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Polymer International
Polym Int 49:302±308 (2000)
In vitro cadmium removal from human serum by
Cibacron Blue F3GA–thionein-complex
conjugated affinity membranes
Fatma Denizli,1 Adil Denizli1 and M Yakup Arıca2*
1
Department of Chemistry, Hacettepe University, Beytepe, Ankara, Turkey
Department of Biology, Kırıkkale University, 71450 Yahşihan – Kırıkkale, Turkey
2
Abstract: A new membrane af®nity biosorbent carrying thionein has been developed for selective
removal of cadmium ions from human serum. Microporous poly(2-hydroxyethyl methacrylate)
(pHEMA) membranes were prepared by photopolymerization of HEMA. The pseudo dye ligand
Cibacron Blue F3GA (CB) was covalently immobilized on the pHEMA membranes. Then, the
cysteine-rich metallopeptide thionein was conjugated onto the CB-immobilized membrane. The
maximum amounts of CB immobilized and thionein conjugated on the membranes were
1.07 mmol cmÿ2 and 0.92 mmol cmÿ2, respectively. The hydrophilic pHEMA membrane had a swelling
ratio of 58% (w/w) with a contact angle of 45.8 °. CB-immobilized and CB-immobilized±thioneinconjugated membranes were used in the Cd(II) removal studies. Cd(II) ion adsorption appeared to
reach equilibrium within 30 min and to follow a typical Langmuir adsorption isotherm. The maximum
capacity (qm) of the CB-immobilized membranes was 0.203 (mmol Cd(II)) cmÿ2 membrane and
increased to 1.48 (mmol Cd(II)) cmÿ2 upon CB±thionein-complex conjugation. The pHEMA
membranes retained their cadmium adsorption capacity even after 10 cycles of repeated use.
# 2000 Society of Chemical Industry
Keywords: poly(2-hydroxyethyl methacrylate); af®nity membrane; Cibacron Blue F3GA; peptide; thionein;
complex conjugation; cadmium; human serum
INTRODUCTION
Membrane separation allows the processing of a large
amount of biological sample in a relatively short time
owing to its structure, which provides a system with
rapid reaction kinetics. The integration of membrane
and af®nity separation provides a number of advantages over normal af®nity separation using columns,
and facilitates activity recovery.1±3 A functionalized
membrane with its immobilized ligand is called an
af®nity membrane because of the speci®c af®nity
between the ligand and the targets ligated, for
example, an antigen and its antibody. Af®nity membranes have a wide variety of applications in biotechnology (e g, downstream processing of bioproducts,
enzyme immobilization) and biomedical sciences
(immunoassays, hemoperfusion and others).4±7
Heavy metals are widely produced and discharged
into the environment. At least 20 metals are known to
be toxic, and all of these, including arsenic, cadmium,
lead, nickel, mercury, silver, selenium, zinc, silver and
copper, are released into the environment in quantities
suf®cient to pose a risk to human health. The heavy
metals and their compounds are adsorbed through the
air passages and alimentary canal from food and
drinking water. Among these heavy metals, the
problem of cadmium overload is well known, espe-
cially because the human body has no specialized
physiological route for cadmium elimination.8±10
Cadmium accumulates mainly in the kidneys and
liver, but is also found in skeletal and muscular
systems, and endocrine glands. The elimination of
cadmium from the body is very slow: its half-life is
7±30 years. The symptoms of chronic toxicity of cadmium compounds include kidney and liver damage,
respiratory dysfunction and osteomalacia. Cadmium
compounds also adversely affect the reproductive
system and reproductive function. No speci®c treatments for acute or chronic cadmium poisoning are
available. However, in addition to haemodialysis,
chelating agents are often used in the treatment of
metal poisoning. Different chelating agents that are
available commercially for the treatment of cadmium
poisoning include dimercaprol (British anti-lewisite)
and calcium disodium EDTA,8,9 but there is histopathological evidence for increased toxicity in animals
when calcium disodium EDTA is utilized. Today, one
of the most promising techniques for blood detoxi®cation is extracoporeal bioaf®nity sorption.8±11 So far,
only a few bioaf®nity sorbents have been reported for
heavy metal detoxi®cation.11±14
This study is the ®rst attempt to use a speci®c
membrane-biosorbent in a haemoperfusion system for
* Correspondence to: M Yakup Arıca, Department of Biology, Kırıkkale University, 71450 Yaşihan – Kırıkkale, Turkey
(Received 8 June 1999; revised version received 18 October 1999; accepted 17 November 1999)
# 2000 Society of Chemical Industry. Polym Int 0959±8103/2000/$17.50
302
In vitro cadmium removal from human serum
the removal of cadmium ions from human serum.
These novel dye±peptide-complex±conjugated-metalchelate-forming biocompatible pHEMA membranes
have several desired properties for biomedical application, such as high porosity, large external surface area,
and high chemical, biological and mechanical stability.
The modi®ed membranes were used in vitro for Cd(II)
removal from human serum overloaded with Cd(II).
This paper presents system parameters such as
thionein loading, adsorption conditions and the
adsorption isotherms of cadmium with biosorbent
membranes.
membranes was determined in distilled water. Dry
membrane pieces were placed in distilled water and
kept at a constant temperature of 25 °C. After reaching
equilibrium (equilibration time 30 min) swollen membranes were removed and weighed by an electronic
balance (Shimadzu, Japan, EB. 280 1 10ÿ3 g). The
swelling ratios of the membranes were calculated using
the expression
EXPERIMENTAL
Materials
Membrane thickness measurements
The thickness of the membrane was determined using
an optical microscope with a micrometer. The swollen
membrane was wrapped around a rubber stopper and
placed under the objective in an upright position. The
measurement was performed at 10 magni®cation.
The metallo peptide thionein (acetate salt, Lys-CysThr-Cys-Cys-Ala, molecular mass 627.8) and 2hydroxyethyl methacrylate (HEMA) were supplied
by Sigma Chemical Co (St Louis, MO, USA). The
latter was distilled under reduced pressure in the
presence of hydroquinone as polymerization inhibitor
and stored at 4 °C until use. a,a'-Azobisisobutyronitrile
(AIBN) was obtained from Fluka AG (Buchs, Switzerland) and was used as received. Cibacron Blue F3GA
(CB) was obtained from Polyscience (Warrington,
USA). All other chemicals were of reagent grade and
were purchased from Merck AG (Darmstadt,
Germany).
Preparation of CB immobilized membranes
Membrane preparation was as previously described.15
The polymerization mixture (5 ml) contained 2 ml
HEMA, 5 mg AIBN as polymerization initiator and
3 ml 0.1 M SnCl4 as pore former. The mixture was
then poured into a round glass mould (9.0 cm in
diameter) and exposed to ultraviolet radiation for
10 min, while a nitrogen atmosphere was maintained
in the mould. The membrane was washed several
times with distilled water and cut into circular disks
(1.0 cm in diameter) with a perforator. The covalent
immobilization of dye-ligand (ie Cibacron Blue
F3GA) was performed under alkaline conditions.
The membrane disks were stirred magnetically at
400 rev minÿ1 in a sealed reactor with 100 ml of the
aqueous CB solution containing 4.0 g NaOH. CB
immobilization was carried out at 80 °C for 4 h. The
initial concentration of the CB in the medium was
3.0 mg mlÿ1. After the immobilization reaction, the
solution was cooled to room temperature and the CBimmobilized membrane disks were collected, and then
washed several times with distilled water and methanol. The membranes were stored at 4 °C with 0.02%
sodium azide until required, to prevent microbial
contamination.
Swelling ratio …%† ˆ
…Ws ÿ W0 †
100
W0
…1†
where W0 and Ws are weights of membrane before and
after swelling, respectively.
Contact angle measurements
The plain and CB-immobilized pHEMA membranes
were characterized by an air-under-water contact
angle measuring technique. This device consists of a
travelling goniometer with x15 eyepieces, a variable
intensity light source and a micrometer-adjustable
X±Y stage vertically mounted on an optical bench.
The polymer sample is held on the underside of the
Te¯on plate stage by means of small Te¯on clips. The
container is then ®lled with triply-distilled water and
the plate with sample is lowered into the container
until the sample is completely immersed. A bubble of
air with a volume of about 0.5 ml is then formed at the
tip of the Hamilton microsyringe below the surface; it
becomes detached and is allowed to rise to the
polymer±water interface. The air bubbles were photographed at 25 °C within 5 min of reaching equilibrium
after contact with the membrane samples. The
equilibrium contact angle (yair) was calculated from
the height (h) and the width (b) of the air bubble at the
pHEMA sample surface by using eqn (2).
air ˆ cosÿ1 ‰…2h=b† ÿ 1Š
for
air < 90
…2†
The mean value of ®ve contact angles measured on
bubbles at different positions is considered. The reproducibility of contact angles is 2%.
Elemental analysis
The amount of Cibacron Blue F3GA immobilized on
the pHEMA membranes was determined using an
elemental analysis instrument CHNS-932, Leco,
USA), by measuring the nitrogen and sulphur
stoichiometry.
Characterization of microporous pHEMA
membranes
Complex conjugation of thionein with the
CB-immobilized pHEMA membrane
Swelling ratio of pHEMA membranes
The swelling ratio of the microporous pHEMA
Thionein-complex conjugation on the CB-immobilized membrane was carried out in phosphate buffer
Polym Int 49:302±308 (2000)
303
F Denizli, A, Denizli, M Yakup Arõca
(pH 7.4, 0.1 M) at 25 °C in a batch system. The initial
concentration of thionein in phosphate buffer was
varied between 0.1 and 3.0 mg mlÿ1. The complexconjugation experiments were carried out for 1 h (ie,
complex formation time) at a stirring rate of
50 rev minÿ1. After the complex formation period,
the membranes were removed from the thionein
solution. The amount of thionein conjugated on the
CB-immobilized membrane was determined by
measuring difference between the initial and ®nal
concentrations of thionein in the medium by radioimmunoassay.16
Adsorption kinetics of cadmium
Cadmium adsorption capacities of the CB-immobilized and CB±thionein-complex conjugated membranes were determined in the batch system. The
blood samples were supplied from a healthy donor at
the University Hospital, Hacettepe, Ankara. Blood
samples were centrifuged at 500 g for 30 min at room
temperature to separate serum. 10 ml of serum was
overloaded with 2 ml of cadmium stock solution
containing different amounts of Cd(II) to obtain
different initial Cd(II) concentrations in the medium
(10±100 mg/ml). Then, Cd(II)-overloaded human
serum was incubated with membrane pieces at 25 °C
(the total external surface area of the wet membranes
used in each batch was about 150 cm2 lÿ1). The time
to reach equilibrium adsorption with continuous
stirring was found to be 20 min, and in the rest of the
study a 60 min adsorption period was therefore
employed. After the equilibrium period (ie, 60 min),
the af®nity membranes were separated from the serum
and the amounts of Cd(II) adsorbed were determined
by using a graphite furnace atomic absorption spectrophotometer (GBC 932 AA, Australia). All instrumental conditions were optimized for maximum
sensitivity as described by the manufacturer. For each
sample, the mean of 10 atomic absorption spectroscopy measurements was recorded. The amounts
adsorbed per unit surface area of the membranes were
calculated by using the expression
qˆ
‰…C0 ÿ C†V Š
A
…3†
where q is the amount of Cd(II) adsorbed onto unit
surface area of the membranes (mmol cmÿ2), C0 and C
are the initial and ®nal concentrations, respectively, of
the Cd(II) ions in the serum for certain period of time
(mmol/lÿ1); V is the volume of the serum (litres); and A
is the outer surface area of the pHEMA membranes
used (cm2).
Stability of affinity membranes in repeated use
Desorption of Cd(II) ions was studied in 25 mM
EDTA solution (pH 4.9). The af®nity membranes
loaded with Cd(II) ions were placed in desorption
medium and stirred magnetically (at a rate of 600 rev
minÿ1) for 1 h at room temperature. The ®nal Cd(II)
304
concentration in the aqueous phase was determined by
a graphite furnace atomic absorption spectrophotometer. The desorption ratio was calculated from the
amounts of Cd(II) ions adsorbed on the af®nity
membranes and the released into the desorption
medium.
To determine the reusability of the CB-immobilized
and CB±thionein-complex conjugated membranes,
the adsorption±desorption cycles were repeated 10
times using the same polymeric biosorbent. Repeated
adsorption processes were carried out in human
serum. It should be also noted that during the desorption of Cd(II) ions with EDTA, CB and thionein
leakage into the medium was also monitored.
RESULTS AND DISCUSSION
Properties of Cibacron Blue-immobilized pHEMA
membrane
The membrane designed for af®nity separation
possesses high porosity, high chemical, biological and
mechanical stabilities, a high degree of hydrophilicity
and the presence of functional groups, which permit
immobilization of a suitable ligand. CB-immobilized
and CB±thionein-complex conjugated membranes
were prepared as a speci®c af®nity biosorbent for
Cd(II) removal from the human serum. pHEMA is a
nontoxic and biocompatible synthetic polymer with
adequate mechanical strength for most biomedical
applications. It is very inert towards microbial degradation and resistant to attack by many chemicals. In
addition, pHEMA contains hydroxyl groups that act
as attachment sites for ligands. The porosity of the
pHEMA membrane provides less diffusion resistance
and facilitates mass transfer because of high external
surface area. This also provides higher dye immobilization and enhances cadmium removal capacity.
These properties are also important in its use as a
support in af®nity separation technology.17±20
Cibacron Blue F3GA was used as the dye ligand for
complex-conjugation of thionein molecules, and was
immobilized covalently to the pHEMA membranes. It
is accepted that ether linkages are formed between the
reactive triazine ring of the dye and the hydroxyl
groups of the pHEMA. CB is a monochlorotriazine
dye, which contains three acidic sulphonate groups
and four basic primary and secondary amino groups.
The binding interactions between CB and peptides or
proteins may result from the cooperative effect of
different mechanisms such as hydrophobic interactions and/or ion exchange effects, caused by the
aromatic structure and sulphonic acid on the CB and
the amino acid side-chain groups of the proteins. The
dye ligand is cheap and stable. It can be easily
immobilized on various types of support. It is also
reported that CB has no adverse effect on biochemical
systems. Therefore, Cibacron Blue F3GA was selected
as a pseudo ligand for conjugation of thionein molecules onto the af®nity membrane.
The equilibrium swelling ratio of pHEMA memPolym Int 49:302±308 (2000)
In vitro cadmium removal from human serum
branes was 58% (w/w). It is worth noting that the
swelling properties of membranes did not change after
CB immobilization. The membrane thickness was
about 0.06 cm. The contact angle represents a
measurement of surface hydrophilicity: equilibrium
values were 45.3 ° and 45.7 ° for the plain and the CBimmobilized pHEMA membranes, respectively. As
seen here, no further change in contact angle was
observed upon immobilization of Cibacron Blue
F3GA.
Dye leakage is a serious problem in column studies
for biomedical applications. However, it is reported
that CB has no side effects on biological systems.21
Dye and thionein leakage was also investigated in
human serum and in phosphate buffer, respectively. In
adsorption media no leakage was observed from the
CB-immobilized and CB±thionein-complex conjugated membranes. Elemental analyses of the plain
pHEMA and the CB-immobilized pHEMA membranes were performed, and the amount of immobilized dye was calculated as 1.07 mmol cmÿ2 from the
nitrogen and sulphur stoichiometry.
The metallothioneins are a group of cysteine-rich
metal-binding proteins. The large number of sulphydryl groups in the peptide permits it to form metal
clusters.22 Metallothioneins and their analogues are
widely distributed among organisms, from bacteria
and fungi to plants and mammals. Metalloproteins
have many important biological functions in humans
and animals, including transport of essential metals,
protection from metal toxicity, free radical scavenging,
storage of metals, metabolism of essential metals,
immune response and genotoxicity and carcinogenicity. Thionein is a very small metallopeptide composed of six amino acids, three of which are cysteine
residues. The thiol groups in the thionein structure
permit it to form metal clusters with Cd(II), Zn(II),
Cu(II), Hg(II) and some other divalent metal ions; this
makes them attractive peptide ligands. Metalloproteins are among the most selective chelators known for
metal ions for biomedical applications.23
Figure 1 shows the loading of thionein onto the plain
and CB-immobilized membranes as a function of
initial thionein concentration. Thionein loading onto
CB-immobilized membranes increased linearly up to
2.0 mg mlÿ1 thionein in the medium, beyond which a
plateau was observed; a maximum loading of
0.92 mmol cmÿ2 was reached at 2.5 mg mlÿ1 thionein
concentration. As seen in Fig 1, a negligible amount of
thionein (0.04 mmol cmÿ2) was adsorbed physically on
the plain pHEMA membranes. After CB immobilization, the thionein binding capacity of the membrane
increased 46-fold. This increase could have resulted
from the cooperative effect of different interaction
mechanisms, such as hydrophobic, electrostatic and
hydrogen bonding (complex conjugation) caused by
the acidic groups and aromatic structures on CB and
by the amino acids (especially lysine residues) in the
thionein molecules. CB is not very hydrophobic
overall, but has aromatic planar surfaces that prefer
Polym Int 49:302±308 (2000)
Figure 1. Effect of initial thionein concentration on thionein adsorption:
amount of immobilized CB, 1.07 mmol cmÿ2, pH 7.4; T, 25 °C.
to interact with hydrophobic groups in the thionein
structure (eg methyl groups in both threonine and
alanine residues and methylene groups in lysine
residues).
Adsorption studies with cadmium from human
serum
Effect of thionein loading on cadmium adsorption
It is well known that the adsorption capacity of an
af®nity sorbent increases with increasing ligand
density. As observed in Fig 2, an increase in thionein
loading on the CB-immobilized membranes led to an
increase in the cadmium adsorption capacity of the
CB±thionein-complex conjugated membranes from
human serum, but this relation levelled off at around
0.92 mmol thionein per cm2 membrane and reached a
plateau of 1.24 mmol cadmium adsorbed per cm2
membrane.
Figure 2. Effect of thionein loading on cadmium adsorption from human
serum: amount of immobilized CB, 1.07 mmol cmÿ2, cadmium initial
concentration, 75g mlÿ1; pH 7.4, T, 25°C.
305
F Denizli, A, Denizli, M Yakup Arõca
Figure 3. Equilibrium adsorption time: amount of immobilized CB,
1.07 mmol cmÿ2, thionein loading, 0.92 mmol cmÿ2; T, 25 °C.
Adsorption time
Figure 3 gives adsorption rates of cadmium onto the
CB±thionein-complex conjugated membranes from
human serum. As seen here, high adsorption rates
were observed at the beginning of the adsorption
process (up to 10 min) and then plateau values were
achieved within 30 min. The cadmium adsorption
onto CB±thionein-complex conjugated membranes
was much faster, especially when the cadmium initial
serum concentration was high. This may be due to the
high driving force (the cadmium concentration difference) between the liquid (serum) and the solid support
(af®nity biosorbent) phases, in this case.
Effect of initial serum cadmium concentration on
adsorption
The adsorption of cadmium from human serum on the
plain, CB-immobilized and CB±thionein-complex
conjugated pHEMA membranes is presented in Fig
4. Cd(II) adsorption increased with an increase in the
initial concentration of Cd(II) in the adsorption
medium. CB immobilization increased the Cd(II)
adsorption capacity of the membranes (up to
0.172 mmol cmÿ2). CB±thionein-complex formation
resulted in signi®cant increases the Cd(II) adsorption
capacity of the membrane (up to 1.24 mmol cmÿ2).
Thus the adsorption capacity of the membrane was
increased about 7.2-fold upon complex-conjugation of
thionein with CB. From the mass stoichiometry, it
seems that one attached thionein molecule interacts
with about twelve cadmium ions. This means that
every cysteine molecule binds four cadmium ions.
Different interaction mechanisms between metal ions
and peptides and proteins have been proposed.24 The
major functional groups on protein contributing to
interactions with metal ions are histidine residues and
thiol groups. In the case of thionein which contains
three cysteine residues each of which has a thiol group,
cysteine plays a dominant role in cadmium adsorption.
306
Figure 4. Cd(II) removal from human serum: amount of immobilized CB,
1.07 mmol cmÿ2, thionein loading, 0.92 mmol cmÿ2; Cd(II) initial
concentration, 75mg mlÿ1; pH 7.4; T, 25°C.
Note that the amount of cadmium adsorbed from
phosphate buffer solution onto the CB±thioneincomplex conjugated membranes was 1.47 mmol cmÿ2;
this was higher than the amounts (1.24 mmol cmÿ2)
adsorbed from human serum. This lower Cd(II)
adsorption capacity for human serum may be the
result of competitive adsorption of serum constituents
such as proteins (especially albumin) and other
divalent cations such as Ca(II) and Mg(II) ions by
the thionein molecules.
Adsorption isotherms
The cadmium adsorption isotherms of the af®nity
membranes are presented in Fig 5. After the value of C
and q had been obtained from experimental data, the
semi-reciprocal plot of C/q versus C was employed to
generate the intercept kd/qm and the slope 1/qm. It is
necessary to determine which theoretical isotherm best
®ts the data. The maximum adsorption capacities (qm)
and the dissociation constants (kd = k2/k1) for the
adsorption of cadmium from human serum to the
CB-immobilized and CB±thionein-complex conjugated membranes were obtained from the semireciprocal plots of the experimental data. The semireciprocal transformation of the equilibrium data gave
rise to linear plots for both af®nity sorbents, and the
respective correlation coef®cients (R) were 0.995 for
CB-immobilized and 0.997 for CB±thionein-complex
conjugated af®nity membranes, indicating that the
Langmuir model could be applied in these systems.
The Langmuir adsorption model assumes that the
molecules are adsorbed at a ®xed number of wellde®ned sites, each of which can only hold a single
molecule. All adsorption sites have the same af®nity
for adsorbate, and there are no lateral interactions
Polym Int 49:302±308 (2000)
In vitro cadmium removal from human serum
Figure 6. Desorption isotherm of Cd(II) from dye–thionein-complex
conjugated pHEMA membranes: Cd(II) loadings 1.05 and 1.24 mmol cmÿ2.
Figure 5. Adsorption isotherms of cadmium with dye-immobilized and
dye–thionein-complex conjugated pHEMA membranes.
between molecules adsorbed to adjacent sites.25 The
model is described by the equation
dq
ˆ k1 C…qm ÿ q† ÿ k2 q
dt
…4†
At equilibrium eqn (4) leads to
qˆ
qm C
…kd ‡ C†
…5†
The maxixmum cadmium adsorption capacities
(qm) were 0.202 mmol cmÿ2 (22.7 mg cmÿ2) and
1.482 mmol cmÿ2 (166.7 mg cmÿ2) for CB-immobilized
and CB±thionein-complex conjugated membranes,
respectively. Complex conjugation of thionein molecules with CB molecules present on pHEMA
membranes leads to a signi®cant increase in the qm
value (about 7.4-fold) of membranes for cadmium. It
is obvious that this increase is due to complex
formation between thionein molecules and cadmium
(ie thionein molecules promote the adsorption of
cadmium). The kd values were found to be 1.5810ÿ7
M and 1.7710ÿ7 M for cadmium with CB-immobilized and CB±thionein-complex conjugated af®nity
membranes, respectively. After thionein loading on
the CB-immobilized membrane, the magnitude of the
order of kd values was not changed as expected, but a
slight increase (about 12%) in the dissociation constants of Cd(II) was observed. Thionein is a heavy
metal chelating peptide and this might lead to a slight
increase in the kd values of cadmium through speci®c
interactions with the metal binding sites of thionein.
Desorption of Cd(II) ions and regeneration of the
affinity membranes
Regeneration of the CB-immobilized and CB±thionein-complex conjugated af®nity membranes was
Polym Int 49:302±308 (2000)
performed in a batch system. The membranes used
in the cadmium adsorption tests from human serum
were placed in desorption medium containing 25 mM
EDTA at pH 4.9. The amount of cadmium desorbed
from the af®nity membranes was determined during a
1 h desorption period. The desorption ratio was
calculated from the ratio between the amounts of
Cd(II) released and adsorbed. The desorption isotherms from CB±thionein-complex conjugated membrane of different Cd(II) loadings are presented in Fig
6. During the desorption period, more than 60% of
adsorbed Cd(II) was desorbed from the biosorbent
within 5 min; as can be seen, the desorption was almost
complete in about 40 min with nearly 97% of the
adsorbed Cd(II) having been desorbed from biosorbent. CB±thionein-complex conjugate works as a good
Cd(II) chelating agent, and is able to both reversibly
bind and release Cd(II). It should be noted that in this
case no thionein was released, showing that thionein
molecules are ®rmly conjugated by strong electrostatic
and hydrophobic interactions to CB molecules present
on the pHEMA membranes. This means that EDTA
scavenges Cd(II) only from its chelates with CB/
thionein molecules. In addition, when EDTA was
used as a desorption agent, the coordination spheres of
chelated cadmium ions were disrupted, and subsequently the thionein molecule changed its conformation and released the bound cadmium ions. With the
desorption data given above, we conclude that EDTA
is a suitable desorption agent, and allows repeated use
of the af®nity membranes investigated in this study.
To test the regeneration of the CB-immobilized and
CB±thionein-complex conjugated af®nity membranes,
the adsorption±desorption cycle was repeated 10 times
using the same membrane; as seen in Fig 7, after 10
cycles, the biosorbent capacity remained relatively
unchanged for both types of membrane. The cadmium
adsorption capacity decreased by about 5% for both
af®nity membranes after 10 cycles, showing that the
307
F Denizli, A, Denizli, M Yakup Arõca
creased to 1.24 mmol cmÿ2. Repeated adsorption±
desorption cycles showed that these novel af®nity
membranes possess good properties for removal of
cadmium from human serum and could be utilized in
blood detoxi®cation processes where serum contains a
speci®c target heavy metal. It is also possible to reuse
this novel metalloprotein af®nity biosorbent without
signi®cant loss of adsorption capacity.
REFERENCES
Figure 7. Reuse number of biosorbent pHEMA membranes: amount of
immobilized CB, 1.07 mmol cmÿ2; thionein loading, 0.92 mmol cmÿ2; Cd(II)
initial concentration; 75 mg mlÿ1; pH 7.4; T, 25 °C.
af®nity membranes are very stable, and hence are
promising matrices for the removal of heavy metal ions
from human serum.
CONCLUSIONS
A biosorbent for cadmium removal from human
serum was prepared by complex-conjugation of a
metallopeptide thionein with CB-immobilized
pHEMA membranes. These hydrophilic pHEMA
membranes (contact angle 45.3 °) with a swelling ratio
of 58% (w/w) and carrying CB and thionein, were used
to remove Cd(II) from human serum. Adsorption±
desorption studies with cadmium on the af®nity
membranes led to the following conclusions. The
cadmium adsorption capacity of the CB-immobilized
membrane was 0.172 mmol cmÿ2. When the thionein
molecules were conjugated to the CB-immobilized
membrane, the cadmium adsorption capacity in-
308
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