Surfactant-stabilized small hydrogel particles in oil: Hosts for remarkable activation of enzymes in organic solvents

Article abstract of DOI:10.1002/chem.200903205

Hydrogels of amino acid based cationic surfactant having C₁₆ tails were used to immobilize heme proteins and enzyme. These hydrogelentrapped proteins/enzyme showed remarkable activation when dispersed in organic solvent. The activation effect (ratio of the activity of the hydrogelentrapped enzyme in organic solvent to the activity of the native enzyme in water) of cytochrome c increased up to 350-fold with varying protein and gelator concentration. Hydrogel-entrapped hemoglobin and horseradish peroxidase (HRP) also showed markedly improved activity in organic solvent. Alteration in the structure of the gelator and its supramolecular arrangement showed that the protein immobilized within amphiphilic networks with larger interstitial space exhibited higher activation. This striking activation of hydrogel-entrapped proteins stems from the following effects: 1) the hydrophilic domain of the amphiphilic networks facilitates accessibility of the enzyme to the water-soluble substrate. 2) the surfactant, as an integral part of the amphiphilic network, assists in the formation of a distinct interface through which reactants and products are easily transferred between hydrophilic and hydrophobic domains. 3) Surfactant gelators help in the dispersion and stabilization of gel matrix into small particles in organic solvent, which enhances the overall surface area and results in improved mass transfer. The activation was dramatically improved up to 675-fold in the presence of nongelating anionic surfactants that helped in disintegration of the gel into further smaller-sized particles. Interestingly, hydrogel-immobilized HRP exhibited about 2000-fold higher activity in comparison to the activity of the suspended enzyme in toluene. Structural changes of the entrapped enzyme and the morphology of the matrix were investigated to understand the mechanism of this activation.

Full text of DOI:10.1002/chem.200903205

FULL PAPER
DOI: 10.1002/chem.200903205
Surfactant-Stabilized Small Hydrogel Particles in Oil: Hosts for Remarkable
Activation of Enzymes in Organic Solvents
Dibyendu Das, Sangita Roy, Sisir Debnath, and Prasanta Kumar Das*[a]
Abstract: Hydrogels of amino acid
based cationic surfactant having C16
tails were used to immobilize heme
proteins and enzyme. These hydrogel-
entrapped proteins/enzyme showed re-
markable activation when dispersed in
organic solvent. The activation effect
(ratio of the activity of the hydrogel-
entrapped enzyme in organic solvent to
the activity of the native enzyme in
water) of cytochrome c increased up to
350-fold with varying protein and gela-
tor concentration. Hydrogel-entrapped
hemoglobin and horseradish perox-
idase (HRP) also showed markedly im-
proved activity in organic solvent. Al-
teration in the structure of the gelator
and its supramolecular arrangement
showed that the protein immobilized
within amphiphilic networks with
larger interstitial space exhibited
higher activation. This striking activa-
tion of hydrogel-entrapped proteins
stems from the following effects: 1) the
hydrophilic domain of the amphiphilic
networks facilitates accessibility of the
enzyme to the water-soluble substrate.
2) the surfactant, as an integral part of
the amphiphilic network, assists in the
3) Surfactant gelators help in the dis-
persion and stabilization of gel matrix
into small particles in organic solvent,
which enhances the overall surface
area and results in improved mass
transfer. The activation was dramatical-
ly improved up to 675-fold in the pres-
ence of nongelating anionic surfactants
that helped in disintegration of the gel
into further smaller-sized particles. In-
terestingly, hydrogel-immobilized HRP
exhibited about 2000-fold higher activi-
ty in comparison to the activity of the
suspended enzyme in toluene. Structur-
al changes of the entrapped enzyme
and the morphology of the matrix were
investigated to understand the mecha-
nism of this activation.
formation of
a
distinct interface
through which reactants and products
are easily transferred between hydro-
philic and hydrophobic domains.
Keywords: enzymes
· heme pro-
teins · hydrogels · immobilization ·
surfactants
Introduction
due to loss of their structural integrity, insolubility, and also
scarcity of essential water around them.[1,2] Researchers over
the past few decades have been striving for superior activity
of enzymes in organic solvents because of technological ad-
vantages.[2,3] To this end, pioneering works of Klibanov and
co-workers showed how enzyme activities can be improved
in organic solvents.[1c,4,5] Still, the activation of enzymes in
nonaqueous media remains a crucial challenge. Among
many other ways, immobilization of enzymes has been em-
ployed to enhance their activity in organic solvents.[6–9] Ide-
ally an immobilization matrix should offer an enzyme-
friendly environment so that enzyme does not encounter
any structural perturbations, remains highly flexible, and can
exhibit similar efficiency in organic solvents as in aqueous
media. In this regard, Tiller and Bruns reported the effects
of amphiphilic networks within polymeric matrix for the ac-
tivation of peroxidase in organic solvents.[6b] Xu and co-
workers subsequently showed improved enzyme activity of
heme proteins (up to eightfold when encapsulated in low
molecular weight two-component hydrogels) in organic sol-
Enzymes are natureꢀs best catalysts due to their fascinating
capability of accelerating a variety of reactions with a high
degree of selectivity and efficiency. Most enzymes perform
best in aqueous environment.[1] On the contrary, these bio-
catalysts suffer considerable deactivation in organic solvents
[a] D. Das, S. Roy, S. Debnath, Dr. P. K. Das
Department of Biological Chemistry
Indian Association for the Cultivation of Science
Jadavpur, Kolkata 700032 (India)
Fax : (+91)33-24732805
E-mail: bcpkd@iacs.res.in
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200903205. It contains Activities
of Hb and HRP, activities of lyophilized proteins; quantitative secon-
dary-structure determination of proteins by FTIR; representative ini-
tial velocities of enzyme-catalyzed reactions; Michaelis–Menten
plots; activities of lyophilized proteins in toluene; and FESEM and
AFM images of hydrogel-entrapped protein.
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vents.[6e] Enzyme activation was realized to be due to the co-
operative effect of amphiphilic nanofibers of the hydrogels,
which helps in mass transfer across the interface.
However, the improvement in the activity of such immo-
bilized proteins could be more distinct if the hydrogelating
molecule is not only an amphiphilic molecule but also a sur-
face-active agent. In this context, surfactants are amphiphilic
molecules that self-aggregate to form different supramolec-
ular structures ranging from micelles of various shapes to
vesicles in water and microemulsions in organic solvents.[10]
Hydrogels formed by supramolecular aggregation of these
surfactants will surely have distinct amphiphilic networks
with large interfacial domains. Consequently, proteins im-
mobilized in the supramolecular networks of these surfac-
tant hydrogels are expected to exhibit superior biocatalysis
due to augmented enzyme–substrate interactions and im-
proved mass transfer across the interface, like the enhanced
activity of enzymes encapsulated in reverse micelles.[11–13]
Additionally, the varying supramolecular arrangements re-
sulting from the aggregation of structurally different surfac-
tant gelators[14,15] can also play a crucial role in the activa-
tion of the hydrogel-entrapped biocatalyst in organic sol-
vents.
Scheme 1. Structure of the surfactant gelators.
In the present work we entrapped heme proteins/enzyme
in the low molecular weight hydrogels prepared by supra-
molecular organization of amino acid based quaternary am-
monium cationic surfactants 1–4 (Scheme 1) having well de-
fined critical micellar concentrations (cmc).[16–20] The surfac-
tant hydrogels were used to ensure that the entrapped pro-
tein enjoys the cumulative advantages of the supramolecular
amphiphilic nanofibers compartmentalized in the milieu of
water with huge interfacial area for the mass transfer. The
hydrogel-immobilized enzymes showed striking activation in
organic solvent. The activation effect (ratio of the activity of
the hydrogel-entrapped enzyme in organic solvent to the ac-
tivity of the native enzyme in water) reached up to about
350-fold for cytochrome c (cyt c) entrapped in gels of 1–4
(Table 1). Importantly, the influence of surfactant amphiphi-
licity in improving the efficiency of biocatalysis was further
established, as the activation of cyt c entrapped in gel 1 was
remarkably enhanced up to 675-fold when sodium dodecyl-
benzene sulfonate (SDBS), a surfactant incapable of gela-
tion, was doped into the enzyme-entrapping hydrogels.
Results and Discussion
The design of an ideal matrix that can immobilize a variety
of enzymes without deactivation, protect them from detri-
mental organic solvents, and activate the entrapped enzymes
by assisting in biocatalytic processes is in great demand. In
this work we utilized surfactant hydrogelators 1–4
(Scheme 1) having a wide range of minimum gelation con-
centrations (MGC) of 0.25–5.0% (w/v) for entrapment of
proteins.[16–18] Quaternary ammonium surfactants 1–4 form
Table 1. Variation of the activity of cytochrome c entrapped in hydrogel in toluene with simultaneous alteration of the gelator concentration and protein
loading.
Loading
(mg)[a]
Concentration of the gelator% (w/v)
15
5
10
20
25
activity[b]
activation
effect[c]
activity[b]
activation
activity[b]
activation
activity[b]
activation
effect[c]
activity[b]
activation
effect[c]
effect[c]
effect[c]
0.5
1
1.5
2
2.5
3
0.037
0.030
0.028
0.016
0.008
0.006
132
107
100
57
29
21
0.063
0.041
0.032
0.016
0.014
0.015
225
146
114
57
50
53
0.069
0.050
0.036
0.019
0.015
0.018
246
179
128
68
53
64
0.070
0.045
0.038
0.019
0.014
0.017
250
161
136
68
50
61
0.091
0.049
0.042
0.024
0.019
0.018
325
175
150
86
68
64
[a] Amount of cyt c added to the hydrogel of 1. [b] The activity is expressed in mmolminÀ1 mgÀ1. [c] Activation effect=ratio of activity of hydrogel-entrap-
ped cyt c in organic solvent toluene to activity of native cyt c in water (2.8ꢂ10À4 mmolminÀ1 mgÀ1). Various substrate concentrations were used throughout
the systems so that the rate of the reaction could be calculated from the plot of the Michaelis–Menten equation. The final concentration of pyrogallol
varied from 0.025 mm to as high as 30 mm. The final concentration of H2O2 was maintained at three times the concentration of pyrogallol. The experi-
mental errors were in the range of 5–15% in duplicate experiments.
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Surfactant-Stabilized Hydrogel Particles in Oil
FULL PAPER
transparent gels instantaneously upon addition of water at
room temperature.
First, a hydrogel of amphiphile 1 was used for immobiliza-
tion of the heme protein cyt c (see Experimental Section),
which is known for its role as electron transporter in redox
reaction. To 75 mL of hydrogel-entrapped cyt c, toluene
(50 mL) was added followed by the addition of the required
concentration of pyrogallol substrate and H2O2. Initially, the
protein loading in the hydrogel was 3.0 mg and the concen-
tration of gelator was 5.0% (w/v). The overall concentration
of protein in the total solution mixture was 60 mgmLÀ1. The
catalytic efficiency of the protein was measured by following
the initial reaction rates within the first minute in a range of
substrate concentrations, and subsequently the activity
(maximum velocity Vmax) was derived from a Michaelis–
Menten plot. The observed activity of free (unbound) cyt c
in water was 2.8ꢂ10À4 mmolminÀ1 mgÀ1, as calculated from
the Michaelis–Menten kinetics curve over a wide range of
substrate concentrations (see Figure S1 in the Supporting In-
formation and the Experimental Section for details). The
calculated Km from the same curve was 0.33 mm. The immo-
bilized cyt c in the hydrogel of 1 exhibited 21-fold improve-
ment in its activity (0.006 mmolminÀ1 mgÀ1, Table 1). Interest-
ingly, the Km value of the immobilized cyt c was found to be
similar (0.35 mm) to that observed in water (see Figure S2 in
the Supporting Information for representative example of a
Michaelis–Menten plot for cyt c at 15% (w/v) of gelator 1 in
toluene).
To understand the importance of the loading of the pro-
tein immobilized within the amphiphilic networks of the hy-
drogels, the protein loading was varied from 0.5 to 3.0 mg at
a fixed concentration of gelator 1. Concentrations of sub-
strates were also proportionately varied. At the lowest pro-
tein loading (0.5 mg) in the hydrogel, the overall concentra-
tion of cyt c in the total reacting solution was 10 mgmLÀ1.
Activation of up to 132-fold was observed in cyt c activity
on decreasing the protein loading from 3.0 to 0.5 mg in the
surfactant gel matrix of 1 at 5% (w/v) (Table 1). A field-
emission scanning electron microscopy (FESEM) image of
the cyt c-immobilizing hydrogel of 1 shows nanoscale fibril-
lar networks of the supramolecular gel (Figure 1A and Fig-
ure S3A in the Supporting Information). However, the loca-
tion of the protein is very difficult to determine from the
FESEM images. At lower protein content, the amphiphilic
network of the hydrogel has sufficient space to localize both
the biocatalyst and the substrate in its hydrophilic domain.
Hence, it facilitates efficient interactions between protein
and substrate. After the reaction, oxidized product can
easily diffuse out to the bulk toluene across the interfacial
hydrophobic domain of the amphiphilic networks formed by
the surfactant gelator. With increasing protein content
(amid concurrent increase of the substrate concentration) in
the hydrogel, the protein gradually saturates the hydrophilic
domain of the networks. This concomitantly decreases
access of the substrate to the protein, which hinders the
available sites for the solubilization of the substrates. The
substrate strives for the same location as is occupied by the
Figure 1. FESEM image of dried gels of 1–4 (A–D) in the presence of cy-
tochrome c.
protein in the supramolecular network. Hence, with increas-
ing amount of cyt c in the hydrogel, the effective interaction
between substrate and protein is impeded, and a reduced ac-
tivation effect of the immobilized protein results (Table 1).
In an effort to enhance the localization sites for both the
protein and the substrate within the supramolecular struc-
ture, the density of the amphiphilic networks was increased
by increasing the gelator concentration. This may lead to
greater access of substrates to the protein and higher activa-
tion of biocatalyst. Indeed, significant activation of cyt c was
observed on increasing the concentration of gelator 1 from
5 to 25% (w/v) at varying protein loadings (0.5–3 mg,
Table 1). The best result was obtained for a protein loading
of 0.5 mg at 25% (w/v) of 1, where the cyt c activity
(0.091 mmolminÀ1 mgÀ1) showed 325-fold enhancement,
which is notably higher than its activity at 5% (w/v) (132-
fold, Table 1). Even at a higher loading of 3.0 mg of protein,
the activity increased from 0.006 mmolminÀ1 mgÀ1 at 5% (w/
v) to 0.018 mmolminÀ1 mgÀ1 at 25% (w/v) with an activation
effect of 64. This improvement in the activity of cyt c re-
flects the importance of the increased supramolecular net-
works in the water phase, which concomitantly offer more
localization sites for both protein and substrate. Thus, the
higher density of the amphiphilic network in hydrogels with
lower enzyme loading is the most favorable condition for
achieving higher activities of the gel-entrapped protein.
At this point we were curious to see whether the amphi-
philic networks of other structurally different surfactant ge-
lators would also exhibit similar activation effects on the im-
mobilized protein. Accordingly, the amino acid at the polar
head of the cationic surfactant of 1 was replaced with other
amino acids (l-phenylalanine, l-tyrosine) and also with a di-
peptide moiety (2–4, Scheme 1). A protein loading of 0.5 mg
was used in hydrogels of surfactants 2–4, as cyt c exhibited
its highest activity at this lowest concentration. Cyt c immo-
bilized in the hydrogel of l-phenylalanine-based surfactant 2
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P. Kumar Das et al.
showed 114- to 196-fold activation with increasing gelator
concentration from 5 to 25% (w/v) (Table 2). Similar en-
philic networks prohibits smooth access of substrates to the
protein, which is easier in the case of 1 and 4. This might be
the probable reason why the extent of the activation effect
for the cyt c was relatively lower in the case of 2 and 3 than
in the case of 1 and 4.
To determine the generality of hydrogel-entrapped
enzyme activity in apolar medium, activity of cyt c immobi-
lized in gel 1 was investigated in toluene and other aromatic
and aliphatic solvents (Table 3). The aromatic solvents o-
hancement
in
cyt c
activity
from
0.043
to
0.069 mmolminÀ1 mgÀ1 with an activation effect of 154 to 246
was observed with increasing concentration of dipeptide-
based gelator 3. Remarkable activation of cyt c was ob-
served in the case of l-tyrosine-based gelator 4, which has
cationic charge at the N terminus and anionic charge at the
aromatic moiety of the tyrosine residue in its polar head. At
25% (w/v) of 4, the thus-far best activity of cyt c
(0.098 mmolminÀ1 mgÀ1) was observed, which is not only sub-
stantially higher than those found for 2 and 3 but also 350-
fold higher than the native activity of the protein in water
(Table 2). In all cases cyt c activity increased with increasing
concentration of gelator. However, despite having structur-
ally different amino acid scaffolds at the polar head, the ac-
tivity of hydrogel-entrapped cyt c was found to be similar in
1 and 4 as well as in 2 and 3. On the other hand, marked dif-
ferences in activation effect were observed when the cyt c
activity of 1 or 4 was compared with those of 2 and 3.
Table 3. Activity of cytochrome c entrapped in hydrogel 1 in different or-
ganic solvents.
Solvent
Activity[a]
Activation effect[b]
[mmolminÀ1 mgÀ1
]
toluene
o-xylene
p-xylene
chlorobenzene
chloroform
dichloromethane
1,2-dichloroethane
carbon tetrachloride
hexane:toluene (1:1)
isooctane:toluene (1:1)
0.091
0.086
0.086
0.089
0.045
0.040
0.034
0.032
0.031
0.030
325
307
307
318
161
143
121
114
111
107
To understand the observed similarity as well as differen-
ces in the activity of cyt c entrapped in hydrogels of 1–4, we
investigated the supramolecular morphology of the protein-
entrapping hydrogels by FESEM (Figure 1). Interestingly in
the morphology of both the gels of 1 and 4 in the presence
of cyt c, fibril networks (Figure 1A and D) were observed.
In case of 1, nanofibrils led to the formation of entangled
networks within which cyt c was entrapped (also see Figure
S3A in the Supporting Information), while the protein was
entrapped within ribbon-shaped fibrils in the case of 4. For-
mation of the fibril network was also confirmed by the
atomic force microscopy (AFM) image of cyt c entrapped in
the hydrogel of 1 (Figure S3B in the Supporting Informa-
tion). These fibrillar morphologies of the aggregates pre-
sumably provided greater exposure of the protein to the
substrate because of the larger interstitial space within the
amphiphilic networks. Consequently, this resulted in higher
activation of the protein entrapped in the gels of 1 and 4.
However, protein entrapped in the hydrogel of 2 exhibited
thick and condensed fibrous structure (Figure 1B), while
that of 3 showed tight porous morphology (Figure 1C). In
both cases constrained interstitial space within the amphi-
[a] 0.5 mg of protein was added to hydrogel 1 (25% w/v). [b] See footno-
te [b] of Table 1. The experimental errors were in the range of 5–15% in
duplicate experiments. Various substrate concentrations were used
throughout the systems so that the rate of the reaction could be calculat-
ed from the plot of the Michaelis–Menten equation. The final concentra-
tion of pyrogallol varied from 0.025 mm to as high as 30 mm. The final
concentration of H2O2 was maintained at three times the concentration
of pyrogallol.
and p-xylene and chlorobenzene were found to be almost as
good as toluene for the cyt c, with an activation effect of
about 310. Improved protein efficiency was also observed in
halogenated solvents like chloroform, dichloromethane, 1,2-
dichloroethane, and carbon tetrachloride, in which activa-
tion effects were 161, 143, 121, and 114, respectively. The
protein had insignificant activation effects in solvents like
hexane and isooctane. However it showed about 110-fold ac-
tivation in its efficiency when the cyt c-entrapping hydrogel
Table 2. Variation of the activity of hydrogel-entrapped cytochrome c in toluene with alteration of the gelator (2–4).
Surfactant
Concentration of gelator [% (w/v)]
15
5
10
20
25
activity[a]
activation
effect[b]
activity[a]
activation
activity[a]
activation
activity[a]
activation
effect[b]
activity[a]
activation
effect[b]
effect[b]
effect[b]
2
3
4
0.032
0.043
0.050
114
154
179
0.042
0.049
0.070
150
175
250
0.045
0.068
0.067
161
243
239
0.051
0.066
0.084
182
236
300
0.055
0.069
0.098
196
246
350
[a] 0.5 mg of the protein was added to the hydrogels. The activity is expressed in mmolminÀ1 mgÀ1. [b] Activation effect=ratio of activity of hydrogel-en-
trapped cyt c in organic solvent toluene to activity of native cyt c in water (2.8ꢂ10À4 mmolminÀ1 mgÀ1). Various substrate concentrations were used
throughout the systems so that the rate of the reaction could be calculated from the plot of the Michaelis–Menten equation. The final concentration of
pyrogallol varied from 0.025 mm to as high as 30 mm. The final concentration of H2O2 was maintained at three times the concentration of pyrogallol. The
experimental errors were in the range of 5–15% in duplicate experiments.
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Surfactant-Stabilized Hydrogel Particles in Oil
FULL PAPER
was dispersed in 1:1 (v/v) mix-
tures of hexane or isooctane
with toluene (Table 3).
To examine the effects of the
amphiphilic networks of surfac-
tant gelators on other proteins,
hemoglobin (Hb) and horse-
radish peroxidase (HRP) were
immobilized in the gel of 1.
The Hb loading was varied
from 1.0 to 3.0 mg with simul-
taneous change in the concen-
tration of gelator from 5 to
25% (w/v). The activity of un-
bound Hb in water was found
to be 6.0ꢂ10À4 mmolminÀ1 mgÀ1,
as calculated from the Michae-
lis–Menten plot with varying
concentrations of substrate
Figure 2. Variation of the activity of Hb entrapped in hydrogel 1 in toluene (50 mL) with varying gelator con-
centration and protein loading. The experimental errors were in the range of 5–15% in duplicate experiments.
Various substrate concentrations were used throughout the systems so that the reaction rate could be calculat-
ed from the plot of the Michaelis–Menten equation. The final concentration of pyrogallol varied from 0.05 mm
to as high as 30 mm. The final concentration of H2O2 was maintained at three times the concentration of pyro-
(see Figure S4 in the Support-
ing Information). However, Hb
immobilized in hydrogel
showed up to 32-fold activation
(Figure 2 and Table S1, Sup- gallol.
porting Information) from
meager 6.0ꢂ
1
a
10À4 mmolminÀ1 mgÀ1 in water to 0.0189 mmolminÀ1 mgÀ1 when
dispersed in toluene at 25% (w/v) gelator concentration.
The observed Km values (ca. 0.9 mm) throughout the activity
measurements for the immobilized Hb in organic solvent
(see Figure S5 in the Supporting Information for a represen-
tative example of Michaelis–Menten plot from which activi-
ty and Km were calculated) were found to be similar to that
observed for unbound Hb in
(from 0.0467 mmolminÀ1 mgÀ1 in water to 0.1795 mmol
minÀ1 mgÀ1 in the matrix) when the gelator concentration
was 25% (w/v). Similar Km values (0.071–0.075 mm) were
observed for the hydrogel-immobilized protein in organic
solvent (see Figure S7 in the Supporting Information for a
representative Michaelis–Menten plot for hydrogel-entrap-
ped HRP). In this context, the activity of HRP in water is
water (Km =0.78 mm). For HRP,
the amount of enzyme was
varied from 0.125 to 0.5 mg
while keeping the gelator con-
centration in the same range
(Figure 3 and Table S2 in the
Supporting Information). The
amount of enzyme loaded in
the gel was 5–6 times lower
than for other heme proteins,
because even at such low con-
centration the activity of HRP
in
water
is
quite
high
(0.0467 mmolminÀ1 mgÀ1, Km =
0.075 mm, see Figure S6 in the
Supporting Information). The
peroxidase also showed im-
proved activity in the entire
range of protein and gelator
concentration. At a loading of
0.125 mg enzyme, the activity of
hydrogel-1-entrapped HRP in-
Figure 3. Variation of the activity of HRP entrapped in hydrogel 1 in toluene (50 mL) with varying gelator
concentration and protein loading. The experimental errors were in the range of 5–15% in duplicate experi-
ments. Various substrate concentrations were used throughout the systems so that the reaction rate could be
calculated from the plot of the Michaelis–Menten equation. The final concentration of pyrogallol varied from
0.05 mm to as high as 30 mm. The final concentration of H2O2 was maintained at three times the concentration
creased up to about 3.8-fold of pyrogallol.
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P. Kumar Das et al.
Table 4. Secondary structure of cytochrome c and hemoglobin in D2O so-
lution and immobilized in the hydrogel of 1, determined by FTIR spec-
troscopy.
superior to the activity of the other used proteins, which
concomitantly results in lesser activation effects for this par-
ticular enzyme in organic solvents. Similar to the trends
shown by cyt c, in the case of both Hb and HRP at a fixed
loading of protein the activation effect increases with in-
creasing concentration of gelator, and at a fixed gelator con-
centration the activity increases with decreasing amount of
protein. The observed activities of hydrogel-1-entrapped
(25% w/v) Hb, cyt c, and HRP (at their lowest loading) are
350-, 1516-, and 2058-fold higher, respectively, compared to
the activity of the corresponding suspended lyophilized pro-
teins in toluene (see Table S3 in the Supporting Informa-
tion). However, this huge activation is due to the very low
activity of the lyophilized proteins in organic solvent, which
further stresses the important role of the amphiphilic net-
works in the enhanced activity of hydrogel-entrapped pro-
tein in organic solvents.
An ideal matrix should immobilize an enzyme without af-
fecting its native structure. To determine the effect of the
surfactant hydrogelators on the structure of the proteins, we
investigated the state of cyt c entrapped in the hydrogel of 1
using UV/Vis, circular dichroism (CD), and FTIR spectros-
copy (Figure 4 and Table 4). Absorbance spectroscopy was
used to monitor the Soret region (390–410 nm), which is
Sample
Secondary structure [%]
a-helix b-sheet turn random
cytochrome c in D2O solution
41Æ2
21Æ1
22Æ2
12Æ3
18Æ3
21Æ1 17Æ3
cytochrome c in the D2O gel of 1 45Æ2
22Æ3 10Æ2
[a]
hemoglobin in D2O solution
78Æ3
8Æ3
–
–
[a]
hemoglobin in the D2O gel of 1
72Æ1
8Æ2
[a] The band due to the random structure is very small and appears as a
shoulder in the a-helix band and therefore is included in the a-helix
value.
sensitive to the tertiary and quaternary structures of heme-
containing proteins. The Soret band of the immobilized cyt c
was found to have the same pattern and was also in the
same position as observed for the native protein (Fig-
ure 4A). This unaltered spectral characteristic reflects the
same state of the heme group upon immobilization. The
higher order structure of unconfined and gel-encapsulated
cyt c was investigated by CD spectroscopy. Retention of the
double minimum in the far-UV region at 210 and 222 nm
(characteristic of the a-helix) for both unconfined and en-
trapped protein indicated preservation of the native secon-
dary structure of cyt c in the immobilized state (Figure 4B).
Furthermore, the structural elements of cyt c and Hb were
quantified by FTIR spectroscopy (Table 4). The a-helical
contents of cyt c and Hb entrapped in the D2O gel of 1 (45
and 72%, respectively) were found to be similar to those of
unconfined protein in D2O solution (41 and 78%, Table 4).
Similarly no notable difference was observed in other struc-
tural elements, such as b-sheet and turn in solution and im-
mobilized state (see Figure S8 in the Supporting Information
for the corresponding raw FTIR spectra of the protein in so-
lution and gel state). Thus, the entrapped protein in this am-
phiphilic surfactant hydrogel retains its structural integrity,
as in the native form in water.
On the basis of the observed activation effect of the heme
proteins entrapped in surfactant hydrogel in organic solvent
and the corresponding microscopic and spectroscopic stud-
ies, it is clear that the amphiphilic networks in the supra-
molecular structure play the crucial role in determining the
superactivity of the protein. First, the hydrophilic domain of
the amphiphilic networks present in the hydrogel aids easy
accessibility of the enzyme to the water-soluble substrate py-
rogallol. Second, the surfactant, as an integral part of the
amphiphilic network, assists in improved mass transfer of
the reactants and products from the gel phase to the bulk
organic phase and vice versa. Furthermore, in the control
experiment [biphasic mixture of toluene (50 mL) and water
(75 mL)] no transfer of the product into the organic phase
was observed in absence of the gelators. Instead, the product
was visually seen to be accumulated in the minute water
phase by monitoring reaction kinetics as were observed in
exclusively aqueous medium. This observation further reiter-
ates the importance of the presence of the gelators. In this
Figure 4. Absorbance spectra of cytochrome c: A) in the Soret region in
water (&) and in the immobilized state in the gel of 1 (*). B) Circular
dichroism spectra of cytochrome c in the far-UV region in water (*) and
in the immobilized state in the gel of 1 (&).
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Surfactant-Stabilized Hydrogel Particles in Oil
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regard, the interstitial space within the supramolecular net-
work has an important role with respect to the both
enzyme–substrate interaction and mass transfer across the
interface. This has an obvious correlation with the supra-
molecular arrangement of the amphiphilic networks, which
depends on the structure of the surfactant gelator. More in-
terstitial space would facilitate mass transfer and result in
remarkable activation of protein, as observed in the case of
hydrogels of 1 and 4 (Figure 1). However, the other impor-
tant reasons behind the striking activation of these immobi-
lized proteins is that these surfactant hydrogelators help in
dispersion of this gel matrix into small particles in oil (or-
ganic solvent) as well as stabilization of these particles sur-
rounded by the surfactants.[7b] Bright-field micrographs of
the dispersed hydrogel of 1 in toluene (see Experimental
Section for details) showed that the sizes of the particles are
in the range of 1.0–1.5 mm (Figure 5A). Dynamic light scat-
tering (DLS) of the same dispersed hydrogel particles in tol-
uene showed similar sizes (ca. 1 mm) to those observed in
the bright-field image. These small hydrogel particles pro-
vide very high surface area for favorable interaction be-
tween enzyme and substrate and also for the easy transfer
of the reactants and the products (shown as an idealized
scheme in Figure 6) that result in the superior activity of
protein entrapped within these particles. This situation is
analogous to the case of reverse micelles, where surfactants
help in the formation of nanometer-scale aggregates due to
their localization at the liquid–liquid interface resulting in
stabilization of the colloidal systems. However, the benefi-
cial effect of the amphiphilic fibers observed in the present
study is missing in reverse micelles.[11–13] In the present
system, the immobilized biocatalyst in the surfactant-based
hydrogel benefits from the amphiphilic fibers confined in
small hydrogel particles having high interfacial area for
mass transfer of extreme polarities.
Figure 5. Bright-field images of the hydrogel particles of 1 (A) and 1 with
SDBS (B) dispersed in toluene (the size of the scale bar in A) and B) is
10 and 5 mm, respectively). C) and D) show the morphologies of the hy-
drogel of 1 in the presence of SDBS and sodium dodecanoate, respective-
ly.
tants with the expectation that these surfactants, incapable
of gelation, will facilitate formation of even smaller hydro-
gel particles with larger surface area through further disper-
sion and stabilization of the gel particles in the organic
phase (Figure 6). Additionally these surfactants can comple-
ment the mass-transfer process of the amphiphilic fibers by
assisting in the formation of greater interfacial domain in
the supramolecular structure. We decided to start with the
negatively charged common surfactant sodium dodecylben-
zene sulfonate (SDBS) to impregnate the 15% (w/v) hydro-
gel of 1 entrapping cyt c (0.5 mg), which will result in a
matrix consisting of both charges. The negatively charged
surfactant was chosen as an additive since it is not known to
inhibit oxidase activity, and also because of the comparative-
It has now become apparent that the presence of a surfac-
tant as a component of hydrogel matrix can be very advan-
tageous for activation of the immobilized enzyme. This en-
couraged us to hybridize the gels with nongelating surfac-
Figure 6. Pictorial representation of the mechanism of enzyme activation entrapped in the supramolecular networks of the hydrogels formed by surfac-
tant gelators.
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4917

P. Kumar Das et al.
rangement in the hydrogel of 1
with increasing concentration of
nongelating surfactant. The am-
phiphilic network possibly loses
its ability to entrap the protein
efficiently, and this results in a
reduced
activation
effect.
Hence, it can be inferred that
the protein shows remarkable
activation effects only when
faced with the optimum pres-
ence of both the amphiphilic
network of the gelating surfac-
tant and the complementary
mass-transfer capability of the
nongelating surfactant. Similar
remarkable improvement in the
Figure 7. Activation effects exhibited by cytochrome c when nongelating additives were doped into the immo-
bilized matrix of 1. The numbers on the top of each bar denote the concentration of the additives in percent
(w/v). The loading of the protein in the gel was 0.5 mg, and the concentration of gelator 1 15% (w/v).
activation
effect
(668-fold:
Figure 7, see Table S4 in the
Supporting Information) was
ly higher activation shown by the heme protein in hydrogel
of 4 that contains both cationic and anionic charges. The
SDBS was doped in the hydrogel of 1 with a varying con-
centration from 1 to 7.5% (w/v). Remarkable improvement
in the efficiency of cyt c was observed, as the activation
effect increased from 246 (in absence of any additive) to 357
in the presence of 1% (w/v) of SDBS (Figure 7 and
Table S4 in the Supporting Information). In the presence of
2.5% (w/v) SDBS, the activation dramatically increased up
to 675-fold, that is, about 175% higher than the cyt c activity
in 1 alone. To investigate the cause of this remarkable acti-
vation we recorded a bright-field micrograph of the dis-
persed hydrogel particles of 1 in the presence of SDBS in
toluene. In accordance with our expectation, smaller hydro-
gel particles (0.4–0.6 mm, Figure 5B) were observed com-
pared to the dispersed hydrogel particles of 1 alone (1.0–
1.5 mm, Figure 5A). Here also the corresponding DLS inves-
tigation showed similar sizes (ca. 0.45 mm) of the dispersed
hydrogel particle in the presence of SDBS. These further
disintegrated particles (Figure 6) would result in a substan-
tial increase in the surface area that enhances the interac-
tion between enzyme and substrate and also facilitates the
mass-transfer process. The FESEM image of hydrogel 1 in
the presence of SDBS showed a wrinkled, soft-sheet like
morphology (Figure 5C) as opposed to the entangled fibril
network of 1 alone (Figure 1A). This transition of the mor-
phology upon addition of the anionic surfactant could also
have played an important role in facilitating the interaction
between protein and substrate. In this context, when SDBS
(15% w/v) alone was used, very low activation was ob-
served.
observed when the nongelating surfactant sodium dodeca-
noate (SDD) was doped at 2.5% (w/v) in the 15% (w/v) hy-
drogel of 1. Here also the FESEM image of the hydrogel 1
in the presence of SDD showed an alteration in the fibril
morphology (Figure 5D) compared to that of 1 alone. Thus,
the amphiphilicity of the nongelating surfactant with distinct
hydrophilic (polar head) and hydrophobic (alkyl chain) moi-
eties play the key role in this striking activation. To establish
the importance of the amphiphilicity of the surfactant as ad-
ditive, we doped the hydrogel with the corresponding acid
forms of negatively charged surfactants, that is, dodecylben-
zenesulfonic acid (DBSA) and dodecanoic acid (DCA), in
which the degree of hydrophilicity at the polar head has
been compromised. We also doped the hydrogel with
sodium p-toluenesulfonate (SPTS), sodium hexanoate
(SHN), and sodium acetate (NaOAc), in which the polar
heads are similar to those of SDBS and SDD but the hydro-
phobic alkyl chain is either truncated or absent. It is expect-
ed that DBSA, DCA, SPTS, and sodium acetate would have
less amphiphilic character than the negatively charged sur-
factants SDBS and SDD. Accordingly, in the case of all of
these additives, the activation effect plummeted below 200
and became lowest (36-fold) in the presence of sodium ace-
tate (Figure 7). This confirms the importance of the additive
being a surfactant for obtaining remarkable performance of
the hydrogel-entrapped protein. Next, we investigated the
effect of cationic surfactants as additives on the activity of
immobilized cyt c. For this purpose cetyltrimethylammoni-
um bromide (CTAB) and cetyl pyridium bromide (CPyB)
were doped at 2.5% (w/v) into the hydrogel of 1 (15% w/v).
However, the activation effect observed for only 1 did not
improve in the presence of these cationic surfactants
(Figure 7). These results underlined the importance of the
anionic surfactants in activation of the protein.
However, this activation effect dropped steadily to 486 as
the concentration of SDBS was increased from 2.5 to 7.5%
(w/v) (Figure 7 and Table S4 in the Supporting Information).
Although this activation effect is almost 100% higher than
that observed with 1 alone, this decline in the activation
effect indicates loss of the supramolecular amphiphilic ar-
Lastly, we were interested in determining the effect of the
amphiphilic networks of the cationic surfactant on the sta-
bility of the immobilized cyt c in presence and absence of
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Surfactant-Stabilized Hydrogel Particles in Oil
FULL PAPER
Figure 8. Percentage retention of cytochrome c activity in the immobilized state (gel of surfactant 1, 15% w/v) when the gel was incubated in entrapped
condition at 258C without SDBS (A), when the gel was incubated under entrapped conditions at 258C with 2.5% (w/v) SDBS (B), when the gel was dis-
persed in toluene with continuous stirring in the absence of SDBS (C), and when the gel was dispersed in toluene with continuous stirring in the pres-
ence of 2.5% (w/v) SDBS (D).
nongelating surfactant SDBS (Figure 8). This investigation
was carried out in two different ways. Firstly, the immobi-
lized protein in 15% (w/v) gelator 1 with or without 2.5%
(w/v) of surfactant SDBS was aged at 258C without addition
of the organic solvent. At different time intervals this pro-
tein-entrapped hydrogel was dispersed in toluene to investi-
gate the residual activity. Both in the presence and absence
of SDBS, more than 90% of the activity (compared to the
initial activity of 0.069 mmolminÀ1 mgÀ1, Table 1) remained
even after 48 h of incubation, and gradually dropped to
40% after 5 d (Figure 8A and C). In the second way, the
protein-immobilizing gel was dispersed in toluene with con-
tinuous stirring conditions and the activity was monitored
over time (Figure 8B and D). In this process more than
70% of the activity was retained up to 5 h in the absence of
SDBS, and the activity gradually dropped to 10% after 8 h.
In presence of SDBS, the rate of the decline of the activity
was slightly faster. Thus, the remarkable activity of the cyt c
is maintained for a substantial period in the immobilized
state.
Conclusion
Proteins and enzyme immobilized in the surfactant hydrogel
matrix show remarkable activation in organic solvents in
comparison to their native activity in water. An optimum
combination of gelator concentration and protein loading is
important for activation of the protein. Upon dispersion in
organic solvents these hydrogels with proteins entrapped in
their supramolecular networks form surfactant-stabilized mi-
crometer-sized hydrogel particles. These surfactant-stabi-
lized hydrogel particles markedly improve the activity of the
immobilized protein by facilitating interaction of hydrophilic
substrates with the enzyme and diffusion of the product in
the organic solvent across the large interfacial domain of the
amphiphilic network. Alteration in the structure of surfac-
tant gelator, the basic building block of these supramolec-
ular matrices, resulted in varied activation profiles of the
protein depending on amphiphilic networks presented by
the supramolecular aggregates. The role of surfactant am-
phiphilicity in the activation effect was further established
when cyt c entrapped in the hydrogel hybridized with non-
gelating surfactants exhibited dramatic activation up to 675-
fold. These nongelating surfactants help to disintegrate the
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4919

P. Kumar Das et al.
teins from these plots were 2.8ꢂ10À4
, , and 0.0467 mmol
6.0ꢂ10À4
gel matrix into smaller particles in oil (organic solvent),
which have substantially increased overall surface area for
superior mass transport. Therefore, heme proteins entrap-
ped within the amphiphilic network of surfactant-stabilized
hydrogel particles in organic solvents show striking activa-
tion that greatly increases further in the presence of nonge-
lating anionic surfactants. These surfactant/hydrogel-based
matrices may lead to the development of proficient hosts for
enzymes in organic solvents.
minÀ1 mgÀ1 for cyt c, Hb, and HRP, respectively. The calculated Km values
for cyt c, Hb, and HRP in water were 0.33, 0.78, and 0.075 mm, respec-
tively. These Km values for the proteins in aqueous solutions are signifi-
cantly lower than the highest substrate concentrations (ca. 10–30 mm)
used for determining the initial rates of the reaction. Thus, the highest
concentration used for determining the activity of protein is at least 40
times higher than Km, and such huge concentration of the substrate is ex-
pected to be sufficiently high to saturate the protein in the bulk water
phase.
Activity measurements of immobilized proteins in organic solvents: Or-
ganic solvent (50 mL) was added to hydrogel-entrapped protein (0.5–
3.0 mg for cyt c and Hb and 0.125–0.5 mg for HRP), and the mixture
stirred for about 3 min, during which the hydrogel matrix breaks down
into small particles. To this stirred solution of pyrogallol (62.5 mL of stock
solution in acetone with concentration varied between 200 mm to 4.8m)
was added. Then, 85 mL of H2O2 in aqueous solution (stock solution with
concentration varied from 441 mm to 8.82m) was added to this stirred so-
lution to initiate the reaction. The overall concentration of protein in the
reaction mixture was varied from 10 to 60 mgmLÀ1 for heme proteins,
and from 2.5 to 10 mgmLÀ1 for HRP. The overall concentrations of pyro-
gallol for heme proteins were proportionately varied from 0.25–30 mm,
and for HRP it from 0.025–10 mm. In all cases, a proportionate concen-
tration of H2O2 was used to maintain an equivalence ratio of pyrogallol:-
H2O2 of 1:3 in the reaction mixture. This was done to minimize the deac-
tivation effects arising from the use of higher concentrations of H2O2.
This has been well documented[21] for peroxidases like HRP, for which it
was shown that, at higher concentrations of H2O2, the enzyme suffers
substrate inhibition. However, it has also been observed that an optimum
concentration of H2O2 is required for maximum activity of the protein.
Consequently, we used a concentration ratio of 1:3 for the substrates py-
rogallol and H2O2. This particular ratio of substrates has been used in a
number of previously reported works. It was assumed that the rate fol-
lows the Michaelis–Menten equation. Furthermore, in a system such as
Experimental Section
Cytochrome c (oxidized) from horse heart, all amino acids, silica gel of
60–120 mesh, IR-grade KBr, n-hexadecylamine, N,N-dicyclohexylcarbo-
diimide, 4-N,N-(dimethylamino)pyridine, N-hydroxybenzotriazole, iodo-
methane, solvents, and all other reagents were procured from SRL,
India. Horseradish peroxidase (HRP, EC 1.11.1.7, Type II, RZ: 2.0) was
purchased from Sigma and used as received. Hemoglobin (Hb) from
bovine blood was procured from Fluka chemical company and was used
as received. Milli-Q Water was used throughout the study. Pyrogallol, the
substrate used for monitoring protein activity, was obtained from Quali-
gens Fine chemical Company, India. Hydrogen peroxide (30% w/v solu-
tion) was purchased from Ranbaxy, India. Thin-layer chromatography
was performed on Merck precoated silica gel 60-F254 plates. Amberlite
Ira-400 chloride ion-exchange resin was obtained from Aldrich Chemical
Company. The UV/Vis absorption spectra were recorded on a Varian
Cary-50 spectrophotometer. Mass spectrometric (MS) data were acquired
by electron spray ionization (ESI) on a Q-tof-Micro Quadruple mass
spectrophotometer, Micromass. 1H NMR spectra were recorded on an
AVANCE 300 MHz (Bruker) spectrometer. FTIR spectra were recorded
on a Perkin-Elmer Spectrum 100 FTIR Spectrometer. Surfactants 1–4
were synthesized by following previously reported protocols.[16–18]
this, Km would actually be closer to the value Km(pyrogallol)+KmACHTUNGTRENNUNG(H2O2)/
3. Aliquots were taken from the upper half of the reaction solvent (the
hydrogel particles are preferentially localized at the bottom of the reac-
tion vessel) to monitor the absorbance of the product. The increase in ab-
sorbance at 420 nm (production of purpurogallin) was measured at cer-
tain intervals. The concentration of purpurogallin was determined from
the molar extinction coefficient at 420 nm.[12c] The initial rates of forma-
tion of the product in the first minute were used to determine the activity
of the immobilized protein (see Figure S9–S12, Supporting Information).
To check that the small hydrogel particles do not interfere through scat-
tering, a control reaction was performed in the exact same way in the ab-
sence of the protein. There was no change in absorbance at 420 nm. Rep-
resentative Michaelis–Menten plots for the calculation of the activity of
the protein entrapped in the hydrogel of 1 (15% w/v) are given as Figur-
es S2, S5, and S7 in the Supporting Information for cyt c, Hb, and HRP,
respectively. The activity of the hydrogel-entrapped protein in the organ-
ic solvent could not be measured at lower loadings of the protein (0.5, 1,
and 0.125 mg for cyt c, Hb, and HRP, respectively) because of experimen-
tal difficulty in monitoring the change in absorbance. Interestingly, the
Km values obtained for the hydrogel-entrapped proteins in organic sol-
vent are similar to that observed for the unbound protein in water. For
cyt c, Km was found to be 0.35 mm, while that of Hb was around 0.9 mm.
For HRP, the observed Km value was 0.071–0.075 mm. This also strongly
indicates that the saturation levels of the proteins both in water and the
organic phase are similar, and that the concentration of the substrates
used is high enough for the saturation of the proteins. In this context, no
transfer of the product into the organic phase was observed in the control
experiment in the organic solvent at similar concentrations of substrates/
enzyme in the absence of the gelators.
Immobilization of proteins in the hydrogel matrix: The required amount
(3.8–18.8 mg) of surfactants 1–4 was dissolved in Milli-Q water (60 mL) in
a 100 mL round-bottom flask. Then, protein (15 mL) from a stock solu-
tion prepared with Milli-Q water was added to this solution to achieve
the required amount of protein (0.125 to 3.0 mg) in the immobilization
matrix. The protein in the gel matrix was then aged for 20–30 min, after
which the activity was monitored. When additives were used, an addi-
tional amount of the additive was added to the gelator, which was then
dissolved in Milli-Q water (60 mL) in a similar way as described above.
Activity measurements on proteins in water: The activities of all three
proteins (cyt c, HRP, and Hb) in water (Milli Q) were monitored spectro-
photometrically with pyrogallol as substrate and H2O2. For cyt c and Hb
activity in water, 3.75 mL of the protein solution (20 mgmLÀ1 in Milli Q
water) and 10 mL of substrate (7.5 mm to 4.5m stock solution in acetone)
were added to 1.5 mL of Milli Q water in a quartz cell. Lastly, 10 mL of
H2O2 (from stock solutions of varying concentration to keep an equiva-
lence ratio of pyrogallol:H2O2 =1:3 in the reaction mixture) was added to
initiate the reaction. Overall protein concentration in the reaction mix-
ture was 50 mgmLÀ1. The absorbance change was monitored instantane-
ously after addition of H2O2. The progress of the reaction was monitored
by formation of purpurogallin, the oxidized product of pyrogallol, at
lmax =420 nm for the initial 1–2 min. For HRP, in a typical experiment,
7.5 mL of the enzyme solution (1.0 mgmLÀ1 in Milli Q water) and 5 mL of
substrate (7.5 mm to 3m stock solution in acetone) were added to Milli Q
water (1.5 mL) in a quartz cell. H2O2 (10 mL; from stocks of varying con-
centration to keep an equivalence ratio of pyrogallol:H2O2 =1:3 in the re-
action mixture) was added to initiate the reaction. Overall concentration
of HRP in the reaction mixture was 5 mgmLÀ1. The activity was moni-
tored by a similar procedure to that described above. For all the proteins
and enzyme the initial velocity was plotted against the varying concentra-
tion of pyrogallol to give the corresponding Michaelis–Menten plot (see
Supporting Information: Figures S1, S4, and S6, for cyt c, Hb, and HRP,
respectively). The calculated activities (maximum velocity Vmax) of pro-
Microscopy studies: Field-emission scanning electron microscopy
(FESEM) was performed on JEOL-6700F. A piece of gel entrapping the
protein was mounted on glass slide and dried for few hours under
vacuum before imaging. The morphology of the dried gel containing the
protein was also studied by atomic force microscopy (AFM) on a Veeco
model AP0100 microscope in noncontact mode. The sample was mount-
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Surfactant-Stabilized Hydrogel Particles in Oil
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ed on silicon wafer and dried under vacuum for few hours before imag-
ing.
Acknowledgements
P.K.D. is thankful to Department of Science and Technology, India for fi-
nancial assistance through Ramanna Fellowship (No. SR/S1/RFPC-04/
2006). D.D., S.R., and S.D. acknowledge Council of Scientific and Indus-
trial Research, India for their Research Fellowships.
Bright-field microscopy: A hydrogel of 1 (75 mL; 15% w/v) and 1 with
SDBS (2.5%,w/v) was prepared. Toluene (50 mL) was added and the
mixture stirred for about 3 min. Aliquots (10 mL) were taken from the re-
action mixture and added to a glass slide. The slide was allowed to dry
for 5–10 min to ensure removal of the organic solvent (toluene). Then
images of the hydrogel particles were taken with a light microscope
(BX61, Olympus).
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Circular dichroism spectra: The CD spectra of cyt c were recorded on a
Jasco J-815 CD spectrometer in the 190 to 350 nm wavelength range. The
concentration of cyt c was kept at 0.05 mgmLÀ1 while the concentration
of 1 was 0.025% (w/v) in Milli Q water. A similar concentration of the
gelator was used as background to eliminate the signals from the surfac-
tant alone. A 1 mm quartz cell was used to record the spectra.
Absorbance spectra: The absorbance spectra of cyt c immobilized in the
gel of 1 were obtained by dissolving 1 (10 mg) in Milli Q water (60 mL)
and adding cyt c (1 mg) in water (15 mL). A portion of this gel was added
to a quartz plate, and a thin layer was prepared by sandwiching the gel
with another quartz plate. The spectrum of unbound cyt c was recorded
in a similar way as a sensible control experiment.
FTIR spectroscopy: For native enzymes, solutions were prepared in D2O
and IR spectra were measured in a cell with CaF2 windows and 100 mm
path length. For each spectrum, an average of 512 scans was obtained at
a resolution of 2 cmÀ1 with an interval of 1 cmÀ1. Reference spectra were
recorded under identical conditions with only D2O without the protein.
For the immobilized cyt c, 2.5 mg of protein was added in water (15 mL)
to gel (60 mL) containing 1 (10 mg). This gel was mounted between CaF2
windows with 100 mm spacers. Spectra of the thin layer of gel containing
the protein were recorded and accumulated for 512 times at a resolution
of 2 cmÀ1 with an interval of 1 cmÀ1. From this spectrum the spectrum of
the gel without the protein (same amount as in the protein-containing
gel), obtained under the same conditions, was subtracted. The raw FTIR
spectra are given in Supporting Information as Figure S8.
Quantification of the secondary structure of cytochrome c in D2O solu-
tion and in the immobilized state: The amide I region from 1600–
1700 cmÀ1 was analyzed to quantify the secondary structural contents of
the enzymes.[5a,11b,22] The difference spectra were smoothed by a nine-
point Savitsky–Golay smoothing function with help of the software sup-
plied with the Perkin-Elmer FTIR Spectrometer. A straight baseline was
obtained in the region from 1750 to 2000 cmÀ1, an important prerequisite
to obtain quantitative structural information. Fourier deconvolution was
performed with the Perkin-Elmer software with a line narrowing factor
of k=2.3 and half-bandwidth of 37 cmÀ1. Then the second-derivative
spectrum was obtained from the deconvoluted spectra by Savitsky–Golay
derivative function software with a five-point window. The a-helical and
b-sheet structures were assigned according to literature reports.[22] Their
relative amounts were determined by computing the areas under the as-
signed bands (see Table S5 in the Supporting Information). Each sample
was measured four times.
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a fixed-angle apparatus (Brookhaven Instruments Corporation, U.S.A.)
equipped with Melles Griot He–Ne laser (Model BI-200SM Goniometer
VER-2.0 plus particle size analyzer operating at 633 nm) with 35 mW
power. The scattering intensities collected were computed by a data pro-
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gel particles of 1, 15% (w/v), of the compound was dissolved in water
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for 1–2 min before the data were collected. For measurements in the
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(w/v) 1 and a similar procedure was used to collect the data.
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Received: November 23, 2009
Revised: January 18, 2010
Published online: March 12, 2010
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ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 4911 – 4922