Affinity adsorption mechanism studies of adsorbents C1-Zn(II) for uremic middle molecular peptides containing Asp-Phe-Leu-Ala-Glu sequence

Article abstract of DOI:10.1007/s11426-010-4193-7

To exploit efficient adsorbents for removing middle molecular peptides containing DFLAE (DE5, a typical peptide sequence accumulated in uremic serum) sequence by hemoperfusion, we designed and synthesized three affinity adsorbents (C1-Zn²⁺, C2-Zn²⁺ and C3-Zn²⁺) that could have high affinity to DE5. Subsequently, we evaluated the corresponding adsorption ability of each adsorbent by static adsorption experiments and isothermal titration calorimetry (ITC). The results showed that C1-Zn²⁺ had the best adsorption ability to DE5-containing peptides and the adsorption capacity for DE5 was 8.52 mg/g. By changing the adsorption conditions, the adsorption mechanism was elucidated. The main driving force of the adsorption is metal-carboxyl coordination and the hydrophobic force affords the cooperative effect. It is expected that our present work can provide basic understanding for the design of adsorbents with high affinity and selectivity towards oligopeptides.

Full text of DOI:10.1007/s11426-010-4193-7

SCIENCE CHINA
Chemistry
February 2011 Vol.54 No.2: 375–379
doi: 10.1007/s11426-010-4193-7
• ARTICLES •
· SPECIAL ISSUE · In Honor of the 80th Birthdays of Professors SHEN JiaCong, SHEN ZhiQuan and ZHUO RenXi
Affinity adsorption mechanism studies of adsorbents C1-Zn(II) for
uremic middle molecular peptides containing
Asp-Phe-Leu-Ala-Glu sequence
LI PingLin, FU LiXue, QIAO YiTao, ZHAO JianXin, WANG Wei & YUAN Zhi*
Key Laboratory of Functional Polymer Materials, Ministry of Education; Institute of Polymer Chemistry,
Nankai University, Tianjin 300071, China
Received September 9, 2010; accepted October 8, 2010
To exploit efficient adsorbents for removing middle molecular peptides containing DFLAE (DE5, a typical peptide sequence
accumulated in uremic serum) sequence by hemoperfusion, we designed and synthesized three affinity adsorbents (C1-Zn2+,
C2-Zn2+ and C3-Zn2+) that could have high affinity to DE5. Subsequently, we evaluated the corresponding adsorption ability
of each adsorbent by static adsorption experiments and isothermal titration calorimetry (ITC). The results showed that C1-Zn2+
had the best adsorption ability to DE5-containing peptides and the adsorption capacity for DE5 was 8.52 mg/g. By changing
the adsorption conditions, the adsorption mechanism was elucidated. The main driving force of the adsorption is
metal-carboxyl coordination and the hydrophobic force affords the cooperative effect. It is expected that our present work can
provide basic understanding for the design of adsorbents with high affinity and selectivity towards oligopeptides.
affinity adsorbent, adsorption mechanism, middle molecular peptides
According to the middle molecule hypothesis [7–10], mid-
1 Introduction
dle molecular weight toxins ranging from 500–5000 Da are
the main toxic substances that accumulated in uremic pa-
tients, and middle molecular peptides have been assumed to
be one type of major uremic toxins. As a complement to
pharmaceutical therapy for uremia, hemoperfusion is an
effective method since it can eliminate many toxins in the
blood of patients. Kaplan et al. reported that, there are 38
middle molecular peptides isolated from the serums of ure-
mic patients containing DE5 sequences [11]. It seems that
DE5 is quite related to the uremia. So, It will be much
helpful for the treatment of uremia if there are some effi-
cient adsorbents to eliminate DE5-containing peptides by
hemoperfusion.
Affinity adsorbents are effective materials for purification or
separation of proteins and peptides owning to their high affinity
and low cost [1, 2]. For example, they have been used in blood
purification for selective elimination of toxin in patients [3].
Effective affinity adsorbent was usually obtained by
screening the candidates using affinity chromatography or
equilibrium binding experiments [4–6]. This process is time
consuming and usually require comparatively large quanti-
ties of samples. Thus, designing effective adsorbent for given
target is a challenge, and basic understanding of the adsorp-
tion mechanism is needed.
Uremia is a common syndrome occurring at the end-
stage of renal failure. In general, it will lead to the accumu-
lation of the toxins and further disorder organ functions.
In this study, we synthesized three polyacrylamide
(PAM)-based adsorbents (Scheme 1, C1–C3) bearing func-
tional monomers which could have high affinity to peptide
DE5. Then the static adsorption experiments were carried
out, and the adsorption mechanism was studied.
*Corresponding author (email: zhiy@nankai.edu.cn)
© Science China Press and Springer-Verlag Berlin Heidelberg 2011
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Li PL, et al. Sci China Chem February (2011) Vol.54 No.2
bisacrylamide (10% molar fraction), AIBN (w = 3%) and
2 Experimental
THF (concentration of monomer: 0.1 g/mL) were placed in
a round-bottom flask under nitrogen atmosphere with a
magnetic stir bar. The reaction was carried out at 60 °C in
an oil bath for 24 h. The yields of three adsorbents were
77.1%, 98.0% and 90.9% respectively. The obtained ad-
sorbents were extracted in a Soxhlet extractor successively
by acetone and distilled water for 48 h, respectively. The
final adsorbents were grinded and sieved (80–100 meshes).
The linear polymers were synthesized and purified as re-
ported method earlier [12]. The mole fraction of the func-
tional monomer in P1, P2, and P3 was 2.7%, 3.6% and
8.6% respectively as determined by 1H NMR.
2.1 Materials
Tetrahydrofuran (THF) was dried over sodium/benzophe-
none and distilled immediately before use. Acrylamide (AM)
was recrystallized from acetone, 2,2′-azobisisobutyronitrile
(AIBN) was recrystallized from ethanol, and 2,6-diamino-
pyridine was recrystallized from toluene. Phenylacetyl
chloride and 3-phenylpropanoyl chloride were prepared by
reaction of thionyl chloride with the corresponding carbox-
ylic acid. Acryloyl chloride was prepared by reaction of
benzoyl chloride with acrylic acid. Peptides were purchased
from GL Biochem (Shanghai) Ltd. 2-Aminopyridine was
purchased from Shanghai Chemicals Co. and recrystallized
in petroleum ether. Other reagents and solvents were com-
mercial available and used without purification.
2.4 Adsorption experiments
The static adsorption experiment was carried out according
to the method in ref. [14]. Briefly, the dry adsorbent (0.1 g) was
incubated with ZnCl2 solution (0.66 mM, 10 mL) and shook
at 30 °C for 24 h. The peptide (0.2 mM) was then added
with further shake. The peptide adsorption was determined
by a Unico UV-4802 spectrometer at 220 nm. The control
experiments were carried out under the same condition. The
adsorption capacity was calculated by the equation:
2.2 Synthesis of functional monomers
The functional monomers M1, M2, M3 were synthesized
from the corresponding acylchloride by the reaction with
2,6-diaminopyridine in tetrahydrofuran (THF) at 10 °C
under nitrogen atmosphere. Typical synthetic procedures
are according to the method in our previous work [12, 13].
The corresponding acylchloride in tetrahydrofuran was
added dropwise to a solution of 2,6-diaminopyridine and
triethylamine in tetrahydrofuran (150 mL) at 10 °C over
night. Then, triethylamine was added to the mixture and
acryloyl chloride was added dropwise, and stirred overnight.
After filtration and decoloration, the residue was purified by
column chromatography, eluting with 8:1 dichloro-
methane/acetic ether.
c  c MV

2 
1
A 
c
m
where Ac is the adsorption capacity, c1 and c2 are respec-
tively the concentration of the target peptides before and
after the adsorption, M is the molar mass of the target pep-
tides, V is the volume of solution used in adsorption, and m
is the mass of the dry adsorbent.
2.3 Synthesis of the adsorbents (C1, C2, C3) and the
liner polymers (P1, P2, P3)
2.5 ITC experiments
ITC was used to evaluate the affinity constants between the
adsorbents and the object peptides. As our earlier study has
demonstrated, linear polymers bearing the same functional
groups can be used as adsorbent models to investigate the
affinity constants [15, 16]. In the present work, the linear
co-polymers (P1, P2, P3) of functional monomers and
acrylamide were used as the adsorbent models.
The functional monomer (80% molar fraction), acrylamide
(10% molar fraction), crosslinking agent N,N′-methylene-
The ITC titration was performed on a VP-ITC micro-
calorimeter (MicoCal Inc., Northampton, MA). In general, a
solution of the peptide was injected in portions to a solution
of the polymer or polymer-Zn2+ under different conditions.
Dilution effects were eliminated by the subtraction of the
data from blank experiment.
3 Results and discussion
3.1 The design of functional monomers
In the present study, peptide DE5 is selected as the target
Scheme 1 Synthesis route of the adsorbents.

Li PL, et al. Sci China Chem February (2011) Vol.54 No.2
377
peptide. Since DE5 is a peptide with multiple carboxyl
groups (Asp and Glu) in the ends and a hydrophobic groups
in the middle (Scheme 2), thus, the designed functional
monomers contain either coordination group (M3 in scheme
1) or hydrophobic group (M2) or both (M1).
used to quantify the interaction between the ligands and
DE5. However, the designed functional monomers (ligands)
are all water-insoluble molecules. Taking the advantages of
copolymerization with acrylamide, it is realizable to intro-
duce hydrophobic functional groups into aqueous solution
for further interaction study. [12] Thus, it is convenient to
study the adsorption mechanism between functional ligands
and the target peptide by PAM-based copolymer (P1, P2,
P3). Affinity constants obtained from ITC were summarized
in Figure 1. Consistent with the adsorption results, C1-Zn2+
showed high affinity to peptide DE5, while C2-Zn2+ and
C3-Zn2+ exhibited much weaker binding affinity under the
same conditions. The agreement of adsorption results with
affinity constant further suggested that the different adsorp-
tion capacities of the adsorbents were mainly determined by
the affinity between functional ligands and DE5.
3.2 Adsorption capacities for DE5
We assessed the adsorption capacities of C1-Zn2+, C2-Zn2+
and C3-Zn2+ for DE5 at room temperature by static adsorp-
tion experiments. As shown in Table 1, C1-Zn2+ had optimal
adsorption to DE5, which was almost six times more than
that of C2-Zn2+ or C3-Zn2+. It has been found that
cross-linked polyacylamide showed little adsorption to DE5
(< 0.1 mg/g), so the adsorption of DE5 onto the adsorbents
should be attributed to the functional groups of adsorbents.
The elemental analyses showed that C1, C2 and C3 had the
similar content of functional groups, which was 29.2%,
22.7% and 32.7%, respectively. Compared with the great
difference in their adsorption capacities, the difference in
functional group content could be neglected.
3.3 Adsorption selectivity toward DE5 and DE5-con-
taining peptides
To examine the adsorption selectivity of C1-Zn2+ to pep-
tides containing DE5 sequence, another five peptides were
selected (Table 1). DE8, EV11 and DG12 were middle mo-
lecular peptides containing DE5 sequence [9]. SH5 is an-
other penta-peptide with double acidic residues but without
the hydrophobic residue. WF4 has multiple hydrophobic
To investigate the adsorption affinity constants, ITC was
Scheme 2 The amino sequence of the three peptides with different hy-
drophobic abilityies.
Figure 1 Affinity constants between the linear polymers and DE5 at 30 °C
(n = 3).
Table 1 The adsorption capacity of three adsorbents to DE5 containing peptides (mg/g)
Peptides
DE5
Amino acid sequence
Asp-Phe-Leu-Ala-Glu
C1-Zn2+
8.52
C2-Zn2+
0.49
0.24
0.72
0.69

C3-Zn2+
1.34
0.77
1.21
0.83

DG8
Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly
Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val
Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly
Ser-Glu-Ala-Asp-His
6.64
EV11
DG12
SH5
7.17
6.99
1.02
WF4
Trp-Met-Asp-Phe
2.90



378
Li PL, et al. Sci China Chem February (2011) Vol.54 No.2
residues but only one acidic residue. We tested the adsorp-
tion capacity of C1-Zn2+ to these peptides by static
adsorption experiments. As shown in Table 1, C1-Zn2+
exhibited good adsorption ability to the peptides containing
DE5 sequence (DE5, DE8, EV11 and DG12). In contrast,
C1-Zn2+ exhibited much weaker affinity to the peptides
without DE5 sequence (SH5, WF4), about one eighth to one
half the capacities of peptides containing DE5. The
adsorption results indicated that C1-Zn2+ could provide
selective adsorption to DE5-containing peptides.
Considering the difference in the structure of the three
adsorbents, it seems that the collaboration of coordination
and hydrophobic interactions led to the good adsorption
ability selectivity of C1-Zn2+. Next, our main job is to con-
firm this deduction.
Figure 3 Adsorption capacity of DE5 onto C1-Zn2+ under different pH values.
decreasing. This phenomenon is mainly due to the protona-
tion of N atoms in the functional ligand of C1 as the acidity
increased, thus the effective N atoms involved in the coor-
dination reduced, finally leading to the reduced the binding
ability of adsorbents.
3.4 Investigation of the adsorption mechanism
The adsorbents with different structures were expected to
generate coordination and hydrophobic interactions with the
DE5 containing peptides. To verify this idea, further ad-
sorption experiments were designed to study the adsorption
mechanism between the adsorbents (C1-Zn2+, C2-Zn2+ and
C3-Zn2+) and DE5 containing peptides.
To determine the coordination site, the adsorption of
peptides by C1-Zn2+ and C2-Zn2+ was compared. Although
the only difference between C1 and C2 is the pyridine-N,
there was almost no adsorption of the four DE5-containing
peptides onto C2-Zn2+, which indicated that the pyridine-N
is the main site for the coordination. In addition, consider-
ing the obvious difference in the adsorption of DE5 con-
taining peptides and WF4, multi-carboxyl structure on the
target peptides probably play another key role in the coor-
dination interaction. In WF4, there is only one acidic amino
acid residue, so it could not provide the effective coordina-
tion interaction, and thus the adsorption amount is much
lower than that of DE5-containing peptides. Hence, we
could conclude that both the structure of adsorbent and pep-
tide could affect the coordination interaction and thus the
adsorption process.
In a word, coordination interaction is one of the main
driving force for the adsorption of DE5-containing peptides
onto C1-Zn2+, and the pyridine-N of C1 as well as the car-
boxyl groups of the peptides are the main binding sites.
The role of hydrophobic interaction in the adsorption was
also studied. Similarly, comparing C1 with C3, the benzene
ring is the main difference. Seen from Table 1, the adsorp-
tion capacity of C3-Zn2+ for the four DE5-containing pep-
tides is very low, which is only 10%–20% of that of
C1-Zn2+ for corresponding peptides. The results indicated
that hydrophobic interaction is also an important driving
force during this adsorption process and the benzene ring is
the main binding site.
As is known, metal ions play a crucial role in coordina-
tion interaction. To understanding the role of metal ions (or
coordination interaction) in the adsorption, the adsorption of
DE5 containing peptides by C1 in the presence or absence
of Zn2+ was investigated. Figure 2 shows that, without the
complex-ion, the adsorption capacity of C1 to four DE5
containing peptides significantly decreased by 86.9% (DE5),
72.0% (DG8), 70.3% (EV11) and 71.8% (DG12) respec-
tively. Thus, it could be seen that the coordination interac-
tion indeed plays crucial role in the adsorption of DE5-
containing peptides onto C1, and it was the main driving
force for the adsorption of the DE5-containing peptides.
To confirm the conclusion above, the adsorption capacity
of C1-Zn2+ for DE5 under different pH was also studied. As
can be seen from Figure 3, the adsorption capacity of
C1-Zn2+ for DE5 dropped significantly with the pH value
Adsorption of peptides with different hydrophobic prop-
eries (Scheme 2) using C1-Zn2+ was utilized to ellucidate the
hydrophobic interaction in adsorption. The hydrophobicity
of the peptides is DFLAE > DGLAE > DGGGE. The ad-
sorption capacities of C1-Zn2+ to DFLAE, DGGGE and
Figure 2 The effect of Zn2+ to adsorption capacity of C1 for the targeted
peptides.

Li PL, et al. Sci China Chem February (2011) Vol.54 No.2
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