cytochrome c in ionic liquids colloidal formulation

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

mailto:arvind@csmcri.

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


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


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


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.


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


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.


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.


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.


Abso

rban

ce

/ nm360 420 480 540 600 660 720 780

0.0

0.5

1.0

1.5

2.0

2.5

(B)


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.


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.


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.

cytochrome c in ionic liquids colloidal formulation

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