Unusually high thermal stability and peroxidase activity of cytochrome c in ionic liquid colloidal formulation

Article abstract of DOI:10.1039/c5cc05722a

Ionic liquid (IL) surfactant choline dioctylsulfosuccinate, [Cho][AOT], formed polydispersed vesicular structures in the IL, ethylmethylimidazolium ethylsulfate, [C₂mim][C₂OSO₃]. Cytochrome c dissolved in such a colloidal medium has shown very high peroxidase activity (～2 times to that in neat IL and ～4 times to that in an aqueous buffer). Significantly, the enzyme retained both structural stability and functional activity in IL colloidal solutions up to 180°C, demonstrating the suitability of the system as a high temperature bio-catalytic reactor.

Full text of DOI:10.1039/c5cc05722a

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Unusually high thermal stability and peroxidase
activity of cytochrome c in ionic liquid colloidal
formulation†
Cite this:DOI: 10.1039/c5cc05722a
Received 10th July 2015,
Accepted 22nd October 2015
a
ab
Pankaj Bharmoria and Arvind Kumar*
DOI: 10.1039/c5cc05722a
www.rsc.org/chemcomm
8
Ionic liquid (IL) surfactant choline dioctylsulfosuccinate, [Cho][AOT], particularly the anionic surfactants, (ii) the high probability of
9
formed polydispersed vesicular structures in the IL, ethylmethylimid- structural alterations and functional deactivation of proteins,
2 2 3
azolium ethylsulfate, [C mim][C OSO ]. Cytochrome c dissolved in such and (iii) the poor/weak self-assembly of surface active molecules.
a colloidal medium has shown very high peroxidase activity (B2 times to Despite these limitations, some ILs have been found suitable to
that in neat IL and B4 times to that in an aqueous buffer). Significantly, dissolve proteins with a slightly perturbed structure with the
10
the enzyme retained both structural stability and functional activity in IL retention of functional activity and the ability to support the
11
colloidal solutions up to 180 8C, demonstrating the suitability of the self-assembly of a variety of amphiphiles. There are a few reports
system as a high temperature bio-catalytic reactor. of supramolecular assemblies, such as ‘‘vesicles’’ of surfactants,
12
lipids, or block copolymers, in an IL medium. However, no
Synthetic vesicular membranes have long been used as mimics report is yet available wherein both the protein and surfactant
to understand the phospholipid bio-membranes synergy with could be dissolved in a common IL, and therefore, the binding
1–3
proteins. The vesicles are colloidal bilayer architectures formed by behavior of proteins with surfactants or surfactant-like ILs in an IL
surfactants upon aggregation beyond the critical aggregation medium is completely unexplored.
4
10
concentration. In living systems, the protein-biomembrane synergy
performs key functions like cell metabolism, signalling, and cell c (Cyt c) could be homogeneously dissolved in [C
Guided by the work of Malvika Bihari et al., wherein cytochrome
2
mim][C OSO ] with
2
3
1
gatekeeping and also act as drugs targets. This synergy has been the retention of peroxidase activity at room temperature and our own
mimicked in vitro for commercial utility with synthetic surfac- previous work on the formation of vesicles and reverse vesicles of an
13
tants as protein–surfactant colloidal formulations in pharma- IL surfactant in an IL medium, herein we report a novel colloidal
ceuticals, cosmetics, drug delivery, biochemical reactions, and formulation comprising Cyt c-[Cho][AOT]–[C mim][C OSO ] as the
2
2
3
5
very recently in bioelectronics. However, most of these for- constituent. The experimental details, materials, and dissolution
mulations are restricted to aqueous medium within the range characteristics of Cyt c in [Cho][AOT]–[C mim][C OSO ] are provided
of room temperature operation. in Fig. S1–S5 (ESI†).
The rise of ionic liquids (ILs—salts with melting points o100 1C ) Initially, we characterised the self-assembling behaviour of
as thermally stable green solvents liaising with an accessible [Cho][AOT] in [C mim][C OSO ] medium from the I /I vibronic
2
2
3
6
2
2
3
1 3
solvating ability and surface activity has generated new oppor- band ratio of pyrene as the polarity probe, Fig. 1A, and using
tunities to utilize them as a medium for the preservation of isothermal titration calorimetry (ITC), Fig. 1B.
biomolecules and to develop thermally stable surfactant–pro-
The I /I ratio (1.20) of pyrene in the native [C mim][C OSO ]
1 3 2 2 3
tein colloidal formulations. However, there are several hurdles lies between highly non-polar and polar solvents and therefore,
in the formulation of such colloidal systems in an IL medium indicates the presence of various bicontinuous microphases
7
14
due to (i) the limited solubility of proteins and surfactants, comprising polar/non-polar domains. Two transitions can be
À1
observed in Fig. 1A. The first transition at 5.5 mmol L (CAC
1
)
a
b
corresponds to the formation of pre-vesicular aggregates, wherein
some of the [C mim][C OSO ] molecules remain embedded in the
aggregates of [Cho][AOT]. Above 5.5 mmol L , the I1/I3 ratio
increases a bit before decreasing in a sigmoidal fashion before
becoming constant in the post-aggregation region. The constancy
AcSIR, CSIR-Central Salt and Marine Chemicals Research Institute,
Bhavangar-364002, India
2
2
3
À1
Salt and Marine Chemicals Division, CSIR-Central Salt and Marine Chemicals
Research Institute, Bhavnagar-364002, India. E-mail: arvind@csmcri.org;
Fax: + 91-278-2567562; Tel: +91-278-2567039
†
Electronic supplementary information (ESI) available: Materials, methods,
1 3
of the I /I ratio indicates that pyrene senses a uniform hydro-
phobic environment in the [Cho][AOT] aggregates. The second
purity specifications (Table S1) and experimental details (Fig. S1–S5), equations
used are given as annexure-I, detailed results. See DOI: 10.1039/c5cc05722a
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medium at a scattering angle of 901 and at 298.15 K was found
to be 164 nm, which is comparable to that of [Cho][AOT]
1
6
vesicles in water (150 nm). Upon comparing the intensity
autocorrelation function of the scattered light by [Cho][AOT]
aggregates in [C mim][C OSO ] and aqueous medium (Fig. S6,
2
2
3
ESI†), it was found that the decay time (t) in [C
t = 1.63 ms) is two order of magnitude higher than that in
water (t = 17.6 ms). Slow relaxation of the scattered light in
mim][C OSO ] can possibly be accounted for due to its high
2 2 3
mim][C OSO ]
(
[
C
2
2
3
17
viscosity (97.58 cP compared to 0.890 cP of water at 25 1C). In sight
of a more transparent understanding of the in situ aggregate size, we
performed multi-angle DLS measurements from 301 to 1501 (Fig. S7,
ESI†). The hydrodynamic diameter D increased from 130 nm to
h
6
00 nm as the angle varied from 301 to 1501, thus indicating
the polydispersed nature of the aggregates. In order to reveal
3
the morphology of the [Cho][AOT] aggregates in [C mim][C OSO ]
2
2
medium, we performed optical microscopy (Fig. 1D and Fig. S8,
ESI†). The microscopy images showed spherical droplets in the size
range of 0.1–1 mm. The droplets observed are the polydispersed
vesicles. Similar observation of formation of vesicles of an IL in an IL
medium was reported by us in our previous communication (Chem.
Fig. 1 (A) Pyrene I
spectra of pyrene in [C
titration calorimetric plot of [Cho][AOT] dilution in [C
dP plot as the inset. (C) D plot with corresponding autocorrelation function as
the inset. (D) Optical images of [Cho][AOT] vesicles in [C mim][C OSO
1
/I
3
vs. [Cho][AOT] conc. Curve along with the fluorescence
mim][C OSO ], shown as the inset. (B) Isothermal
mim][C OSO ], with the
2
2
3
2
2
3
h
13
2
2
3
]
2 2 3
Commun., 2013). [Cho][AOT] self-assemblies in [C mim][C OSO ]
medium (the black spots in the figure are due to lens artefacts).
were found to be kinetically stable at room temperature as no
precipitation or phase separation was observed for 10 months,
even at a concentration as high as 300 mmol L . The proposed
À1
À1
transition observed at 13.52 mmol L corresponds to the critical structure of the [Cho][AOT] vesicles in [C
2 2 3
mim][C OSO ] medium
vesicular concentration (CAC ). Such an aggregation behavior is depicted in Fig. S9 (ESI†).
2
has been previously observed for CTAB in [C mim]Br/H O and
We explored the application of [Cho][AOT]–[C mim][C OSO ]
2 2 3
colloidal system as a high temperature preservative for the
6
2
1
5
[
C
4
mim]Br/H
The ITC curve (Fig. 1B) also confirms the aggregation of enzyme Cyt c by investigating its binding behavior, conforma-
Cho][AOT] in the [C mim][C OSO ] medium. The negative tional stability, and functional activity. The binding isotherm of
mim][C OSO
that in the aggregation process of [Cho][AOT], van der Waal’s (Fig. 2A) was defined by subtracting the dilution enthalpogram
interactions dominate over the electrostatic interactions between of [Cho][AOT] in [C mim][C OSO ] medium from that of Cyt
Cho][AOT] and [C mim][C OSO ]. It is to be noted that the c-[C mim][C OSO ] solution (Fig. S10, ESI†). In surfactant–protein
2
O mixtures.
[
2
2
3
enthalpy changes observed from the enthalpogram indicates [Cho][AOT] to Cyt c (15 mM) dissolved in [C
2
2
3
]
2
2
3
13
[
2
2
3
2
2
3
Gordon parameter (G), an indicator of the solvophobic effect of a systems, the surfactant interacts with protein in different forms
1
1a
solvent to support self-assembly, is 0.846 for [C
which is higher than the 0.463 of [C mim] [Tf N] reported earlier the mid-concentration region, and as micelle or vesicles, post
to support the self-assembly of [ProC
solvophobicity of [C mim][C OSO ] can be accounted as one of an endothermic process, whereas the binding of [Cho][AOT],
the reasons to support the self-assembly of [Cho][AOT]. The Gibbs aggregates, and vesicles to cytochrome is an exothermic process
2
mim][C
2 3
OSO ],
(monomers at low concentration, protein-induced aggregates in
4
2
13
18
3
][LS]. Therefore, the good CAC). The binding of [Cho][AOT] to Cyt c at low concentration is
2
2
3
ꢀ
À1
free energy of aggregation, DG , (À14.4 kJ mol ) was calculated with a small change in enthalpy. It is to be noted that small enthalpy
using the mass action model (Annex 1, ESI†) by considering the
ionisation degree of aggregation (a) as 1 due to the exclusively
ionic shell surrounding the aggregates in the IL medium.
agg
changes upon binding to proteins occur when the interactions are
19
electrostatic in nature. Therefore, the small enthalpy changes
indicate that [Cho][AOT] binds to Cyt c mainly through electrostatic
19
ꢀ
interactions.
A negative value of DG indicates the feasibility of [Cho][AOT]
agg
Alterations in the structure of Cyt c upon [Cho][AOT] binding
at different concentrations were measured from the changes in
UV-Vis spectra (Fig. 2B). In the neat [C mim][C OSO ], Cyt c
2 2 3
showed an intense Soret band at 408 nm and weak Q bands at
self-assembly in [C
2
2 3
mim][C OSO ]. The entropic contribution
ꢀ
ꢁ
ꢀ
ꢀ
TDS
to the DG
was determined from the standard
agg
agg
Gibbs free energy equation (Annex 1, ESI†) and was found to
À1
ꢀ
ꢀ
be 14.82 kJ mol . The high TDS value compared to DH
agg
agg
528 and 556 nm due to p–p* transitions in the heme group
À1
10a
(
À0.42 kJ mol ) indicates that the aggregation process is buried deep inside the hydrophobic core of the protein. Upon
mainly entropy driven.
The formation of aggregated structures was confirmed from c was observed in low concentrations, followed by a continuous
the addition of [Cho][AOT], a slight increase in absorbance of Cyt
DLS measurements (Fig. 1C). The hydrodynamic diameter (D
h
) of decrease in absorbance at higher concentrations. The decrease
À1
the [Cho][AOT] (50 mmol L ) aggregates in the [C mim][C OSO ] in absorbance (hypsochromic shift) occurs due to the lowering in
2
2
3
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samples outside the spectrophotometer using a silicon oil bath.
The CD and UV-Vis spectra of the incubated samples were then
obtained. The comparative CD and UV-Vis spectra of Cyt c
dissolved in [C mim][C OSO ] and [Cho][AOT]–[C mim][C OSO ]
2
2
3
2
2
3
measured at different temperatures and time periods are pro-
vided in Fig. S12 and S13 (ESI†). Retention of the CD Soret band
at 408 nm and the UV Soret and Q bands indicated the structural
stabilisation of Cyt c around the heme cleft. It has been observed
that Cyt c remains stable up to 180 1C for a maximum of 5 min,
whereas at 130 1C it remains stable for a time period of 1 h.
These conclusions were arrived at by checking the structural
stabilisation of Cyt c for more than 1 h at 130 1C and for 1 h at
140 1C, wherein Cyt c was found to lose its activity. It has also
been observed that the time period for structural stabilisation of
Cyt c increased with the decrease in temperature, as evident from
its spectra at 100 1C after 6 h of heating (Fig. S12A, B and S13A,
ESI†). Comparison of the stabilisation of Cyt c in [C
2 2 3
mim][C OSO ]
Fig. 2 (A) Binding isotherm of [Cho][AOT] binding to Cyt c (15 mM) in neat
and [Cho][AOT]–[C mim][C OSO ] solutions showed that Cyt c
2
2
3
[
[
[
C
2
mim][C
Cho][AOT] concentrations; (C and D) Mid-UV CD spectra of Cyt c in
Cho][AOT] (100 mM) + [C mim][C OSO ].
2 3
OSO ]; (B) UV-Vis spectra of Cyt c (15 mM) in different
remains more stable in water, which is evident from the higher
ellipsity of the CD Soret band at 408 nm (Fig. S12, ESI†) and the
higher absorbance of the UV Soret and Q bands (Fig. S13, ESI†).
Therefore, the [Cho][AOT] vesicles imparted extra thermal stability
2
2
3
energy of the p* orbital upon exposure to the surrounding polar to Cyt c in the colloidal formulation.
environment, which is possible due to the unfolding of the protein The activity of Cyt c (30 mM) in [C mim][C
around the heme cleft. Though an unfolding was observed around [C mim][C OSO ] was investigated by incubating the Cyt c solutions
2
2 3
OSO ] or [Cho][AOT]–
2
2
3
the heme cleft, unlike the aqueous formulation, the oxidation state of for 10 min in the temperature range from 60 to 180 1C. After
Cyt c did not change at any concentration, as indicated by the incubation, the catalytic activity was observed using guaiacol as a
retention of Q bands at 528 and 556 nm. Due to the absorption of substrate and H O as an oxygen donor at room temperature. Cyt c
2
2
imidazolium in the far UV-region, we could not observe the secondary catalyses the oxidation of guaiacol to tetraguaiacol, giving an orange
structure of Cyt c from the CD-measurements. However, when we colour and showing an absorption band at 470 nm (Fig. S14 and
20
compared the mid-UV CD spectra, the bands due to Tyr and Trp were S15, ESI†). Qualitatively, the activity was monitored by visual
observed at 263 and 287 nm, but the Phe band was missing (Fig. S11, inspection of the appearance of the orange colour (Fig. 3).
ESI†). Therefore, compared to water, the Tyr and Trp residues
Cyt c retained its functional activity up to 180 1C for a maximum
experienced a more non-polar environment, which can be due to of 5 min and 160 1C for a maximum of 10 min, both in neat
14
the presence various non-polar domains in [C mim][C OSO ]. Upon [C mim][C OSO ] and [Cho][AOT]–[C mim][C OSO ] vesicular
2
2
3
2
2
3
2
2
3
the addition of 100 mM [Cho][AOT], a bathochromic shift of 4 nm solution. 180 1C is considered as the highest activity temperature
in the band at 263 nm and a hypsochromic shift of 2 nm in the band on account of the fact that the maximum product formation
at 287 was observed; this indicated that the conformation of the occurs within five minutes of the reaction inception (Fig. S16,
protein in the vesicles is such that Trp, which is present around the ESI†). Quantitatively, the tetraguaiacol formation was calculated
heme pocket, faces the hydrophobic tail part of the vesicle. The rise in from the absorption changes at 470 nm and the molar extinction
4
À1 À1
ellipsity (y) of the Tyr and Trp residues indicated the stabilisation of coefficient of 2.66 Â 10 cm .M (Fig. S17, ESI†). The activity
the tertiary structure of the protein. of Cyt c in the [Cho][AOT]–[C mim][C OSO ] vesicular solutions
2
2
3
We also checked the thermal stability of Cyt c in the vesicular was found to be B2-fold higher than that observed in neat
regime by measuring the temperature-dependent CD spectra of [C mim][C OSO ] at all temperatures (Fig. S18A, ESI†).
2
2
3
the Tyr and Trp residues (Fig. 2C) and the Soret band at 408 nm
Fig. 2D). The band due to Tyr at 267 nm disappeared with the
(
rise in temperature, which indicated conformational changes in
the protein backbone, as three of the Tyr residues are usually
present in the protein backbone. The overall y of the bands
(
at 286 nm due to Trp and the Soret band at 408 nm) increased
with their respective signs upon increasing the temperature from
0 to 95 1C without deformation of the CD bands, which
3
indicated the thermal stability of the protein’s tertiary structure
in the region around the heme cleft. Because of the inbuilt
temperature limitations of CD and UV-Vis spectrophotometers,
Fig. 3 Functional activity of Cyt
c
in [C
2
mim][C
2
OSO
3
]
and
2 2 3
the measurements above 100 1C were carried out by heating the [Cho][AOT](100 mM)–[C mim][C OSO ] from 60 to 180 1C.
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The higher activity in vesicular solution can be attributed to preservation of enzymes and as enzymatic catalytic reactors at
i) exposure of the catalytic iron centre of the enzyme due to a elevated temperatures.
(
1
0a
slight perturbation in conformation around the heme cleft
Financial support from DST, India for this work (No. SB/S1/
as observed from the UV-Vis and CD spectra (Fig. 2B–D), (ii) the PC-104/2012) is acknowledged. We thank the Centralized
higher thermal stability imparted by [Cho][AOT] vesicles in the Instrumental Facility, Dr. Santlal Jaiswar, Praveen Singh Gehlot
2 2 3
[Cho][AOT]–[C mim][C OSO ] system (Fig. S12 and S13, ESI†). and Damarla Krishnaiah for assistance in various capacities.
Since Cyt c retained its activity in buffer solution until 80 1C, we
did a comparative analysis of the activity of Cyt c in buffer, in neat Notes and references
2 2 3 2 2 3
[C mim][C OSO ] and in [Cho][AOT] (100 mM)–[C mim][C OSO ]
vesicular solutions by incubating the enzyme at 80 1C (Fig. S18B,
ESI†). It was found that the activity of Cyt c in [Cho][AOT]–
1
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C mim][C OSO ] vesicular solution after 5 min of incubation
2 2 3
is B4 fold higher than in buffer and B2-fold higher than in
neat [C mim][C OSO ]. The initial rates of enzymatic reactions
2
2
3
were calculated from the maxima of the first derivatives of the
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2
2
3
À1
4
-fold higher than that in neat [C
and 13-fold higher than in buffer (12.17 mM min ). A high
activity of Cyt c has earlier been reported in ILs ([C mim][Tf N],
mim][PF ] and in [C mim][PF ]) solutions of methanol/crown
2 2 3
mim][C OSO ] (39.2 mM min )
9
R. M. Vrikkis, K. J. Fraser, K. Fujita, D. R. MacFarlane and
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1
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maximum temperature (130 1C) of stabilisation for 1 h and at 100 1C
for 6 h, wherein Cyt c was found to be redox active (Fig. S19, ESI†).
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1
1
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23
one of the reason for protein solubilisation and stabilisation.
b
1
1
of [C mim][C OSO ] is 0.71 and therefore must be one of the
2
2
3
reasons for Cyt c solubilisation and stabilisation. Furthermore,
the decrease in stabilisation time with temperature can be
1
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24
[
C OSO ] anion begins to happen, thus introducing the volume
2 3
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´
´
1
effects towards structural and functional stability.
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2
2
3
] is a unique IL, which 18 D. Otzen, Biochim. Biophys. Acta, 2011, 1814, 562–591.
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2
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2
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b = 0.71, and good solvophobicity (G = 0.846) of [C mim][C OSO ] can
2
2
3
be accounted for as reasons for Cyt c’s solubility and [Cho][AOT] self-
assembly in [C mim][C OSO ]. Cyt c thus dissolved in [Cho][AOT]–
2
2
3
2
2
3 K. Tamura, N. Nakamura and H. Ohno, Biotechnol. Bioeng., 2012,
2 2 3
[C mim][C OSO ] vesicular solutions retains its functional activity up
to very high temperatures for different time periods. The results
demonstrate the potential of IL colloidal solutions as media for the
109, 729–735.
4 A. Fern ´a ndez, J. S. Torrecilla, J. Garc ´ı a and F. Rodr ´ı guez, J. Chem.
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