Affinity chromatography with collapsibly tethered ligands

Article abstract of DOI:10.1021/ac0263768

We introduce a novel affinity chromatography mode in which affinity ligands are secured to the media surface via collapsible tethers. In traditional affinity chromatography, the immobilized ligands act passively, and their local concentration is static. In collapsibly tethered affinity chromatography, the ligand can move dynamically in response to external stimuli, a design that enables marked changes in both the local concentration of the ligand and its surrounding environment without exchange of solvent. Using the thermoresponsive polymer poly(N-isopropylacrylamide) (PIPAAm) as a scaffold for ligand and hapten attachment, we were able to achieve controlled mobility and microenvironment alteration of the affinity ligand Ricinus communis agglutinin (RCA₁₂₀). The glycoprotein target, asialotransferrin, was loaded onto a column in which PIPAAm was partially substituted with both RCA₁₂₀ and lactose. At 5 °C, the column retained the glycoprotein, but released most (95%) of the asialotransferrin upon warming to 30 °C. This temperature-induced elution was much greater than can be explained by temperature dependency of sugar recognition by RCA₁₂₀. The simplest explanation is that upon thermally induced dehydration and collapse of the PIPAAm chains, coimmobilized RCA₁₂₀ ligand and lactose hapten are brought into closer proximity to each other, enabling immobilized lactose to displace affinity-bound asislotransferrin from the immobilized RCA₁₂₀ lectin.

Full text of DOI:10.1021/ac0263768

Anal. Chem. 2003, 75, 1658-1663
Affin it y Ch ro m a t o g ra p h y w it h Co lla p s ib ly Te t h e re d
Lig a n d s
Hide nori Ya m a na ka ,* Kim ihiro Yos hiza ko, Yos hika ts u Akiya m a , Hiroyuki Sota , Yukio Ha s e ga w a , a nd
Ya s uro Shinoha ra
Department of Research and Development, Amersham Biosciences K.K., 3-25-1 Hyakunincho, Shinjuku-ku,
Tokyo 169-0073, Japan
Akihiko Kikuc hi a nd Te ruo Oka no*
Institute of Advanced Biomedical Engineering & Science, Tokyo Women’s Medical University, 8-1 Kawadacho, Shinjuku-ku,
Tokyo 162-8666, Japan
pH, ionic strength, and solvent) or by use of competitive elution.2
In any case, the diffusion of immobilized ligand is always strictly
limited, and therefore, the local concentration of immobilized
ligand is constant. The environment surrounding the immobilized
ligand changes to modulate the ligand-analyte affinity.
An important question is whether similar elution can be
induced if the immobilized ligand physically moves to produce
changes in its local concentration and surrounding environment.
Despite numerous elution studies, the concept of such a locally
mobile ligand has never been described.
We introduce a novel affinity chromatography mode in
which affinity ligands are secured to the media surface
via collapsible tethers. In traditional affinity chromatog-
raphy, the immobilized ligands act passively, and their
local concentration is static. In collapsibly tethered affinity
chromatography, the ligand can move dynamically in
response to external stimuli, a design that enables marked
changes in both the local concentration of the ligand and
its surrounding environment without exchange of solvent.
Using the thermoresponsive polymer poly(N-isopropyl-
acrylamide) (P IP AAm) as a scaffold for ligand and hapten
attachment, we were able to achieve controlled mobility
and microenvironment alteration of the affinity ligand
Ricin u s com m u n is agglutinin (RCA120). The glycoprotein
target, asialotransferrin, was loaded onto a column in
which P IP AAm was partially substituted with both RCA120
and lactose. At 5 °C, the column retained the glycoprotein,
but released most (9 5 %) of the asialotransferrin upon
warming to 3 0 °C. This temperature-induced elution was
much greater than can be explained by temperature
dependency of sugar recognition by RCA1 2 0 . The simplest
explanation is that upon thermally induced dehydration
and collapse of the PIPAAm chains, coimmobilized RCA120
ligand and lactose hapten are brought into closer proxim-
ity to each other, enabling immobilized lactose to displace
affinity-bound asislotransferrin from the immobilized
RCA1 2 0 lectin.
In the present stud,y we utilized poly(N-isopropylacrylamide)
PIPAAm),3-6 a well-investigated thermoresponsive polymer, as
(
a microactuator to induce significant environmental changes of
immobilized ligand. Thermoresponsive polymers exhibit a ther-
moreversible phase transition in aqueous solution at a lower
critical solution temperature (LCST). The polymer chain hydrates
to form an extended chain conformation below the LCST, whereas
it dehydrates to form a shrunken globule structure above the
LCST. The hydrodynamic size of the PIPAAm chain is reported
to shrink by about one-half above the LCST, which corresponds
7,8
to an ∼10-fold hydrodynamic volume reduction. It was, therefore,
expected to provide a unique system in which local ligand
concentrations could be regulated dynamically by external stimuli.
To our knowledge, no such attempts have been previously
described, though thermoresponsive polymers have been used
for the development of new separation systems coupled with
9
traditional separation techniques (e.g., precipitation, aqueous two-
phase system,10 chromatography ).
11
Affinity chromatography has been used widely in biomedical
1
research and biotechnology. It is based on molecular recognition
(
2) Firer, M. A. J. Biochem. Biophys. Methods 2 0 0 1 , 49, 433-442.
where one recognition partner is immobilized on a base matrix,
and soluble target molecules can be retained from a crude mixture.
The target molecule can then be released and recovered in a
functional form. The basis of elution is to reduce the affinity
between immobilized ligand and analyte. This is most often
accomplished by use of a bond-breaking buffer (i.e., by changing
(3) Heskins, M.; Guillet, J. E. J. Macromol. Sci., Chem. 1 9 6 8 , 2, 1441-1455.
(
(
(
4) Galaev, I.; Mattiasson, B. Enzyme Microb. Technol. 1 9 9 3 , 15, 354-366.
5) Schild, H. G. Prog. Polym. Sci. 1 9 9 2 , 17, 163-249.
6) Wang, K. L.; Burban, J. H.; Cussler, E. L. Adv. Polym. Sci. 1 9 9 3 , 110,
67-79.
(
7) Tiktopulo, E. I.; Uversky, V. N.; Lushchik, V. B.; Klenin, S. I.; Bychkova,
V. E.; Ptitsyn, O. B. Macromolecules 1 9 9 5 , 28, 7519-7524.
8) Wu, C.; Zhou, S. Macromolecules 1 9 9 5 , 28, 5388-5390.
9) Fong, R. D.; Ding, Z.; Long, C. J.; Hoffman, A. S.; Stayton, P. S. Bioconjugate
Chem. 1 9 9 9 , 10, 720-725.
(
(
*
Address correspondence to either author. (Yamanaka) Fax: +81-3-5331-
361. E-mail: hidenori.yamanaka@jp.amershambiosciences.com. (Okano) Fax:
81-3-3359-6046. E-mail: tokano@abmes.twmu.ac.jp.
1) Wilchek, M.; Chaiken, I. Methods Mol. Biol. 2 0 0 0 , 147, 1-6.
9
+
(10) Kumar, A.; Kamihira, M.; Galaev, I. Y.; Mattiasson, B.; Iijima, S. Biotechnol.
Bioeng. 2 0 0 1 , 75, 570-580.
(11) Miyata, T.; Asami, N.; Uragami, T. Nature 1 9 9 9 , 399, 766-769.
(
1658 Analytical Chemistry, Vol. 75, No. 7, April 1, 2003
10.1021/ac0263768 CCC: $25.00 © 2003 American Chemical Society
Published on Web 03/01/2003

We employed immobilized lectin affinity chromatography12,13
as a model system, using Ricinus communis agglutinin (RCA120).
Immobilized-RCA120 affinity chromatography is widely used for
the fractionation and characterization of glycopeptides and oligo-
saccharides.14,15 RCA120 binds specifically to nonreducing-end
galactose residues.16 Competitive elution using lactose, a hapten
sugar, is generally used to elute the target molecule from the
RCA120-immobilized column. In this study, we introduced both
RCA120 and lactose onto a column matrix via PIPAAm and
investigated the effects of controlled mobility and microenviron-
ment alteration on the binding behavior of immobilized lectin with
target.
Synthesis of P oly[N-(acryloyloxy)succinimide] (P NAS). A
mixture of N-(acryloyloxy)succinimide (2.21 g, 13.0 mmol) and
AIBN (14 mg, 0.007 molar equivalent) in benzene (100 mL) was
heated at 60 °C for 24 h. A white precipitate was formed after
cooling the solution to room temperature. This precipitate was
filtered and washed four times with tetrahydrofuran (THF, 20 mL).
Drying in vacuo yielded poly[N-(acryloyloxy)succinimide] (PNAS)
(2.16 g, 12.7 mmol, 98% yield) as a white solid. The polymer was
taken up in dry THF (300 mL), stirred vigorously overnight,
filtered, and dried in vacuo. The molecular weight of PNAS was
determined by GPC (as poly(acrylic acid) (PAA)) after complete
hydrosis by treatment of a PNAS solution with 6 N HCl at 100 °C
for 24 h. GPC analysis of PAA was performed on a Shimadzu LC-
EXPERIMENTAL SECTION
Materials. N-Isopropylacrylamide (IPAAm; Wako Pure Chemi-
cals, Osaka, Japan) was purified by recrystallization from a
1
0AD with a Waters Ultrahydrogel linear column in water at 40
C. The following standards were used: PAA of M 130 kD, 390
of PAA determined thereby was 121
°
w
kD, and 1100 kD. The M
w
toluene-hexane mixture and dried at room temperature in vacuo.
0
00, and the degree of polymerization was 1680.
P olymerization of IP AAm. PIPAAm was prepared by radical
polymerization of IPAAm in THF. A mixture of IPAAm (20.0 g,
77 mmol), 2-aminoethanethiol hydrochloride (201 mg, 1.77
2
-Aminoethanethiol (Wako Pure Chemicals, Osaka, Japan) was
distilled under low pressure. Acryloyl chloride, N-hydroxysuccin-
imide, isopropylamine, triethylamine, 2,2′-azobis(isobutyronitrile)
1
(
(
AIBN), lactose, tetrahydrofuran (THF), N,N-dimethylformamide
DMF), toluene, hexane, ethanolamine, hexylamine, and ethyl-
mmol), and AIBN (290 mg, 1.77 mmol) in THF (200 mL) was
degassed by subjecting it to freeze-thaw cycles, and the sample
containing the mixture was sealed under reduced pressure. Then
the mixture was heated at 70 °C for 2 h. After evaporation of the
solvent, the reaction mixture was poured into diethyl ether to
precipitate the polymer. The polymer was further purified by
repeated precipitation from THF poured into diethyl ether. The
LCST of PIPAAm was 32 °C, which agreed well with the previously
enediamine were purchased from Wako Pure Chemicals. Human
transferrin and sialidase from Arthrobacter ureafaciens were
obtained from Sigma Chemical Co. (St. Louis, MO). Caster bean
lectin RCA120 (CAS 172304-66-4) was obtained from Honen Corp.
(
×
Tokyo, Japan). A HiTrap NHS-activated HP column (1 mL; 0.7
2.5 cm) was obtained from Amersham Biosciences (Bucking-
hamshire, U.K.).
reported value.3 The molecular weight (M
) of the obtained
) 2.2) by GPC
P reparation of Asialotransferrin (AST). Enzymatic digestion
of transferrin by sialidase was performed according to the method
described previously.17 Briefly, human transferrin (25 nmol) was
incubated with A. ureafaciens sialidase in 1 mL of 50 mM sodium
citrate buffer (pH 5.0) at 37 °C for 8 h.
n
PIPAAm was determined to be 4160 (M / M
w
n
(TSKgel R-3000 column) at 40 °C using DMF as an eluent.
P artial Substitution of P NAS. Isopropylamine (157.4 mg,
2
.66 mmol, 90 mol % relative to the succinimide groups of the
PNAS) was added to a stirred solution of PNAS (0.5 g, 2.99 mmol)
in DMF (3 mL) at room temperature. The solution was allowed
to stir for 24 h, and then dried in vacuo. The product was
redissolved in THF and poured into diethyl ether to precipitate
the polymer, yielding a white powder designated PIPAAm-90 (“90”
indicates the molar ratio of substituted isopropylamine) in
quantitative yield. The same procedure, adjusted for the appropri-
ate amount of isopropylamine, was used for the other substitution
ratios described below.
Synthesis of N-(Acryloyloxy)succinimide. Acryloyl chloride
(
33.4 g, 30 mL, 369 mmol) was added dropwise to a stirred solution
of N-hydroxysuccinimide (42.5 g, 369 mmol) and triethylamine
41.0 g, 56.5 mL, 409 mmol, 1.1 equiv) in CHCl (300 mL, 1.23
(
3
M) at 0 °C on an ice bath. The solution was allowed to stir for 3
h at 0 °C, and then it was washed with water (2 × 300 mL) and
dried over MgSO4. The product was recrystallized from a solution
of ethyl acetate/ hexane (1:1) to yield 46.1 g (213 mmol) of
colorless crystal with 72% yield (mp 69 °C).
Synthesis of â-Lactosylamine. Lactosylamine was synthe-
sized according to a modification of the method of Lihkosherstov
et al.18 Briefly, lactose monohydrate (1.0 g, 3.0 mmol) was
dissolved in 50 mL of saturated ammonium hydrogen carbonate.
Additional solid ammonium hydrogen carbonate was then added
to maintain a saturated state during the reaction. The mixture
was stirred at 37 °C for 24 h and then desalted by direct
lyophilization. Lyophilized â-lactosylamine was stored at -20 °C.
P reparation of RCA120 and Lactose Co-Immobilized P oly-
mer. Since attachment of hydrophilic lactose residues to the
PIPAAm-90 polymer will alter the LCST, their effect must be
balanced by additional of hydrophobic residues. For this purpose,
we chose hexylamine. â-Lactosylamine (0.2 mg, 1 mol % relative
to the succinimide groups of the PNAS) and hexylamine (0.5 mol
% relative to the succinimide groups of the PNAS) were added to
a solution of RCA120 (5 mg in phosphate-buffered saline [PBS],
pH 7.2) at 4 °C, and the mixture was left for 30 min at 4 °C. Then,
to this premixed solution, a solution of PIPAAm-90 (10 mg) in 0.2
mL of PBS was added, and the mixture was left for 12 h at 4 °C.
This reaction product was applied directly for subsequent im-
mobilization onto the column resin, as described below.
Modification of Sepharose Resin. Short spacer arms were
attached to an N-hydroxysuccinimide HiTrap NHS-activated HR
column (1 mL) of highly cross linked agarose-based Sepharose
(
(
(
12) Kobata, A. Biochem. Soc. Trans. 1 9 9 4 , 22, 360-364.
13) Endo, T. J. Chromatogr., A. 1 9 9 6 , 720, 251-261.
14) Shinohara, Y.; Kim, F.; Shimizu, M.; Goto, M.; Tosu, M.; Hasegawa, Y. Eur.
J. Biochem. 1 9 9 4 , 223, 189-194.
(
(
(
15) Merkle, R. K.; Cummings, R. D. Methods Enzymol. 1 9 8 7 , 138, 232-259.
16) Wei, C. H.; Koh, C. J. Mol. Biol. 1 9 7 8 , 123, 707-711.
17) Uchida, Y.; Tsukada, Y.; Sugimori, T. J. Biochem (Tokyo). 1 9 7 9 , 86, 1573-
1
585.
(
18) Lihkosherstov, L. M.; Novikoya, O. S.; Derevitskaja, V. A.; Kochetkov, N.
K. Carbohydr. Res. 1 9 8 6 , 146, C1-C5.
Analytical Chemistry, Vol. 75, No. 7, April 1, 2003 1659

resin by reaction with ethylenediamine solution. Subsequently,
the NHS polymer derivatized with RCA120 and lactose as described
above was added to the column at 4 °C and allowed to attach to
the spacer arms. The procedures for coupling, washing, and
measuring of coupling efficiency were carried out according to
the protocol provided by the manufacturer. Briefly, the coupling
efficiency was determined by 280-nm UV monitoring of N-hydroxy-
succunimide elution, which is released during the coupling
reaction. The resulting column (column P) contained RCA120 lectin
along with lactose haptens attached independently onto PIPAAm
chains, which were in turn attached to the agarose-based resin.
The column was stored at 4 °C until use.
Ta b le 1 . Typ ic a l Re c o ve ry Ra t io s o f As ia lo t ra n s fe rrin
fro m Co lu m n s R, P , a n d I b y S e q u e n t ia l Th e rm a l a n d
Co m p e t it ive Elu t io n
recovery ratioa
elution
temperature
thermal elution
(%)
lactose elution
(%)
column
(°C)
R
R
R
P
P
P
I
20
30
40
20
30
40
20
30
40
10.9
23.6
35.7
92.7
94.8
95.1
9.1
89.1
76.4
64.3
7.3
5.2
4.9
90.9
76.1
68.9
I
I
23.9
31.1
The conventional immobilized-RCA120-lectin column (column
R) was prepared by direct injection of RCA120 solution onto a
HiTrap NHS-activated HR column. The amount of immobilized
RCA120 was estimated to be 40.1 nmol by the method described
above. A column with RCA120 and PIPAAm immobilized indepen-
dently (column I) was prepared by sequential injection of RCA120
and amino-terminated PIPPAm onto the NHS-activated column.
The amounts of immobilized RCA120 and PIPAAm were estimated
to be 40.5 and 457.7 nmol, respectively, by the method described
above.
a
The recovery ratios were calculated on the basis of the areas of
the UV absorbance peaks observed upon thermal and subsequent
competitive elution during the experiments shown in Figures 2 and 3.
We employed a strategy of sequential substitution reactions
onto poly[N-(acryloyloxy)succinimide] (PNAS), which was itself
synthesized via homopolymerization of N-(acryloyloxy)succinim-
ide. Modification of the PNAS side chain was achieved by
sequential reactions with isopropylamine and then a mixture of
RCA120 and â-lactosylamine (Figure 1). The effect of the isopro-
pylamine substitution ratio on the solubility of the otherwise water-
insoluble PNAS was investigated by varying the feed ratio from
Measurement of P olymer LCST. The LCST of all poly-
mers was determined as the temperature resulting in a 50%
decrease in optical transmittance of polymer aqueous solutions
(
1 wt %), as measured at 500 nm using a Shimadzu UV-240 spectro-
photometer.
Chromatography. Chromatography was performed using a
5
0 to 90 mol %. The substitution reaction proceeded quantitatively,
and we found that 90 mol % substitution sufficed to make this
polymer water-soluble. This partially substituted, water-soluble,
temperature-responsive PIPAAm-90 was then successfully reacted
with the mixture of RCA120 and â-lactosylamine (0.07, and 1.0 mol
Shimadzu LC-10AD pump and SPD-10AD UV detector (280 nm).
The mobile phase was 0.01 M PBS, pH 7.4. The flow rate through
the various HiTrap columns was held constant at 1.0 mL/ min.
Column temperature was controlled by submersion in water baths
held at the specified temperatures using thermostated circulators
%
). The number of molecules introduced onto each polymer main
chain was calculated to be 1.2 and 16.8 for RCA120 and lactose,
respectively. Since RCA120 possesses two carbohydrate-binding
sites per molecule,21 this comes to a 7-fold excess of hapten to
binding site. Finally, the LCST-controlled polymer was im-
mobilized onto an NHS-activated HiTrap column, as described in
the Experimental Section.
(
CL-80F, TAITEC, Tokyo, Japan).
Elution of Asialotransferrin by Temperature Changes.
Twenty microliters of a 2.0 mg/ mL asialotransferrin solution was
injected onto the column at 5 °C, and subsequent elution of the
protein was performed by rapid transfer of the column into a
second water bath set to 20, 30, or 40 °C. Following 13 min of
elution at the specified temperature, residual adsorbed materials
were eluted with a linear gradient of 0-50 mM lactose in 0.01 M
PBS buffer, pH 7.4. The amount of eluted glycoprotein was
estimated from the area of the UV (280 nm) absorbance peak.
Intrinsic Temperature Dependence of Sugar Recognition
by RCA1 2 0 . Interactions between lectins and carbohydrates are
often affected by temperature. Therefore, it was essential to clarify
the intrinsic temperature-dependence of sugar-recognition by
RCA120 under the conditions employed in the current study.
The chromatographic behavior of AST, whose major carbo-
hydrate structure is asialobiantennary oligosaccharides,22 was
evaluated using a traditional immobilized RCA120 column. We
prepared this column by immobilizing RCA120 onto an NHS-
activated HiTrap column (column R). After injection of AST (0.5
nmol, 20 µL) at 5 °C, the temperature was increased to 20, 30, or
40 °C, and the extent of AST leakage from the column was
monitored. Following each chromatographic analysis, all remain-
ing bound species were eluted with an excess of lactose. As shown
in Figure 2, temperature-dependent AST elution was observed at
all temperatures tested, and it tended to increase with temperature.
The quantitative values of recovery at each step are summarized
in Table 1. Recoveries with temperature elution at 20, 30, and 40
RESULTS AND DISCUSSION
Design and P reparation of a Matrix with Collapsibly
Tethered Ligands. A galactose-specific lectin, RCA120, and its
hapten sugar, lactose, were selected for incorporation onto
PIPAAm as a model system, and the complex was attached onto
Sepharose beads. Though copolymers of IPAAm can be synthe-
sized easily by copolymerization with other component(s),19 it is
generally difficult to achieve homogeneous polymerization, since
the rate constant of polymerization for each component can be
quite different.20 Here, we strove for homogeneous incorporation
of RCA120 and lactose in order to simplify the system and its
evaluation.
(
(
19) Spaltenstein, A.; Whitesides, G. M. J. Am. Chem. Soc. 1 9 9 1 , 113, 686-687.
20) Mammem, M.; Dahmann, G.; Whitesides, G. M. J. Med. Chem. 1 9 9 5 , 38,
(21) Baenziger, J. U.; Fiete, D. J. Biol. Chem. 1 9 7 9 , 254, 9795-9799.
(22) Spik, G.; Bayard, B.; Fournet, B.; Strecker, G.; Bouquelet, S.; Montreuil, J.
FEBS Lett. 1 9 7 5 , 50, 296-299.
4
179-4190.
1660 Analytical Chemistry, Vol. 75, No. 7, April 1, 2003

Fig u re 1 . Synthetic procedure for preparation and immobilization of collapsibly tethered ligands onto a chromatographic matrix (column P).
Fig u re 3 . Elution profile comparison between columns P and R.
Asialotransferrin was injected and eluted as in Figure 2, except that
the same temperature shift to 20 °C was applied to both columns.
Fig u re 2 . Chromatograms showing the intrinsic temperature de-
pendence of sugar recognition by RCA120. Flow rates were held
constant at 1 mL/min. Asialotransferrin (2.0 mg/mL, 20 µL) was
injected into an immobilized RCA120 column (1 mL) at 5 °C. Two
minutes after injection, the temperature was shifted to 20, 30, or 40
is consistent with such interaction. On the other hand, the
°
C for 13 min. Finally, residual adsorbed material was eluted with a
temperatures tested were insufficient to elute the majority of
bound molecules.
linear (0-50 mM) lactose gradient.
Feasibility Study of Collapsibly Tethered Ligand Affinity
Chromatography. Twenty microliters of AST (40 µg, 0.5 nmol)
was injected onto column P at 5 °C, and 2 min later, the
temperature was shifted to 20, 30, or 40 °C for 13 min. Each test
was terminated by lactose elution of residual bound material. A
15 °C temperature shift (5 f 20 °C) eluted most of the AST
(Figure 3). As summarized in Table 1, 93-95% of bound AST was
°
C were calculated to be 10.9, 23.6, and 35.7%, respectively. This
is consistent with the previous finding that the binding efficiency
of RCA120 decreases slightly with increasing temperature.23 As is
generally true for carbohydrate-protein interactions, hydrogen
bonding is the primary interaction force between RCA120 and
carbohydrate.24 The observed temperature dependence of elution
(
23) Lotan, R.; Beattie, G.; Hubbell, W.; Nicolson, G. L. Biochemistry 1 9 7 7 , 16,
787-1794
(24) Solis, D.; Fernandez, P.; Diaz, M. T.; Jimenez, B. J.; Martin, L. M. Eur. J.
Biochem. 1 9 9 3 , 214, 677-683.
1
Analytical Chemistry, Vol. 75, No. 7, April 1, 2003 1661

Although the crystal structure of RCA120 has not been reported,
it consists of two ricin-like heterodimers, and these subunits share
>
80% sequence homology between the two lectins. Crystal-
lographic analysis of ricin indicated the largest distance between
two amino acid residues on the heterodimeric protein to be ∼80
26
Å. The estimated size of extended PIPAAm (M ) 4160) based
n
upon correlation function determined from previously reported
values25 is 56 Å.
On the basis of the above discussions, the greatly enhanced
temperature elution observed with column P should be attributable
to extrinsically promoted environmental change(s). Considering
that the temperature shift was the sole stimulus applied, the 10-
fold reduction in hydrodynamic volume reported for PIPAAm
above its LCST4,5 most likely played a key role in this phenom-
enon. The dramatic increase in local concentrations of immobilized
ligands would alter the local environment for the two immobilized
molecular species. As a consequence, the immobilized lactose
hapten will compete more effectively with AST for binding to
RCA120 (Figure 5). In effect, local movement of the immobilized
ligands alters their environment and in lieu of injection of affinity-
modulating or competing buffers.
Fig u re 4 . Chromatograms of two successive analyses on column
P. The analytical conditions are the same as described in Figure 3.
eluted by temperature shift alone. This high recovery is remark-
able, as compared with ordinary immobilized RCA120 affinity
chromatography (column R). As shown in Figure 4, two successive
analyses gave essentially the same chromatographic behaviors,
demonstrating that all of the covalently bound components were
stable under these chromatographic conditions and that response
to external stimulus (temperature shift) is reversible.
To examine the effect of the introduction of PIPAAm itself on
the interaction of AST with immobilized RCA120, a column was
prepared on which RCA120 and PIPAAm were independently
immobilized (column I). The difference between columns I and
R is that the former contains PIPAAm, but the latter does not. As
indicated in the Table 1, the amount of AST recovered by
temperature elution was almost the same or even less than that
obtained with column R using the same injection and elution
protocol. This result indicated that the effect of PIPAAm introduc-
tion on the interaction between AST and RCA120 is negligible.
We have previously observed thermoresponsive affinity regula-
tion in dye-affinity chromatography through a column in which
PIPAAm and Cibacron blue F3GA had been independently
incorporated.25 The interaction of Cibacron blue with proteins was
regulated by masking and steric expulsion mediated by PIPAAm.
That these effects were not observed with column I is most easily
explained by the molecular sizes of RCA120 and PIPAAm employed.
Although the present study was limited to the examination of
one combination of lectin and its hapten sugar, the concept of
collapsibly tethered ligands was successfully demonstrated. Simple
thermal modulation to achieve elution provides significant advan-
tages over ordinary elution conditions, which require manipulation
of the liquid mobile phase (i.e. pH, ionic strength or solute
content). Since the required temperature change is an extremely
mild elution condition, damage to biomolecules is minimized. The
fact that the eluted fraction contains neither displacement agents
nor salt makes the eluent very clean, providing great benefits for
any subsequent assays. One promising application lies in the field
of proteome research, in which such prefractionation techniques
based, for example, on attached glycan moieties could remove or
select a subset of proteins. The extremely mild and clean nature
of the described technique is ideal for use in conjunction with
other modes of chromatography and mass spectrometry.
Affinity regulation based on this concept would be applicable
to other lectins as well as other specific protein-protein, drug-
protein or nucleic acid-protein interactions when an appropriate
hapten-like molecule is available.
Fig u re 5 . Schematic diagram of temperature-modulated affinity control.
1662 Analytical Chemistry, Vol. 75, No. 7, April 1, 2003

CONCLUSIONS
concept is widely applicable to any case in which hapten-like
molecules are available for a target.
A novel concept of affinity chromatography modulated by
collapsibly tethered ligands was proposed and demonstrated. In
the current study, temperature-responsive PIPAAm was used as
a scaffold for the coimmobilization of RCA120 and lactose. This
architecture enabled facile thermal manipulation of the mobility
and microenvironment of the immobilized ligands. The designed
column could reversibly bind asialotransferrin in a purely tem-
perature-controlled manner. It was suggested that environmental
change surrounding the immobilized ligand could be brought
about by temperature shifts in which local movements of ligand
and hapten altered their effective local concentrations. This simple
ACKNOWLEDGMENT
The authors thank Dr. R. F. Whittier for useful discussions in
the preparation of this manuscript. A part of this work was
performed under the management of the Research Association
for Biotechnology as part of the R&D Project on Basic Technology
for Future Industries supported by NEDO (New Energy and
Industrial Technology Development Organization) in the Ministry
of International Trade and Industry.
(
25) Yoshizako, K.; Akiyama, Y.; Yamanaka, H.; Shinohara, Y.; Hasegawa,
Received for review December 2, 2002. Accepted January
Y.; Carredano, E.; Kikuchi, A.; Okano, T. Anal. Chem. 2 0 0 2 , 74,
30, 2003.
4
160-4166.
26) Cartellieri, S.; Helmholz, H.; Niemeyer, B. Anal. Biochem. 2 0 0 1 , 295,
6-75.
(
6
AC0263768
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