The heavy metal ions adsorption of protein crystal, a
natural porous material
Junyan Zhang+, Joshua C. Falkner+, Sujin Yean#,
Amy Kan#, Mason Thomson#, Vicki L. Colvin+*
+Department of chemistry, #Department of Civil and environmental engineering,
Rice University, Houston, TX 77005
It is of increasingly concerned about the contamination and pollutants in groundwater,
especially the contamination of heavy metal ions in groundwater and wastewater. How to
effectively and efficiently remove heavy metal ions in water is therefore becoming more
and more serious and crucial to our daily life and the future of ecosystem. To look for
efficient materials to remove heavy metal ions from water is always of great interest to
environmental researchers, and a number of materials have been investigated such as
polymer ion exchange resin1, polymer adsorbent2, carbon black, and so on. Of them,
carbon black has been extensively using for many years to remove heavy metal ions as
well as organic contaminants from water as a typical water cleaner compared with other
materials because of its porous structure that bestow a large specific surface area and a
high adsorption capacity 3,4. Especially, activated carbon fibers are attractive because of
their uniform micro pore structure and faster adsorption kinetics than granular activated
carbons5,6. The idea of introducing functional group to these artificial inorganic materials
to improve their heavy metal ions adsorption capacity has been paid much attention. It
has been shown that electrochemical oxidation treatment on activated carbon fibers
probably created more oxygen containing functional groups which accommodate more
heavy metal ions although some pore structures have been damaged during the oxidation
process7.
More recently, the light was shed on artificial porous materials such as silica based
mesoporous8,9, porous polymer beads, ceramic hollow microspheres10 as well as
microporous polymer membrane11. It is reported that methacrylamidocysteine-containing
porous poly(hydroxyethlmethacrylate)chelating spherical beads with size of 150-200 m
possess significant high adsorption capacity to heavy metal ions, for instance, 1.058g/g
for Cd2+, 0.123g/g for As3+, 0.639g/g for Pb2+12. Based upon the ability of DNA to
intercalate heavy metal ions, DNA modified porous polysulfone beads were found to be
stable in water and can selectively remove heavy metal ions such as Ag+, Pb2+, Cd2+,
Cu2+, and Zn2+13. Lacking of functional sites on the pore walls of porous silica
materials, generally organic functionalities are immobilized onto solid porous substrate to
serve as trapping sites to form complex with heavy metal ions through. Due to sulfur is a
very strong ligand to most heavy metal ions, thiol group typically was introduced to
modify the surface properties of porous materials in order to attract metal ions from
solution based on acid-base reaction. Hybrid macroporous materials with thiol functional
groups attached to titania and zirconia frameworks have been prepared through colloidal
crystal templating techniques for the use to remove heavy metal ions. Since thiol groups
were incorporated into the material framework during the preparation, therefore, thiol
groups are attached to metal oxide firmly through sulfonate linkages. It demonstrated that
the hybrid macroporous materials were effective adsorbents fopr the removal of heavy
metal ions such as mercury and lead from solution14.
Protein crystals have also recently been identified as valuable materials beyond their
applications in biological research. Central to their use is the fact that these biomolecular
crystals contain well-ordered interpenetrating micro- and, in some cases, mesoporous
solvent channels. These channels provide a chemically heterogeneous and chiral
environment which comprises 30-65% of the total crystal volume15,16. In lysozyme, the
model system for this work, the pore diameter ranges from 1 to 2.5 nm in a series of
linked major and minor pores. Figure 1 shows the major porous structure of a hen egg
white lysozyme (HEWL) crystal generated from standard crystallographic information
for the tetragonal crystal structure, where the pores are aligned along the c-axis of the unit
cell within the crystal. With unique aspects: (1) three dimensional pores throughout
whole crystal; (2) a lot of active groups on pores wall; (3) insoluble, so it is easy to be
removed and recovered to reuse; (4) metal can be easily attached on pores wall through
active sites, they can be considered analogous to inorganic zeolites.
This paper reports the use of crosslinked HEWL protein crystals as substrate for the
adsorption of metal ions from aqueous solution, the investigation of the location of
adsorbed metal ions within the crystal. The results indicated clearly that protein crystals
have significant potential in the application of heavy metal ions removal.
Hen Egg White Lysozyme (HEWL) crystals are grown with a batch method. The typical
procedures for large crystals are as following: 4 g of HEWL are dissolved completely in
50 mL sodium acetate buffer at concentration of 0.04 M at pH 4.7 followed by filtration
through a syringe filter. Then 50 mL of 10 % (w/v) sodium chloride solution is added
into above solution with stirring. A few hours later the crystals appear and grow big in a
few days. As grown crystals are washed with 50 mL 0.1 M sodium acetate buffer
containing 2.0 M NaCl at pH 4.8 three times and are finally stored at 50 mL this solution.
The preparation of small protein crystals was described in elsewhere. Most applications
for protein crystals require a secondary cross-linking step that is necessary due to the
poor mechanical properties and stability of un-cross-linked crystals. Crystals are treated
with compounds such as glutaraldehyde to form covalent bonds between key amino acid
functionalities within the structure17,18. In the case of HEWL, this treatment only slightly
degrades the crystal quality and provides samples that can be easily handled and even
dehydrated. Cross-linked protein crystals have also been shown to be resilient under high
shear conditions encountered in both fluid transfer and agitation19.
Glutaraldehyde then is added to a final concentration of 0.5% to cross-linking the protein
crystals, after 2 days, the crystals show color of yellow with complete cross-linking
(Figure 2). Cross linked HEWL crystals are washed with Millipore water 10 times in
order to remove free glutaraldehyde as well as chemicals in buffer exist within crystals
pores and then are dried at air for days.
The adsorption of Pd ions to cross linked protein crystals is carried out as following, 0.1g
dried cross-linked HEWL crystals are put into 10 mL of K2PdCl4 at concentration of
0.1% to infiltrate metal at a certain concentration. The color of crystals became to orange
as shown in Figure 2. UV-vis spectrophotometer was applied to monitor the change of
K2PdCl4 solution concentration in the presence of HEWL protein crystals. Since the
unique pore structure of HEWL protein crystal, there is a hypothesis that it is easier for
metal ions to diffuse into protein crystal pores when smaller protein crystals are used
compared with large protein crystals because the metal ions’ diffusion path is short in the
case of small crystal. To verify this point, two size HEWL protein crystals are applied,
500 μm and 3μm (Figure 3), respectively. Upon UV-vis results, a figure is given as
Figure 4 that, at first few hours, the adsorption rate of Pd(II) ions into small protein
crystal is faster than that to large protein crystal; then both adsorption rates go to become
almost the same. This confirms above hypothesis that small protein crystal would have
faster metal ion adsorption rate due to short diffusion path compared with large protein
crystal.
X-ray photoelectron spectroscopy (XPS) was employed to solve where the adsorbed
metal ions are within protein crystals. In order to have a comparison, both cross-linked
lysozyme crystal with and without Pd loading were investigated (Figure 5, Table 1). C1s
at 284.6 eV was utilized as reference. No binding energy shift was found to N1s at 399.9
eV and O1s at 532.5eV of lysozyme after Pd (II) loading (not shown here). This clearly
imply that nitrogen and oxygen atoms of amino acids within the lysozyme crystal did not
interact with Pd(II). As for sulfur atoms of lysozyme, two sets of Gaussian doublet
appeared with regard to two types of sulfur in lysozyme, the sulfur of methionine is at
162.8 eV, and the sulfur of cysteine is at 166.88 eV20. Once Pd(II) adsorbed into
lysozyme crystal, S2p signals clearly shifted toward higher binding energy from 162.59
eV to 163.54 eV and from 166.88 eV to 167.49eV, respectively; while the binding energy
of Pd(II) shifted to 337.3 eV from 338.2 eV20 in K2PdCl4. Above information of Pd and S
apparently clues that Pd(II) interacted with the sulfur atoms of lysozyme. Pd(II) draws
partial charge from sulfur atom that resulted in higher binding energy shifting to sulfur
while lower binding energy shifting to Pd(II). This explores the exact positions of loaded
Pd (II) within the lysozyme crystal. Figure 6 displays a cartoon of Pd(II) bound to sulfur
atoms on the wall of lysozyme crystal pore. Additionally, when HEWL crystals were
treated in the atmosphere of air with heating to 800C, whole crystals were burnt off with
nothing left. This implies that the adsorbed heavy metal ions into HEWL crystals can be
easily recovered or collected just through burning off the crystals. Upon this point, the
study of thermo-gravimetric analysis (TGA) combined with Inductively Coupled Plasma
Atomic Emission Spectrometry (ICP-AES) indicates that about 19% or 8.5% of the Pd(II)
infiltrated HEWL crystals is Pd complex or atomic Pd content, respectively.
The adsorption of cadmium (II) to HEWL crystal was also performed in a batch system.
A set of concentrations of Cd solutions diluted from 1000 ppm Cd stock solution were
utilized and electrolyte solution containing 0.01 NaCl, 0.01 M THAM, and 0.01 M NaN3
at pH 8 was applied. Twenty milliliters of solution was added to 0.1 g protein. Let the
sample equilibrate on a tumbler for 24 h, and then filtrate the supernatant with 0.45 μm
nominal membrane filter. All filtrate were acidified with trace metal grade HNO3 acid to
contain 1 % HNO3 in solution. Cadmium concentration was measured with ICP-MS.
Cadmium adsorption isotherms to two different protein particles were conducted. The Kd
values for large protein crystals and 3µm protein are 142 and 123 L Kg-1, respectively.
The Cd adsorption constants (Kd) for both large and small lysozyme crystals are similar
to those of Cd-anatase adsorption reported by Gao et al21. Cadmium adsorption behaved
almost identically on both large crystals and small crystals. The results are with good
agreement to what expected based on protein crystal structure that the surface area of
HEWL crystal does not vary with the size of protein crystal, therefore, the total
adsorption of metal of a given amount protein crystals should be the same, independent
on crystal size. In order to confirm that if in this case the sulfur atoms of lysozyme also
acted as binding sites for trapping Cd(II) ions, XPS was also applied to investigate the
binding energy change of sulfur and cadmium atoms with data from literature as
identification reference for sulfur of lysozyme and cadmium of salt (Table 1). After
Cd(II) infiltrated into HEWL crystals, the binding energy of S2p shifted to 163.5 and
167.7 eV from 162.6 and 166.9 eV, respectively, corresponding to sulfurs in methionine
and cysteine, a higher binding energy shifting. To Cd3d5/2, the binding energy shifted to
405 from 406.1 eV of Cd in CdCl2, clearly a lower binding energy shifting. This
convinced that sulfur atoms of lysozyme play the role of trapping heavy metal ions.
The adsorption of Ag(I) to HEWL crystals has also been conducted, and, finally, 7% of
Ag(I) loaded HEWL crystals is Ag(I) verified by TGA.
In summary, as natural porous material, protein crystal worked well as novel material for
the recovery of heavy metal ions, Pd(II) and Cd(II), from aqueous solution. With XPS,
the exact location of adsorbed metal ions was figured out that sulfur atoms of lysozyme
acted as binding sites to trap heavy metal ions via strong coordination effect. There is no
significant difference in final adsorption amount of heavy metal ions to protein crystals
for large (500μm) and small protein crystals (3μm) although at first few hours the
adsorption rate of heavy metal ions to small protein crystal is faster than that to large
crystals.
Acknowledgement
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Caption
Figure 4 Schematic drawing of Pd(II) bound to sulfur atoms on the surface of protein
crystal pore walls. Pod-like yellow area stands for a typical pore shape within the
lysozyme crystal. On the right side of the figure, some residues on the pore’s wall are
displayed on unfolded pore wall. Orange large spheres represent bound Pd (II); red spots
are oxygen atoms; blue spots are nitrogen atoms; green spots adjacent large orange balls
are sulfur atoms; yellow parts are carbon atoms and bonds
Figure 1 Computer generated pore structure of Hen egg white lysozyme crystal
Figure 2 The images of HEWL crystals (left: native HEWL crystals, middle: cross-linked
HEWL crystals, right: Pd(II) adsorbed cross-linked HEWL crystals). Scale bar: 1mm
Figure 3 SEM of 3 μm HEWL crystals
1.2
500 μm crystal
3 μm crystal
1
Intensity
0.8
0.6
0.4
0.2
0
0
8
16
24
32
40
48
Time (Hour)
Figure 4 Adsorption of Pd(II) to large and small HEWL crystals monitored by UV-vis
S of lysozyme
88
0
86
0
84
0
82
0
80
0
78
0
S of lysozyme with Pd(II)
70
0
68
0
66
0
64
0
62
0
60
0
58
0
17
8
17
4
17
0
16
6
16
2
15
8
Binding energy (eV)
Figure 5 XPS spectra of S2p of HEWL crystal before and after Pd(II) adsorption
Table 1 XPS data of S, Pd, and Cd before and after metal infiltration to lysozyme crystal
S2p3/2 (eV)
Pd 3d5/2 (eV)
Cd 3d5/2 (eV)
a
a
Original
162.80 ;
338.20
406.10a
a
166.88
After metal
163.54;
337.30
405.00
infiltration
167.49
* a: Source from Chastain, J.; King Jr, R.C. edited, Handbook of X-ray photoelectron
spectroscopy, Physical Electronics, Inc. 1995
Figure 6 The cartoon of HEWL crystal pore structure and the location of adsorbed Pd(II)
ions within the crystal
4000
3 μm crystal
500 μm crystal
Linear (3 μm crystal)
Linear (500 μm crystal)
q(ug/kg)
3000
2000
1000
0
0
10
20
30
Cd (ug/L)
Figure 7 Cd(II) adsorption isotherm on two sized HEWL crystals