cytochrome c in ionic liquids colloidal formulation
TRANSCRIPT
1
Unusually High Thermal Stability and Peroxidase Activity of Cytochrome c in Ionic Liquids Colloidal Formulation
Pankaj Bharmoria,a Arvind Kumara,b*
aAcSIR, CSIR-Central Salt and Marine Chemicals Research Institute.
bSalt and Marine Chemicals Division, CSIR-Central Salt and Marine Chemicals Research Institute,
Bhavnagar-364002, India. Fax: +91-278-2567562.; Tel: +91-278-2567039;
E-mail: [email protected]
Supporting Information
1. Materials, Methods and Experimental Details 1.1. MaterialsIonic Liquid (IL), 1-Ethyl-3-methylimidazolium ethylesulfate ([C2mim][C2OSO3]) with
stated purity higher than >95% mass fraction was purchased from Sigma Aldrich. Choline
dioctylsulfosuccinate [Cho][AOT] was synthesized in our laboratory. All the chemicals used
for synthesis of ILs were of AR Grade. Choline Chloride (>98%) was purchased from Sigma
Aldrich. Sodium dioctylsulfosuccinate (>99 %) was purchased SDFCL. Brief procedure of
synthesis is as follows: Equimolar amounts of choline chloride and [Na][AOT] were
dissolved in water and stirred for 24 h at room temperature. The progress of the reaction was
monitored by TLC. After completion of the reaction the product was extracted into DCM
layer, followed by washing of DCM layer with water for several times to completely remove
the chloride ion present. The washing was performed until the aqueous layer gave a clear
solution even with the addition of excess 1M AgNO3. The DCM layer is then distilled to get
pure [Cho][AOT]. Further, [Cho][AOT] is dried under vacuum for several hours to remove
the moisture present and stored in desiccator. The synthesized [Cho][AOT] was characterized
by using 1HNMR (Fig. S1), LC-MS, CHNS and DSC (Fig. S2).
Electronic Supplementary Material (ESI) for Chemical Communications.This journal is © The Royal Society of Chemistry 2015
2
1H-NMR: 200MHz (DMSO-d6): δH (ppm) 0.86 (m, 11H), 1.24 (m, 15H), 1.49 (m 2H), 2.85 (m, 2H), 3.10 (m, 5H), 3.34 (s, 9H), 3.65 (m, 1H), 3.88 (m, 5H), 5.26 (t, 1H)
ESI-MS: [C5H14NO]+ m/z:104.14, [C20H37O7S]- m/z = 421.2208.
CHNS Analysis:
Atom Theoretical Experimental
C 57.11% 56.20%
H 9.78% 9.043%
N 2.66% 2.59%
S 6.10% 5.77%
Fig. S1. 1H-NMR spectra of [Cho][AOT]
3
Melting point of [Cho][AOT] was checked using differential scanning calorimetry (DSC)
and was found to be -47.4oC (Fig. S2).
Table S1. Purity Specification of Ionic Liquids
Ionic Liquid Purity (mass fraction)
Chloride Content (ppm)(Volhard Method)
Moisture content (ppm)(Karl Fischer Titration)
[C2mim][C2OSO3](Sigma Aldrich)
95%* < 25 ppm < 75 ppm
[Cho][AOT](Synthesized)
99% < 10 ppm < 50 ppm
* [C2mim][C2OSO3] was further purified by washing with toluene several times. The volatile
impurities were removed by heating the sample at 80oC in a rotary evaporator at zero vacuum
for 24 h which also ensured removal of moisture. The purity of ionic liquids was determined
by using 1H NMR.1
The water content of the ionic liquid was measured from Karl Fischer Titration. The halide
contents of ionic liquids were measured using the standard Volhard titration method.1
Fig. S2. Differential scanning calorimetric plot of [Cho][AOT].
4
Enzyme Cyt c from horse heart was purchased SRL Pvt. Ltd. and was characterized for
purity using spectroscopic techniques. The prominent bands at 409, 528 and 549 nm in the
UV-Vis spectra and 40.1% α-helical content in far-UV CD spectra in Millipore water and
phosphate buffer pH 7.0 validated the purity of enzyme (Fig. S3).
Guaiacol with stated purity of 98% was purchased from Sigma Aldrich. Hydrogen peroxide
(H2O2) 30% (w/v) was purchased from Fischer Scientific. Buffer solution (50 mM, phosphate
buffer) for activity analysis was prepared in degassed Millipore grade water using AR-grade
potassium di-hydrogen phosphate (99%) and di-potassium hydrogen phosphate (99%)
purchased from sd fine-chem Ltd.
Molecular Structure of the studied ionic liquids and structure of Cyt c with amino acid
sequences are given in below. Structure of Cyt c is regenerated from reference 2 with
permission.2
N+
N
SO OO
O-
[Cho][AOT][C2mim][C2OSO3]
O OOO
S O
O-O
HO
N+
190 200 210 220 230 240 250-20
-15
-10
-5
0
5
10
15
20
25
CD /
(mde
g)
/ nm
Water Buffer
(A)300 360 420 480 540 600 660
-0.20.00.20.40.60.81.01.21.41.61.82.0
Abso
rban
ce
/ nm
Water Buffer
(B)
Fig. S3 (A) Far-UV CD spectra of Cyt c (B) UV-Vis-NIR spectra of Cyt c in
water and phosphate buffer solution.
NHCO
CH3
SS
FeMET80
SN
N HIS18
TRP59
TYR
CYS
THR THRTHR
PHEGLY
PRO
ALA GLN
GLY
THRLYS
ARGGLYPHE
GLYHIS
LEU
LEU
ASNPRO
GLY THR
LYS HIS
LYSGLY
GLY
LYS
GLU
GLU
THRVAL
GLN
ALACYSLYS
GLN
VALPHE
ILE
LYSLYS GLY
LYS
GLU
GLU
VAL
ASP
ASPGLY
ASP
ALA
ASN
ASN
LYSASNLYS
GLY
ILE
LYSGLU
THR
LEUMET
GLU
TYR
LEUGLU
ASNPROLYS LYS
TYR
ILEPRO
GLY
TYR LYS
ILEPHE
ALAGLY
ILE
LYSLYS
LYS
THR
GLUARG
LEU
ILE
ALATYR
LEU
LYS
LYS
ALA
THR
GLUCOOH
10
15
20
5
2530
4045
35
50
55
60
65
70
75
85
90
95
100
Cyt c
5
1.2.Methods
1H NMR: The 1H NMR spectra for both the pure ILs and their mixtures were recorded using
a Brüker 500MHz spectrophotometer. The proton chemical shifts were referenced with
respect to an external standard TMS (δ = 0.000 ppm) in C6D6 (deuterated benzene).
ESI-MS: Electrospray ionization mass spectrometry (ESI-MS) was done with Q-Tof
microTM 23 Micromass, U.K.
Differential Scanning Calorimetry (DSC): The melting point of [Cho][AOT] was
determined by using differential scanning calorimetry measurements using a Mettler Toledo
DSC822 thermal analyser at a scan rate of 5oC/minute. Uncertainty in DSC measurements
has been found to be ±0.06%.
CHNS Analysis: CHNS analysis of [Cho][AOT] was carried out using CHNS analyser
(Elementer Vario Micro cube). The uncertainty in measurements is found to be ±0.6.
Karl-Fischer Analaysis: The Karl-Fischer analysis was done in Mettler Toledo DV705, T50
Autotitrator using dry methanol as solvent and Karl-Fischer reagent. The moisture content of
[C2mim][C2OSO3] and [Cho][AOT] were found to be, < 75 ppm and < 50 ppm.
Pyrene Fluorescence: Pyrene fluorescence spectra in [C2mim][C2OSO3] as a function of
[Cho][AOT] concentration were taken in Fluorolog (Horiba Jobin Yvon) spectrometer. In a
typical experiment 20 μl of 10-4 M pyrene solution in DCM was taken in a quartz cuvette.
The DCM was evaporated at room temperature for 30 minutes followed by addition of 2 ml
of [C2mim][C2OSO3]. The solution was then stirred for 12 hours for complete mixing. The
emission spectra of pyrene were recorded at with continuous addition of 75 nmex 334
mM [Cho][AOT]. At least 4 spectra were recorded at each concentration after solution
stirring for 5 minutes. The final I373 / I383 value was taken as an average of four recorded
spectra.
Isothermal Titration Calorimetry (ITC): Enthalpy changes (dH) due to aggregation of
[Cho][AOT] in [C2mim][C2OSO3] and Binding to Cytc was measured using MicroCal
ITC200 microcalorimeter, with an instrument controlled Hamiltonian syringe having volume
capacity of 40 μL. The titration was done by adding 2μL aliquots of [Cho][AOT] stock
solution into sample cell containing 200 μL [C2mim][C2OSO3] or 15 μM Cyt c solution in
[C2mim][C2OSO3] with continuous stirring (500 rpm). The parameters like time of addition
and duration between each addition were controlled by software provided with the
instrument. The enthalpy change at each injection was measured and plotted against
concentration by using origin software provided with the instrument. The concentrations of
6
stock solutions taken were 75mM of [Cho][AOT] in [C2mim][C2OSO3]. The Uncertainty in
ITC measurements was found to be ±0. 5%.
Dynamic Light Scattering (DLS): DLS measurements were performed at 298.15 K, using a
NaBiTec SpectroSize300 light scattering apparatus (NaBiTec, Germany) with a He−Ne laser
(633 nm, 4 mW). The hydrodynamic measurements were carried out in quartz cuvette of 1
cm path length. For accurate measurement both viscosity and refractive index of the samples
were considered. The analysis was carried out at multi-angle (30o to 150o) to validate the
accuracy of the results. The error in the measurements was found to be ±10 nm.
Optical Microscopy: Bright field optical microscopic images of [Cho][AOT] colloidal
particles in [C2mim][C2OSO3] were taken from AXIO Imager M1 Carl Zeiss (Germany)
Light Microscopy equipped with AxioCam HRc. Images were recorded at the magnification
of 10 x 100X.
UV-Vis Spectroscopy: The changes around the heme cleft of Cyt c upon binding with
[Cho][AOT] in different concentration regimes in [C2mim][C2OSO3] were measured using a
UV 3600 Shimadzu UV-vis-NIR spectrophotometer at 298.15 K. The concentrated solution
of [Cho][AOT] was titrated against a 15 μM Cytochrome c solution in a quartz cuvette of 1
cm path length. The measurements were taken 3 minutes after addition of [Cho][AOT] at
each concentration in order to match the time gap of the energy changes after each addition in
the ITC analysis. The temperature dependent UV-Vis studies were carried out by heating the
sample solutions {Cyt c (30 μM) dissolved in [C2mim][C2OSO3] or [Cho][AOT] (100
mM)+[C2mim][C2OSO3]} in silicon oil bath on a hot plate equipped with temperature sensor
for a specific time period and subsequently measuring the spectra. Prior to the measurements
the baseline was corrected using neat [C2mim][C2OSO3] as blank.
Circular Dichroism Spectroscopy. Alterations in the tertiary structure and region around
heme cleft of Cyt c in vesicles of [Cho][AOT] at different temperatures were monitored using
a Jasco J-815 CD spectrometer from 30oC to 95oC. The temperature of the measurement cell
was controlled with a Julabo water thermostat within ±0.1 K. Spectra were collected in a 1
mm path length quartz cuvette at a scan rate of 100 nm per minute and a sensitivity of 100
mdeg. The response time and the bandwidth were 2 s and 0.2 nm, respectively. For
temperature greater than 95oC the measurements were carried out by heating the sample
solutions {Cyt c (30 μM) dissolved in [C2mim][C2OSO3] or [Cho][AOT] (100
mM)+[C2mim][C2OSO3]} in silicon oil bath on a hot plate equipped with temperature sensor
for a specific time period and subsequently measuring the spectra.
7
1.3. Experimental Details: Cyt c was dissolved in [C2mim][C2OSO3] by stirring for 3-4 days
at 60oC. The concentration of Cyt c used for this work was from 15μΜ to 30μΜ. The images
of Cyt c dissolved in [C2mim][C2OSO3] and [Cho][AOT] vesicles in [C2mim][C2OSO3] are
shown in Fig. S4. Cyt c did not precipitate out even at a concentration as high as 300 mM.
The retention of the structure in [C2mim][C2OSO3] was confirmed from UV-Vis experiments
(Fig. S5).
Activity Assay of Cyt c: The thermal activity of Cyt c in [C2mim][C2OSO3] or
[Cho][AOT] (100mM)-[C2mim][C2OSO3] was investigated by incubating the Cyt c solution
for 10 minutes at different temperatures (60-180oC) and observing the catalytic activity using
guaiacol as substrate and H2O2 as an oxygen donor at room temperature. In a typical
experiment the 100 μL solution of 30 μΜ Cyt c in buffer, [C2mim][C2OSO3] or
[C2mim][C2OSO3][Cho][AOT] (100 mM) was taken in glass vial and heated at different
(A) (B)
Fig S4. (A) Native Cyt c in [C2mim][C2OSO3]. (B) 15μΜ Cyt c +300
mM [Cho][AOT] in [C2mim][C2OSO3]
350 400 450 500 550 600 650
0.0
0.3
0.6
0.9
1.2
1.5
1.8
556 nm526 nm
(A)
Abs
orba
nce
/ nm
408 nm
Fig S5. UV-Vis spectra of native Cyt c (15μΜ) in
[C2mim][C2OSO3].
8
temperatures for 10 minutes. It was followed by the addition of 900 μL buffer solution + 100
μL of guaiacol (20 mM) + 100 μL of H2O2 (50 mM). H2O2 was added in the end as it can also
oxidize the enzyme. It was followed by the UV-Vis measurement which could be started after
minimum of 30 seconds due to the instrumental formalities to start the experiment like
baseline correction and sample code etc. Cyt c catalyzes the oxidation of guaiacol to
tetraguaiacol which gives orange colour and show absorption band at 470 nm. The activity at
100oC for 6 hrs. and at 130oC for 1 hrs. was measured by heating the samples for a specific
period followed by mixing of 100 μL sample with 900 μL buffer solution + 100 μL of
guaiacol (20 mM) + 100 μL of H2O2 (50 mM). The activity was observed qualitatively from
the appearance of orange colour.
Thermal stability of Cyt cThe thermal stability of the Cyt c in both [C2mim][C2OSO3] and [Cho][AOT] (100mM)+
[C2mim][C2OSO3] was measured from CD and UV-Vis spectra. Since the in situ CD spectra
can be measured only up to 95oC, we have carried out measurements above 95oC by heating
the sample solutions {Cyt c (30 μM) dissolved in [C2mim][C2OSO3] or [Cho][AOT] (100
mM)+[C2mim][C2OSO3]} immersed in silicon oil bath on a hot plate, equipped with
temperature sensor for a specific time period and subsequently measuring the spectra. Similar
procedure was followed to measure the UV-Vis spectra.
Annexure 11. Standard Gibbs free energy of calculation using mass action model is given by equation
1.3
)1..(..............................ln)2( CACoagg xRTG
In the equation 1 is standard free energy of aggregation, α is free energy of counterion oaggG
dissociation, R is gas constant, T is temperature and x is the mole fraction of the [Cho][AOT]
at critical aggregation concentration.
2. The entropic contribution to the free energy of aggregation was calculated from standard
Gibbs free energy equation.
)2.(..............................oagg
oagg
oagg HGST
Where is standard entropy of aggregation is standard enthalpy of aggregation oaggS o
aggH
calculated from ITC experiments.
9
3. )3....(........................................2
sin4 rnq
Where q is scattering vector of light, λ is wavelength of laser light, nr is refractive index
of solution, θ is scattering angle.
2. Results
Fig. S6 Comparative autocorrelation functions of [Cho][AOT] vesicles in (A)
Water and (B) [C2mim][C2OSO3]. Red symbols in (B) showing intensity
autocorrelation function of native [C2mim][C2OSO3] which is indicating that
[C2mim][C2OSO3] do not contribute to the scattered light by vesicles
1E-6 1E-5 1E-4 1E-3 0.01 0.1
1.0
1.1
1.2
1.3
1.4
1.5
1.6
1.7
= 17.6 s
(A)
g(1) ()
log10()
ChoAOT Vesicles in Water1E-6 1E-5 1E-4 1E-3 0.01 0.1
0.9
1.0
1.1
1.2
1.3
1.4
1.5
1.6
= 1.63 ms
Pure [C2mim][C2OSO3]
g(1) ()
log10 ()
[Cho][AOT] in [C2mim][C2OSO3]
(B)
20 40 60 80 100 120 140 160
50
100
150
200
250
300
Variation in Hydrodynamic Radii at different angle
R h / n
m
Scattering Angle () / o
(B)1E-8 1E-7 1E-6
0.0
0.2
0.4
0.6
0.8
1.0
Norm
alize
d In
tens
ity
Rh / m
Angle / o 30 45 60 75 90 105 120 135 150
(A)
Fig. S7 (A) Hydrodynamic radii profile of [Cho][AOT] aggregates at
different angle. (B) Hydrodynamic radii vs angle plot.
10
Increase in aggregate size with an increase in scattering angle in ILs is an unusual observation since the scattering vector (equation 3 annexure 1 ESI†) has the units of rad.m-1
so the large size must be observed at small angle which is true for [Cho][AOT] aggregation in water.4 Such an unusual angular dependence of aggregate size in IL medium needed to be explored by some expert photophysicist.
5μm
Fig. S8 (A) Optical microscopic images of polydispersed [Cho][AOT]
vesicles in [C2mim][C2OSO3].
Fig. S9 Proposed structure of [Cho][AOT] vesicle in [C2mim][C2OSO3] medium.
11
0 10 20 30 40 50 60
-16-14-12-10-8-6-4-202
dP / W
atts
Time / minute
0 2 4 6 8 10 12 14 16-2.0
-1.5
-1.0
-0.5
0.0
0.5
1.0
dH /
kJ.m
ol-1
C / mmol.L-1
250 260 270 280 290 300
-0.8
-0.6
-0.4
-0.2
0.0
0.2
0.4
0.6
Trp
Phe
Cyt c in Water Cyt c in EmimEtSO4 Cyt c in 100 mM ChoAOT in EmimEtSO4
CD /
mde
g
/ nm
Tyr
Fig. S10 Enthalpograms of [Cho][AOT]
aggregation in Cyt c solution along with
dP plot.
Fig. S11 Comparative mid-UV CD
spectra of Cyt c in water,
[C2mim][C2OSO3] and vesicles.
12
Fig. S12. CD spectrum of Cyt c in [C2mim][C2OSO3]; and in
[Cho][AOT] (100mM) in [C2mim][C2OSO3] in the near UV and Far-
VISIBLE regions after heating at different temperatures and time
periods (A) 100oC for 6 h (B) 130 oC for 1 h (C) 160 oC for 10 min
and (D) 180oC for 5 min.
380 390 400 410 420 430 440 4500.0
0.5
1.0
1.5
2.0
2.5 At 130 oC for 1 hour
Cyt c in [C2mim][C2OSO3] Cyt c in [Cho][AOT]+[C2mim][C2OSO3]
CD
/ m
deg
/ nm
(D)
250 260 270 280 290 300 310-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
Cyt c in [C2mim][C2OSO3] Cyt c in [Cho][AOT]+[C2mim][C2OSO3]
At 100 oC for 6 hours
CD
/ m
deg
/ nm
(A)
380 390 400 410 420 430 440 4500.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Cyt c in [C2mim][C2OSO3] Cyt c in [Cho][AOT]+[C2mim][C2OSO3]
At 100 oC for 6 hours
CD
/ m
deg
/ nm
(B)
250 260 270 280 290 300-1.0
-0.5
0.0
0.5
1.0
1.5
2.0At 130 oC for 1 hour
Cyt c in [C2mim][C2OSO3] Cyt c in [Cho][AOT]+[C2mim][C2OSO3]
CD
/ m
deg
/ nm
(C)
380 390 400 410 420 430 440 450
-0.5
0.0
0.5
1.0
1.5
2.0
2.5 At 160 oC for 10 min.
Cyt c in [C2mim][C2OSO3] Cyt c in [Cho][AOT]+[C2mim][C2OSO3]
CD
/ m
deg
/ nm
(F)
250 260 270 280 290 300-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
Cyt c in [C2mim][C2OSO3] Cyt c in [Cho][AOT]+[C2mim][C2OSO3]
At 160 oC for 10 min.
CD
/ m
deg
/ nm
(E)
380 390 400 410 420 430 440 450
-0.5
0.0
0.5
1.0
1.5
2.0
2.5 At 180 oC for 5 min.
Cyt c in [C2mim][C2OSO3] Cyt c in [Cho][AOT]+[C2mim][C2OSO3]
CD
/ m
deg
/ nm
(H)
250 260 270 280 290 300-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0At 180 oC for 5 min.
Cyt c in [C2mim][C2OSO3] Cyt c in [Cho][AOT]+[C2mim][C2OSO3]
CD
/ m
deg
/ nm
(G)
13
Fig. S13. UV-Vis spectra of Cyt c in [C2mim][C2OSO3]; and in [Cho][AOT]
(100mM) + [C2mim][C2OSO3] after heating at different temperatures and
time periods (A) 100 oC for 6 hrs. (B) 130 oC for 1 hrs. (C) 160 oC for 10 min
and (D) 180 oC for 5 min
360 420 480 540 600 660 720 7800.0
0.5
1.0
1.5
2.0
2.5
3.0
(A)
After heating at 100 oC for 6 hrs.
Cyt c in [C2mim][C2OSO3] Cyt c in [Cho][AOT]+[C2mim][C2OSO3]
Abso
rban
ce
/ nm360 420 480 540 600 660 720 780
0.0
0.5
1.0
1.5
2.0
2.5
(B)
Cyt c in [C2mim][C2OSO3] Cyt c in [Cho][AOT]+[C2mim][C2OSO3]
After heating at 130 oC for 1 hour
Abso
rban
ce
/ nm
360 420 480 540 600 660 720 780
0.0
0.5
1.0
1.5
2.0
2.5
After heating at 160 oC for 10 min.
Cyt c in [C2mim][C2OSO3] Cyt c in [Cho][AOT]+[C2mim][C2OSO3]
Abso
rban
ce
/ nm
(C)360 420 480 540 600 660 720 780
-0.20.00.20.40.60.81.01.21.41.61.8
After heating at 180 oC for 5 min.
Cyt c in [C2mim][C2OSO3] Cyt c in [Cho][AOT]+[C2mim][C2OSO3]
Abso
rban
ce
/ nm
(D)
14
420 455 490 525 560 595
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
After Incubation For 5 minutes at 80oC
Abso
rban
ce
/ nm
Buffer [C2mim][C2OSO3] [Cho][AOT]+[C2mim][C2OSO3]
470 nm
450 475 500 525 550
0.2
0.3
0.4
0.5
0.6
0.7
(A)
600 s
Abso
rban
ce
/ nm
30 s
100 200 300 400 500 600
0.35
0.42
0.49
0.56
0.63
0.70
(B)
A 47
0 nm
Time / sec.
Steady phase
Fig. S16 (A) UV-Vis spectra of tetraguaiacol at room temperature for
10 min. (B) Changes in absorbance of tetraguaiacol at 470 nm at
different time intervals for 10 min.
Fig. S15. Comparative UV-Vis spectra of tetraguaiacol
after enzyme incubation at 80oC for 10 min and 5 min
after the start of reaction in various solvent media.
Fig. S14. Peroxidase reaction of tetraguaiacol formation from guaiacol
15
1 2 3 4 58
10
12
14
16
18
20
22
24
[C2mim][C2OSO3]
60oC 80oC 100oC 120oC 140oC 160oC 180oC
Tetra
guai
acol
/ M
Time / minute
(A)
1 2 3 4 5
15
20
25
30
35
40
45 (B)
60oC 80oC 100oC 120oC 140oC 160oC 180oC
Tetra
guai
acol
/ M
Time / minute
[Cho][AOT]+[C2mim][C2OSO3]
Fig. S17. Plots showing the amount of tetraguaiacol formed in (A)
[C2mim][C2OSO3]; (B) in [Cho][AOT]-[C2mim][C2OSO3] vesicular
solution at various temperatures
0 1 2 3 4 50
7
14
21
28
35
42
49
Buffer [C2mim][C2OSO3] [Cho][AOT]+[C2mim][C2OSO3]
Tetra
guia
col /
m
ol.L
-1
Time / minute
(B)
60 80 100 120 140 160 18010
15
20
25
30
35
40
45
[C2mim][C2OSO3] [Cho][AOT]+[C2mim][C2OSO3]
Tetra
guai
acol
/ M
T / oC
(A)
Fig. S18. (A) Temperature dependent activity of Cyt c (30 µM) in neat
[C2mim][C2OSO3] and [Cho][AOT] (100mM) + [-[C2mim][C2OSO3] solutions; (B)
activity of Cyt c in buffer, neat [C2mim][C2OSO3] and
[Cho][AOT](100mM)+[C2mim][C2OSO3] vesicular solutions after incubation at 80oC.
16
References:
1. A. Stark, P. Behrend, O. Braun, A. Muller, J. Ranke, B. Ondruschka, B. Jastorff, Green Chem., 2008, 10, 1152.
2. G. C. Mills, J. Theor. Biol., 1991, 152, 177-190.3. K. C. Tama and E. W. Jones, Chem. Soc. Rev., 2006, 35, 693-709.4. P. Bharmoria, T. J. Trivedi, A. Pabbathi, A. Samanta, A. Kumar, Phys. Chem. Chem.
Phys. 2015, 17, 10189-10199.
(A) (B) (C) (D)
Fig. S18. Images showing functional activity of Cyt c in
[C2mim][C2OSO3] (A), (C) and in [Cho][AOT]-[C2mim][C2OSO3]
vesicles (B), (D) at incubation at 130oC for 1 h and 100oC for 6 h.
At 130oC for 1 hrs. At 100oC for 6 hrs.