tensively with 6 M guanidineaHC1. To further ensure that the biological
function being measured is due to material covalently attached, the
effect of varying the concentration of the insoluble derivative should
be studied, and the possible release of the material must be examined
after incubation in buffers containing high concentrations of albumin
or other substances that simulate the biological environment.40

Example 1: Purijication of Antibodies. Protein-agarose and haptene-
agarose derivatives appear to be nearly ideally suited for use as immuno-
sorbents.41-43 For example, porcine insulin linked to agarose 2B through
its single lysyl residue (B-29) can completely remove insulin antibodies
from crude mixtures.41 The very strong binding of antibody by this

"P. Cuatrecasas, Biochetn. Biophys. Res. Commun. 35,S3 1 (1969).
42L. Wofsv and B. Burr.J. Imm&ol. 103,380 (1969).
43G. Omenn, D. Ontjes, and C. B. Anfinsen, Nafure (London) 225, 189 (1970).


376          SEPARATION BASED ON SPECIFIC AFFINITY       r311

derivative is reflected by the need to use strong acid conditions (1 N HCI)
to achieve elution of the major fraction of the antibody from the column.
Recovery of antibody applied to such a column approaches 80%.
Large-scale purification of sheep antiporcine insulin is readily achieved
with these adsorbents. A column containing 20 ml of packed insulin-
Sepharose described in Fig. 7 removed all the insulin antibody present in
200 ml of crude immune sheep serum. The combined washes of equili-
brating buffer (500 ml) and of 0.1 M sodium acetate, pH 5.5 (1 liter),
contained only 5% of the total antibody applied to the column. About
65% (150 mg) of the total insulin antibody applied was eluted sharply
with acetic acid at pH 2.8 in a volume of 29 ml. Subsequent elution with
6 M guanidinemHC1 removed about 15% of the antibody applied.
Example 2: Immunoglobulin-Sepharose for Purification of Antigens?4 Puri-
fied insulin antibodies, obtained by affinity chromatography as described
above, can in turn be covalently attached to agarose to prepare de-
rivatives capable of isolating the corresponding antigen and other
immunologically cross-reacting proteins. The studies on anti-insulin
shown in Fig. 7 illustrate the importance of attaching large proteins by
few linkage points. The immunoglobulin coupled to activated agarose
at pH 9 could adsorb only 7% of its theoretical capacity for insulin,
whereas that coupled at pH 6.5 could bind 77% of its theoretical ca-
pacity for insulin. Since the total protein content of both derivatives
is the same, the former derivative must contain immunoglobulin which
is incapable of effectively binding antigen.

Example 3: Polypeptide Hormones and Isolation of Cellular Receptor Struc-
tures. Insolubilized ligands or proteins can also be used to study inter-
actions of various molecules with intact cells, and to explore membrane
phenomena. Although the macromolecules involved were not attached
covalently to the supporting matrix, the recent studies of Wigzell and
Andersson45 on the fractionation of immunologically active cells on
columns of glass or plastic beads to which antigen coatings were tightly
adsorbed indicate the power of the approach. Similar studies on the
immunoadsorption of cells on reticulated polyester polyurethane
foam-coated with antibodies have been reported by Evans, Wage, and
Peterson.46

Insulin-agarose derivatives are capable of stimulating several dif-
ferent biological properties of intact isolated fat cells nearly as ef-
fectively as native insulin (Fig. 8). 4o Columns prepared with these
insulin-agarose derivatives,  in contrast to those with unsubstituted

"P. Buatrecasas; unpublished observations (1970).
UH. Wigzell and B. Andersson,]. Exp. Med. 129,23 (1969).
`@W. H. Evans, M. G. Wage, and E. A. Peterson,]. Immunol. 102,899 (1969).


r311                AFFINITY CHROMATOGRAPHY            377

Insulm concentrotlon, pU/ml

F~ti. 8. Effect of insulin-agarose concentration on the oxidation of [U'"C] glucose
by isolated adipose cells. Fat cells (3-8 pmoles of triglyceride) were incubated for 2 hours
at 37" in 2 ml of medium containing 0.5 mM [IJ"C] (0. I &i/pmole). The data presented
are for native insulin (e), insulin coupled to Sepharose 2B (at pH 9) through lysine B29
(0). insulin coupled (at pH 5) through phenylalanine Bl (A), and insulin acetylated at
positions Al and Bl and coupled to agarose by lysine B29. Data from P. Cuatrecasas,
Proc. Naf. Acad. Sci. U. S. 63.450 (1969).

agarose, bind intact fat cell "ghosts" very strongly.44 The latter can
be specifically eluted intact fram the insulin-agarose columns with
concentrated insulin solutions. These studies indicate that such insoluble
hormone derivatives interact very effectively with membrane structures,
and thus are promising tools for the isolation of "receptor" proteins
(hormone, drug, etc.).

Example 4: PuriJication of Histidyl-tRNA with Enzyme-Sepharose?7 Five
milliliters of packed Sepharose 4B, activated with 1.3 g of cyanogen
bromide, is reacted with I.8 mg of purified histidyl-tRNA-synthetase
(lacking other acyl tRNA-synthetase activities) in 50 mM phosphate
buffer, pH 6.5. Virtually all the protein is coupled to Sepharose, since
the washes contain essentially no absorbance at 280 rnp and only
l-3% of the enzyme activity is present. Columns containing this ad-
sorbent strongly adsorb histidyl-tRNA, which can be eluted with
1 M NaCl (Fig. 9).

Example 5: PuriJication qf` Peptides Prebared by Organic Synthesis. A
polypeptide corresponding to the amino acid sequence from residue
6 through 47 in staphylococcal nuclease (149 amino acids), obtained by
organic synthesis, can be purified by passing the crude synthetic poly-

"F. Blasi and R. Goldberger, personal corntrlLlrlicatiol~ (1969).


378          SEPARATION BASED ON SPECIFIC AFFINITY      [311

Effluent (ml)

FIG. 9. Purification of histidyl-tRNA on a 0.6 X 8 cm column containing agarose
coupled with purified histidyl-tRNA-synthetase (0.36 mg/ml Sepharose). The column was
equilibrated with 66 mM potassium cacodylate, pH 7.0, containing 8 mM reduced gluta-
thione and IO mM MgCI,. About 0.4 optical density unit of [3H]histidyl-tRNA was applied
to the column in 0.2 ml of the above buffer. Elution was obtained with 3.0 M NaCI, acetic
acid pH 3; it is also possible to elute with I N NaCI. Data from F. Blasi and R. Coldberger,
unpublished.

peptide mixture through a column containing a Sepharose-linked
native peptide which comprises residues 49-149.48 In solution these
two peptides separately have no discernible tertiary structure or en-
zymatic activity, but upon mixing there is regeneration of enzymatic
activity. Advantage of the affinity of these two peptides is taken to ob-
tain a "functional" purification of the synthetic peptide. In a similar
manner, it is possible to purify synthetic RNase-S-peptide derivatives
on agarose columns containing RNase-S-protein (Fig. 1).5 The specific
activity of the purified peptide is increased by this procedure, but the
chemical and catalytic properties of the purified materials suggest
the presence of closely related synthetic side products which bind
tightly to the S-protein conjugate but which yield enzymatically in-
active complexes with RNase-S-protein.

`sD. Ontjes and C. B. Anfinsen,J. Biol. Chm. 244,6316 (1969).