A graphene oxide/hemoglobin composite hydrogel for enzymatic catalysis in organic solvents

Article abstract of DOI:10.1039/c1cc10412h

A graphene oxide/hemoglobin (GO/Hb) composite hydrogel was prepared for catalyzing a peroxidatic reaction in organic solvents with high yields, exceptional activity and stability.

Full text of DOI:10.1039/c1cc10412h

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A graphene oxide/hemoglobin composite hydrogel for enzymatic catalysis
in organic solventsw
Cancan Huang, Hua Bai, Chun Li and Gaoquan Shi*
Received 21st January 2011, Accepted 3rd March 2011
DOI: 10.1039/c1cc10412h
A graphene oxide/hemoglobin (GO/Hb) composite hydrogel
was prepared for catalyzing a peroxidatic reaction in organic
solvents with high yields, exceptional activity and stability.
communication, we prepared a GO/hemoglobin (Hb) composite
supramolecular hydrogel by directly mixing the dispersions of
both components and explored its application as a catalyst for a
peroxidatic reaction in organic solvents. The activity and stability
of the enzyme-containing hydrogel were tested to be much higher
than those of Hb itself in organic solvents.
Enzymes have been widely used as catalysts for organic reactions
in organic solvents.1 However, these enzymatically catalyzed
reactions usually suffer from some inherent shortcomings,
such as poor solubility of reactants and products in organic
media, difficulty in recovering the enzymes from reaction
systems and unwanted side reactions.2 Moreover, the catalytic
activities of enzymes in organic solvents are strongly weakened
by several factors including unfavourable substrate desolvation,
suboptimal pH and reduced conformational mobility.3 To
improve the activities of enzymes, several strategies have
been implemented.1,3,4 Among them, the immobilization of
enzyme in an aqueous microenvironment was tested to be
effective for retaining its activity in organic media. For this
purpose, chemical or physical hydrogels were chosen as the
supports of enzymes to provide the enzymes with an aqueous
microenvironment.4ꢀ7 Interestingly, it was found that the enzymes
bound in these hydrogels showed even higher catalytic
activities in organic media than those in water.6 However,
the preparation of these hydrogels usually involves chemical
reactions (for chemical hydrogels) or heating treatments
(for physical hydrogels). These processes may damage the
structures of enzymes and decrease their activities. Therefore,
a mild, convenient and cheap method for immobilizing enzymes
is required. Recently, our group found that graphene oxide
sheets (GO, a precursor of chemically converted graphene)
could be easily assembled into a hydrogel by just shaking the
mixed dispersion of GO and a crosslinker under ambient
condition.8 The GO based hydrogels were formed by various
supramolecular interactions including hydrogen bonding,
coordination, electrostatic interaction and p–p stacking.8,9
Therefore, it is expected that biomacromolecules such as
DNA and proteins can act as physical crosslinkers of GO
sheets to form multifunctional composite hydrogels.9 In this
GO was prepared by a modified Hummers’ method (ESIw),10
and the single-layered GO sheets have topographic thicknesses
of 0.6ꢀ0.8 nm and lateral dimensions of 2ꢀ5 mm (Fig. S1, ESIw).
To prepare a GO/Hb composite hydrogel, 4 mg of Hb was
dissolved into 0.1 mL deionized water, in which 0.8 mL of GO
solution (9 mg mL–1) was added. The blended solution was
shaken violently for about 15 s to form a hydrogel (Fig. 1A).
The water content of this gel was calculated to be 98.5%.
The rheological properties of the composite hydrogel were
studied by small-deformation oscillatory tests (Fig. 1B). The
results revealed that the storage or elastic modulus (G0) of the
hydrogel is about 3 times its loss or viscous modulus (G00) over
the entire tested range of angular frequencies (1–100 rad sꢀ1).
This phenomenon implies that elastic response is predominant
and the hydrogel has a permanent network.8 The G0 and G00 of
the hydrogel are slightly sensitive to angular frequency and
do not cross over each other in the tested frequency range,
Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical
Biology, Department of Chemistry, Tsinghua University,
Beijing 100084, People’s Republic of China.
E-mail: gshi@tsinghua.edu.cn
w Electronic supplementary information (ESI) available: Experimental
procedures and supplementary figures. See DOI: 10.1039/c1cc10412h
Fig. 1 The preparation of GO/Hb composite hydrogel (A) and its
rheological behaviour (B).
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4962 Chem. Commun., 2011, 47, 4962–4964
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which is a characteristic of gels with a high degree of non-
covalent cross-linking. The G0 of the gel at 10 rad sꢀ1 was
measured to be about 4.1 kPa and this value is much higher
than that of the GO solution with a concentration of 9 mg mLꢀ1
(Fig. S2, ESIw). The viscosity of the GO/Hb composite
hydrogel is also much higher than that of the GO solution
at a given shear rate (Fig. S3, ESIw). The SEM images of both
lyophilized GO solution and GO/Hb hydrogel exhibit a 3D
network with pore sizes of several micrometres, indicating that
the addition of Hb did not change the morphology of GO
sheets (Fig. 2). The formation of GO/Hb hydrogel can be
ascribed to the strong electrostatic interaction between its
both components. The pH of the GO aqueous solution was
measured to be 1.87, and this value is much lower than the
isoelectric point of Hb (pH E 6).5,11 Therefore, there is an
electrostatic attraction between positively charged Hb molecules
and negatively charged GO sheets, which is responsible for the
formation of the composite hydrogel. Hb in the composite
hydrogel acted as a crosslinking reagent of the GO 3D
network. This is different from immobilization of enzyme in
other conventional hydrogels, in which enzyme is not an
essential component. Since the electrostatic attraction between
Hb and GO is a weak intermolecular interaction, the structure
of Hb has not been changed significantly in the hydrogel. As a
result, the hydrogel kept the catalytic activity of the enzyme.
To test the catalytic activity of the composite hydrogel, the
oxidation of pyrogallol by H2O2 (Fig. 3A) was chosen as a
model reaction. We evaluated the catalytic performance of the
GO/Hb composite hydrogel by measuring the reaction rate,
which can be calculated from the absorbance changes of
the product, purpurogallin, at 425 nm (Fig. S4, ESIw).12 Since
purpurogallin is liposoluble, we chose several hydrophobic
solvents including methylene dichloride, toluene, and chloroform
as the reaction media. Taking the reaction in methylene
dichloride as an example, one can see that the GO/Hb composite
gel exhibits higher stability than those of pure Hb and the GO
in methylene dichloride (Fig. 3B). The absorbance at 425 nm
in the system catalyzed by GO/Hb gel increased continually in
the first 1.5 h, indicating that the gel has high catalytic activity
in this period (Fig. 3B). However, the activity of the GO/Hb
gel gradually decreased during the successive reaction process,
showing that the product and surrounding residues inhibited
the catalytic activity of the hydrogel (Fig. S5, ESIw).16 For the
system with free Hb molecules, the enzyme lost its activity
after 30 min and the product concentration reached a
plateau. Pure GO showed a stable but very low activity in the
whole experiment period. The high stability of the composite
hydrogel provided an aqueous microenvironment for protecting
Fig. 3 (A) Scheme of the peroxidatic reaction; (B) the extended
90 min reaction courses in methylene dichloride with different catalysts;
(C) the reaction courses for the first 5 min in methylene dichloride with
different catalysts.
Hb from deactivation induced by an organic solvent. Moreover,
the yield of the reaction catalyzed by the GO/Hb hydrogel was
about 4 times that catalyzed by free Hb after reaction for 1.5 h.
Thus, the catalytic activity of the hydrogel is much higher than
that of pure Hb or GO in a long reaction time. However, in the
first 5 min, the reaction rate of the system containing Hb is
higher than that of the system with the GO/Hb hydrogel or
GO (Fig. 3C). After the addition of the catalyst (e.g. GO/Hb
hydrogel) into the reaction system, a microinterface between
the water phase and organic solvent was formed. Since the
substrate, pyrogallol, is hydrophilic and insoluble in organic
solvents, its molecules were accumulated at the surfaces of the
gel, and then crossed the interface to enter the gel for reaction.
Therefore, this reactant diffusion process took time and limited
the initial composite gel. However, the free Hb lost its activity
in a short time because of the organic circumstance. In summary,
free Hb molecules have higher initial catalytic activity but a
shorter reaction rate. However, for the reaction catalyzed by
free Hb, a small amount of Hb and substrate dissolved in
H2O2 solution immediately for the reaction to occur. This is
a relatively fast process. Thus, its initial reaction rate is higher
than that catalyzed by the lifetime. On the other hand, the
GO/Hb composite hydrogel has a lower apparent activity in
the initial stage of the reaction, while it can maintain its
activity for a much longer time, which results in a higher
average activity in the whole reaction period. The interrelated
catalytic performances of the GO/Hb hydrogel in toluene
Fig. 2 SEM images of freeze dried GO solution (9 mg mLꢀ1) (A) and
GO/Hb composite hydrogel (B).
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Table 1 The apparent Michaelis–Menten constant (Km) and maximum
initial velocity (Vmax) of a GO/Hb hydrogel catalyzed oxidation of
pyrogallol in different organic solvents
showed higher activity and stability than free Hb or GO when
they were utilized for catalyzing oxidation of pyrogallol in
organic solvents. The catalytic activity of the composite
hydrogel could be preserved even after it was stored at room
temperature for a long time. In the system, the hydrogels
provide aqueous microenvironment to protect the enzyme
from deactivation, and it acted as the transit station to
attract the substrate and exclude the product. Considering
the easy separation of the catalyst and the biocompatibility
of GO, our composite hydrogel has great potential in
industrial applications, such as organic synthesis and enzymatic
biotransformations.
Solvent
Km/mM
Vmax/minꢀ1
Methylene dichloride
Toluene
Chloroform
0.1255
0.3697
0.5049
0.05016
0.03940
0.04579
and chloroform are similar to that in methylene dichloride
(Fig. S6, ESIw), indicating the applicability of the hydrogel in
different solvents.
As shown in Fig. 3B, we also observed a low catalytic activity
of pure GO, which is also reported by other researchers.13
Therefore, the catalytic activity of GO/Hb hydrogel can be
partly attributed to the synergetic effect of both components.
Except the synergetic effect, several other factors also have
contributions to the high activity of the GO/Hb hydrogel.
First, hydrophilic pyrogallol molecules entered the aqueous
microenvironment of the hydrogel to form a micro-reaction
system.6,14 Second, the GO/Hb gel has a 3D network constituted
by 2D GO sheets, and the pore sizes of the network are as large
as 5–10 mm (Fig. 2). Thus, the molecules of reactants or product
could be easily diffused to or released from the reaction
system. Third, pyrogallol is hydrophilic and purpurogallin is
hydrophobic, which promoted the indiffusion of a reactant to
the hydrogel and the outdiffusion of the reaction product.
Moreover, the outdiffusion of the product reduced catalyst
inhibition, improving the activity of GO/Hb hydrogel.
We also investigated the catalytic kinetics of the hydrogel in
different organic solvents. It is assumed that the reaction occurred
only in the gel, and the product diffused out the gel as soon as
it was produced. The reaction rates in the first five minutes
were measured to evaluate the activity of the catalyst. Two kinetic
parameters, maximum initial velocity (Vmax) and Michaelis–
Menten constant (Km), calculated from the Lineweaver–Burk
plots15 are list in Table 1 (see ESIw). According to this table,
the Vmax measured in methylene dichloride is higher than those
observed in toluene and chloroform. However, the apparent
Km of the GO/Hb hydrogel in the first solvent is lower than
those in the latter two media. These results indicate that
methylene dichloride is the best among these three solvents
for the reaction, most possibly due to its suitable polarity. It
should be noted here that the reaction product can be collected
and purified simply by decantation to remove the hydrogel. It
was also found that the GO/Hb composite gel had an improved
thermal stability. Hb usually should be stored in relatively low
temperatures to keep its activity. In contrast, the GO/Hb
composite gel can maintain its activity even after keeping it at
room temperature for more than two weeks (Fig. S7, ESIw).
Thus, the reaction system developed here has potential industrial
application.
This work was supported by natural science foundation of
China (91027028, 50873092).
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In summary, we have demonstrated that 2D GO sheets are
able to form stable composite hydrogels with Hb. The hydrogels
c
4964 Chem. Commun., 2011, 47, 4962–4964
This journal is The Royal Society of Chemistry 2011