Effects of charge balance and hydrophobicity of the surface of cytochrome: C on the distribution behaviour in an ionic liquid/buffer biphasic system

Article abstract of DOI:10.1039/c9ob00900k

Factors contributing to the different distribution behaviour of cytochrome c were investigated in a biphasic tetrabutylphosphonium 2,4,6-trimethylbenzenesulfonate and potassium phosphate buffer system, which shows a lower critical solution temperature. To change charge balance and hydrophobicity of cytochrome c, surface modification with a few modifier molecules was applied. Surface charge and hydrophobicity affected the distribution behavior of chemically modified cytochrome c in the tetrabutylphosphonium 2,4,6-trimethylbenzenesulfonate and potassium phosphate buffer biphasic system. The distribution ratio into tetrabutylphosphonium 2,4,6-trimethylbenzenesulfonate decreased with decreasing isoelectric point of cytochrome c. Furthermore, cytochrome c possessing a low isoelectric point showed different distribution ratio depending on surface hydrophobicity. Taken together, these findings indicate that isoelectric point and surface hydrophobicity of cytochrome c are important factors controlling the distribution behavior in temperature sensitive biphasic systems.

Full text of DOI:10.1039/c9ob00900k

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OPrgleanasice&dBoionmotoaledcjuulsatr mChaermgiinsstry
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DOI: 10.1039/C9OB00900K
Organic & Biomolecular Chemistry
ARTICLE
Effects of Charge Balance and Hydrophobicity of the Surface of
Cytochrome c on the Distribution Behaviour in an Ionic
Liquid/Buffer Biphasic System
Received 00th January 20xx,
Accepted 00th January 20xx
Kazuma Ikeda,a Kyoko Fujita,b* Hiroyuki Ohno a and Nobuhumi Nakamura a*
DOI: 10.1039/x0xx00000x
Factors contributing to the different distribution behaviour of cytochrome
c were investigated in a biphasic
www.rsc.org/
tetrabutylphosphonium 2,4,6-trimethylbenzenesulfonate and potassium phosphate buffer system, which shows a lower
critical solution temperature. To change charge balance and hydrophobicity of cytochrome c, surface modification with a
few modifier molecules was applied. Surface charge and hydrophobicity affected the distribution behavior of chemically
modified cytochrome c in the tetrabutylphosphonium 2,4,6-trimethylbenzenesulfonate and potassium phosphate buffer
biphasic system. The distribution ratio into tetrabutylphosphonium 2,4,6-trimethylbenzenesulfonate decreased with
decreasing isoelectric point of cytochrome c. Furthermore, cytochrome c possessing a low isoelectric point showed different
distribution ratio depending on surface hydrophobicity. Taken together, these findings inidicate that isoelectric point and
surface hydrophobicity of cytochrome c are important factors controlling the distribution behavior in temperature sensitive
biphasic systems.
cooling. This reversible phase behaviour, classified as a lower critical
Introduction
solution temperature (LCST)-type phase change of the IL/water
10
mixture, has been previously reported.9,
The phase change
Ionic liquids (ILs) have excellent physicochemical properties such as
negligible vapour pressure and high thermal stability.1 ILs have been
gaining interest as new solvents in processes such as organic
synthesis, catalysis and electrochemistry.2 Recently, the application
of ILs as media for biochemistry has received attention because ILs
also possess interesting characteristics as enzyme solvents.3
IL-based aqueous two phase systems, such as hydrophobic
IL/water systems and IL/inorganic salt solution systems, have been
reported and have garnered much interest as solvent extraction
media for many compounds including biopolymers.4-7 Kragl et al.
have investigated the partition coefficients of several proteins in
IL/inorganic salt solution systems.8 They have suggested that the
charges on proteins are a major factor governing the dissolution of
the proteins in the IL-rich phase.
Previously, we studied the remarkable properties of ILs upon
mixing with water,9 and reported the preparation of ILs with amino
acid anions bearing a trifluoromethanesulfonyl group on the amino
group. These ILs underwent a temperature sensitive phase change
after mixing with water. For example, tetrabutylphosphonium N-
trifluoromethanesulfonyl-leucine ([P4444][Tf-Leu]) was found to be
miscible with water; however, a clear phase separation was observed
in the mixture upon heating, which became miscible again upon
between the homogeneous phase and the separated liquid/liquid
biphasic system was controlled by temperature; thus, the LCST-type
phase transition of the IL/water mixture permits applications such as
the extraction of biopolymers including proteins.11
We have also reported that the surface charge of the proteins
and their isoelectric point (pI) should influence the distribution
value.12 Proteins which have pI over around 7.0 showed extraction
into IL phase, such as hemoglobin (pI 7.0), chymotrypsin (pI 8.6) and
Lysozyme (pI 11.4). However, protein extraction in the biphasic
system is not so simple. For example, horseradish peroxidase (HRP,
pI 7.2) was expected to be extracted into the IL phase, but it was
remaining in the water phase. On the other hand, albumin (BSA, pI
4.9) was transfer to IL phase. The distribution value of HRP and BSA
cannot be explained only by pI value. Other factors could influence
the partition coefficients of proteins including hydrophobic
interactions13 and the salting-out phenomenon.14 However, the
major driving force governing the extraction of proteins from the
aqueous to the IL phase remains unclear. As previous studies utilised
individual proteins, which have a distinct sequence, size and
structure, the basic characteristics, such as hydrophobicity and pI,
were also different. Thus, discussion about factors related to the
protein distribution in IL/water systems was difficult in a systematic
matter. Although there are some reports about distribution of
proteins into ILs, these did not investigate the effects on distribution.
In this study, we used surface modification on a single protein which
is expected to enable an investigation of proteins with approximately
the same size and basic features, except for surface modifier
characteristics, in order to determine factors governing the
a.Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-
24-16 Koganei, Tokyo 184-8588, Japan.
b.Department of Pathophysiology, Tokyo University of Pharmacy and Life Sciences,
1432-1 Horinouchi, Hachioji, Tokyo, 192-0392, Japan
† Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
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Journal Name
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DOI: 10.1039/C9OB00900K
1.0
0.5
Fig. 1 Chemical modification of cyt.c for the preparation of
acetylated, TEG and succinylated cyt.c.
0.0
3
4
5
6
7
8
9
10
extraction of proteins from the aqueous to the IL phase. We
formulate analysed the effect of potential factor on the distribution
value of the protein without influence by other factors.
pI
Fig. 2 Relationship between D IL and pI for (●) native cyt.c, (◆)
acetylated cyt.c, (▲) methyl-TEG-cyt.c and (■) succinylated cyt.c.
In this study, cytochrome c (cyt. c) from horse heart is chosen
as a model protein for study since it is one of the most thoroughly
physicochemically characterized metalloproteins.15 It is easy to
confirm the effect of the surface modification on the redox state and
coordination state of active site. Furthermore, modification
technique of cyt.c by using lysine residue has been established.16
Systematize examination is expected by surface modification to cyt.c
with appropriate modifier. In addition, we have reported the
extraction of proteins using such IL/water biphasic systems, and
found that cyt.c was selectively transferred into the IL-rich phase.7
Furthermore, the distribution behaviour of cyt.c differed with redox
state17; oxidized cyt.c showed extraction from the aqueous phase to
the IL phase via LCST transition, while reduced cyt.c remained in the
aqueous phase even after LCST transition. With additional control of
cyt.c redox state by applied electrochemical potential, phase transfer
of cyt.c in an LCST type IL/ water biphasic system has been
confirmed. We analysed controlling factors related to the distribution
ratio of proteins using different chemical modifications of the
protein, cyt.c. We investigated the effect of cyt.c surface charge and
hydrophobicity on the distribution behaviour in a [P4,4,4,4][TMBS] /
potassium phosphate buffer (PKB) biphasic system. The present
study provides insight into the partition of proteins in IL-based
aqueous two phase systems.
residues. The modifications included acetylation, triethylene glycol
(TEG) modification and succinylation, resulting in the loss of a
positive charge, the addition of an amphipathic unit and the
exchange of a positive charge to a negative charge, respectively. The
average chemical modification number and pI of the three types of
synthesised chemically modified cyt.cs, acetylated cyt.c, succinylated
cyt.c and methyl terminal TEG cyt.c (Fig. 1), were analysed as shown
in Table 1. Modification numbers on cyt.c were related to the molar
ratio of the modifier. Chemically modified cyt.c showed a decreasing
pI with increasing modification number, as the positive charge of the
lysine residues on cyt.c was used for modification, as shown in Fig. 1.
By using these chemically modified cyt.cs and native cyt.c, the
relationship between DIL and pI were evaluated. The observed DIL of
native cyt.c was nearly 1.0, indicating that cyt.c transferred into the
IL phase from the water phase following LCST biphasic behaviour. In
contrast, the DIL of chemically modified cyt.c decreased with
decreasing pI (Fig. 2). This observation suggests that the pI of cyt.c is
correlated with the distribution behaviour of cyt.c in the
[P4444][TMBS]/ PKB biphasic system. Interestingly, the DIL of
chemically modified cyt.c differed around pI 4 according to the
modifier. All these chemically modified cyt.cs with a pI around 4 had
modifications in over half of all lysine residues (10 – 14). This suggests
that the positive lysine residues of cyt.cs were similarly used for
modification. Of the modified cyt.cs, succinylated cyt.cs showed the
lowest DIL at around pI 4; the succinyl modifier may dissociate at pH
8. Succinylated cyt.c has been suggested to have a more hydrophilic
surface than other modified cyt.cs with undissociated modifiers.
Thus, the degree of cyt.c surface hydrophobicity could cause
differences in DIL. Therefore, the hydrophobicity order of chemically
modified cyt.cs was compared.
Results and discussion
The surface of cyt.c was modified using three different modifiers with
different electric charge and hydrophobicity. Chemical modifications
were targeted to the lysine residues on cyt.c, which has 19 lysine
Table 1 Association between modifier molar ratio, average
chemical modification number and pI
Table 2 Association between average chemical modification
number and pI of TEG modified cyt.c
2 | J. Name., 2012, 00, 1-3
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suggested as a preferential control factor of distribution behaviour
View Article Online
DOI: 10.1039/C9OB00900K
1.0
0.5
0.0
when cyt.c has a certain pI. These findings strongly support the role
of cyt.c surface hydrophobicity as a key factor affecting distribution
behaviour at the low pI region (around 4) in the [P4444][TMBS]/ PKB
biphasic system.
Conclusions
Factors related to the different distribution behaviour of cyt.c
were investigated in the [P4,4,4,4][TMBS] / PKB buffer biphasic
system using chemical modification of cyt.c. By using the
surface modification on a single protein, effect of focused
factor i.e. charge balance and hydrophobicity of the surface on
the distribution of protein was investigated. Influence from
other factors were eliminated. Surface charge was controlled
by changing the modification number of the 19 lysine residues
of cyt.c. The pI of modified cyt.c was related to the modification
number. Decreased pI was correlated with decreased DIL values.
Furthermore, chemically modified cyt.c with a similar low pI
(e.g. a pI around 4) showed different DIL values depending on
the surface hydrophobicity based on the TEG modifier. The
influence of the surface hydrophobicity of cyt.c on the
distribution behaviour was observed more clearly with cyt.cs
possessing a lower pI. The pI of chemically modified cyt.c and
the surface hydrophobicity were revealed as important factors
controlling the distribution behaviour of cyt.c in the
[P4,4,4,4][TMBS] / PKB biphasic system. By tuning the surface
charge and hydrophobicity, the distribution behaviour of a
specific protein may be more easily controlled in the LCST
biphasic system. This might provide a great advantage in the
complete separation for recycling use of proteins and enzymes
in reaction separation platforms in temperature sensitive
aqueous and non-aqueous biphasic systems.
3
4
5
6
7
8
9
10
pI
Fig. 3 Relationship between D IL and pI for (●) native cyt.c, (◆)
butyl-TEG cyt.c, (■) ethyl-TEG-cyt.c and (▲) methyl -TEG cyt.c.
The expected hydrophobicity order of the modified proteins was
acetylated > TEG > succinylated cyt.c. Hydrophobic interaction
chromatography was used to evaluate the degree of surface
hydrophobicity of the modified cyt.cs. The chemically modified cyt.c
was loaded onto a phenyl column, and the retention time was then
measured by applying a concentration gradient of ammonium
sulphate (0.8 ~ 0 M). The retention time of acetylated cyt.c, TEG cyt.c
and succinylated cyt.c was 600 ~ 650 min, 400 ~ 450 min and 0 min
(shortly after protein loading), respectively (Fig. S1 in Supporting
information). Based on these results, the predicted hydrophobicity
order was confirmed as acetylated cyt.c, TEG cyt.c and succinylated
cyt.c. The predicted hydrophobicity order was quite consistent with
the observed DIL at pI 4L. Succinylated cyt.c had the most hydrophilic
surface of these chemically modified cyt.cs. Succinylated cyt.c with a
pI of 4 showed a DIL < 0.3. These cyt.cs remained in the aqueous
phase even after LCST transition. The hydrophobicity derived from
each modifier was suggested to affect the distribution ratio of cyt.c
in the LCST biphasic system at the lower pI region.
Experimental
Both the pI and surface hydrophobicity of cyt.c were suggested
above as factors associated with the DIL of cyt.c in the LCST biphasic
system. The effect of these two factors, pI and surface
hydrophobicity, on DIL was further considered using TEG modifiers on
cyt.c. Three different TEG-based modifiers with different terminal
alkyl chain lengths, methyl, ethyl and butyl (Fig. S2 in Supporting
information), were used to modify cyt.c. These TEG modifiers were
expected to have a different hydrophobicity depending on the alkyl
chain length. Table 2 shows the modification number and pI of the
modified cyt.cs. pI decreased with increasing modification number
because of the use of the positively charged lysine residues. Fig. 3
shows the observed relationship between DIL and pI depending on
the modifier. When methyl-TEG and ethyl-TEG were used to modify
cyt.c, the DIL decreased with decreasing pI of the modified cyt.c,
which was based on the cyt.c modification number. In contrast, no
change in DIL was observed for cyt.c modified with butyl-TEG showed,
regardless of pI, i.e. modification number. In particular, at the low pI
region (pI 4 – 5) of TEG-modified cyt.c, DIL differences dependent on
terminal alkyl length were clearly observed. The DIL value of methyl-
TEG, ethyl-TEG and butyl-TEG modified cyt.c was 0.55, 0.80 and 0.92,
respectively. Surface hydrophobicity based on TEG modifier was
Materials
Sodium 2,4,6-trimethylbenzenesulfonate (Na[TMBS]), succinic
anhydride, triethylene glycol monomethyl ether, triethylene glycol
monoethyl ether, triethylene glycol monobutyl ether, N-
hydroxysuccinimide,
1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride were purchased from Tokyo Chem.
Ind. Co. Hydrochloric acid (HCl) and pyridine were purchased from
Wako Chem. Co. Cytochrome c and partially acetylated cytochrome c
from horse heart were purchased from Sigma Aldrich.
Tetrabutylphosphonium hydroxide ([P4444]OH) were provided by
Hokko Chem Co. A pI broad kit 3-10 was purchased from GE
healthcare. All of the chemicals and proteins were used as received.
Synthesis of tetrabutylphosphonium 2,4,6-
trimethylbenzenesulfonate ([P4,4,4,4][TMBS])
An aqueous solution of [P4444]OH was first neutralised with
hydrochloric acid, to prepare [P4444]Cl. Next, [P4444]Cl was dissolved
in water, and a slight excess of Na[TMBS] was added. This solution
was stirred for 24 h at room temperature. The product was extracted
with dichloromethane, and the dichloromethane layer was washed
repeatedly with water. The dichloromethane layer was evaporated
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ARTICLE
and the product was dried under reduced pressure for 24 h. δΗ (400 Tetrabutylphosphonium
Journal Name
2,4,6-trimethylbenVzieewneAsrtuicllfeoOnnalitnee
MHz, CDCl3) 0.95 (t, J 14.4, 12H, CH2CH3), 1.49–1.50 (m, 16H, CH2), ([P4,4,4,4][TMBS]) was used as the IL that uDnOdeI:r1g0o.1e0s39th/Ce9OLCBS0T0-9t0yp0Ke
2.30 (s, 3H, ArCH3), 2.32–2.38 (m, 8H, PCH2), 2.70 (s, 6H, ArCH3), 6.79 phase transition. [P4,4,4,4][TMBS] was dissolved in 100 mM phosphate
(s, 2H, ArH)
buffer (pH 8.0), and induced phase separation by heating. After
phase separation, obtained bottom phase was used as an IL phase.
cyt.c were dissolved in 100 mM phosphate buffer (pH 8.0), and the
solution was added to the IL phase. The mixture formed a
homogeneous phase. This homogeneous solution was then heated
and left to stand until the phases were separated clearly. The
absorbance of the buffer phase was measured by UV-vis
spectroscopy. The DIL of cyt.c was calculated using following
Synthesis of N-Hydroxysuccinimide activated methyl, ethyl and
butyl terminal triethylene glycol modifiers
Triethylene glycol monomethyl ether, triethylene glycol monoethyl
ether and triethylene glycol monobutyl ether were dissolved in 1,2-
dichloroethane under a Ar atmosphere each other. Next succinic
anhydride was added into these solvents. To these reaction mixtures,
pyridine was added dropwise. The reaction mixtures was then stirred
o
equation: DIL =(Absbuffer-before -Absbuffer-after
)
Absbuffer-before, where
/
at 40-50 C for 24 h under Ar reflux. These were poured into water
Absbuffer denote the absorbance of the buffer phase. The maximum
and neutralized with 1N concentrated HCl. The organic layer was then
washed with brine solution. The 1,2-dichloroethane layer was absorption of the Soret band around 408.0 nm was used for cyt.c.
evaporated and the product were dried under reduced pressure for
2h. These products were dissolved in acetonitrile. Next N-
hydroxysuccinimide was added into these solvents. 1-(3-
Conflicts of interest
There are no conflicts to declare.
dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride
was
added drop wise to these reaction mixtures. The reaction mixtures
were then stirred at r.t. for 48 h. The dichloromethane layer was then
washed with brine solution. The dichloromethane layer was
evaporated and the product were dried under reduced pressure for
2h. These were purified by passing through a silica gel column using
methanol : chloroform, 1:9 (v/v).
Acknowledgements
This research was supported by a Grant-in-Aid for Scientific
Research from the Japan Society for the Promotion of Science
(KAKENHI, No. 17H01225 and 18H01719)
N-Hydroxysuccinimide activated methyl terminal triethylene glycol
δΗ (400MHz,CDCl3) 2.72-2.84(m,6H,OC(=O)CH2,CH2-
C(=O)NC(=O)CH2), 2.97 (t, J 14.2, 2H, C(=O)CH2) ,3.38 (s, 3H,
CH3O),3.54-3.56(m,2H,OCH2) ,3.63-
Notes and references
1.
P. Wasserscheid, T. Welton and Editors, Ionic Liquids in
Synthesis, 2003.
M. Armand, F. Endres, D. R. MacFarlane, H. Ohno and B.
Scrosati, Nature Materials, 2009, 8, 621-629.
S. Oppermann, F. Stein and U. Kragl, Applied microbiology
and biotechnology, 2011, 89, 493-499.
A. Cizova, J. Korcova, P. Farkas and S. Bystricky, Int J Biol
Macromol, 2017, 98, 314-318.
C. P. Song, P. E. Liew, Z. Teh, S. P. Lim, P. L. Show and C. W.
Ooi, Frontiers in chemistry, 2018, 6, 529.
P. Xu, Y. Wang, J. Chen, X. Wei, W. Xu, R. Ni, J. Meng and Y.
Zhou, Talanta, 2018, 189, 467-479.
Y. Ito, Y. Kohno, N. Nakamura and H. Ohno, International
journal of molecular sciences, 2013, 14, 18350-18361.
S. Dreyer, P. Salim and U. Kragl, Biochemical Engineering
Journal, 2009, 46, 176-185.
K. Fukumoto and H. Ohno, Angewandte Chemie, 2007, 46,
1852-1855.
Y. Kohno and H. Ohno, Physical chemistry chemical physics
: PCCP, 2012, 14, 5063-5070.
A. M. Ferreira, H. Passos, A. Okafuji, A. P. M. Tavares, H.
Ohno, M. G. Freire and J. A. P. Coutinho, Green chemistry,
2018, 20, 1218-1223.
Y. Kohno, S. Saita, K. Murata, N. Nakamura and H. Ohno,
Polym. Chem., 2011, 2, 862-867.
Y. Pei, J. Wang, K. Wu, X. Xuan and X. Lu, Separation and
Purification Technology, 2009, 64, 288-295.
Z. Du, Y.-L. Yu and J.-H. Wang, Chemistry – A European
Journal, 2007, 13, 2130-2137.
G. W. Pettigrew, Cytochromes C. Biological Aspects, 1987.
K. Wada and K. Okunuki, Journal of biochemistry, 1969, 66,
249-262.
3.72(m,8H,CH2OCH2CH2OCH2),4.73(t, J 9.6, 2H, CH2C(=O))
N-Hydroxysuccinimide activated ethyl terminal triethylene glycol
δΗ (400 MHz,CDCl3) 1.21(t, J 14.2,CH3CH2), 2.76-
2.83(m,6H,OC(=O)CH2,CH2-C(=O)NC(=O)CH2), 2.96 (t, J 14.2, 2H,
C(=O)CH2) ,3.38 (s, 2H, CH2O)、3.50-3.54(m,2H,OCH2) ,3.58-
3.60(m,2H,OCH2)、3.63-3.72(m,8H,CH2OCH2CH2OCH2),4.28(t, J 9.2,
2H, CH2C(=O))
2.
3.
4.
5.
N-Hydroxysuccinimide activated butyl terminal triethylene glycol
δΗ ( 400 MHz,CDCl3) 0.91(t,J 14.6, 3H,CH3CH2),1.31-1.60(m, 4H,
CH2CH2O), 2.76-2.83 (m,6H,OC(=O)CH2,CH2-C(=O)NC(=O)CH2), 2.96
(t, J 13.7, 2H, C(=O)CH2), 3.38 (s, 2H, CH2), 3.50-3.54(m,2H,OCH2),
3.57-3.60(m,2H,OCH2), 3.63-3.66(m,8H,CH2OCH2CH2OCH2), 4.28(t, J
9.6, 2H, CH2C(=O))
6.
7.
8.
Preparation of chemically modified cyt c
9.
Lysine residues of cyt.c was modified by adding succinic anhydride,
acetic anhydride and N-Hydroxysuccinimide activated triethylene
glycols (succinylated cyt.c, acetylated cyt.c, triethylene glycol
modified cyt.c (TEG cyt.c)) in boric acid buffer (100 mM, pH 9.5) at
35oC for 2 h. After the reaction, the solvents were replaced with pure
water by using Amicon (Mw 5000) at 5000 g centrifugation. After
centrifugation, these chemically modified cyt.cs were stored by using
freeze dry procedure.
pI of chemically modified cyt.c was measured by isoelectric focusing
(GE healthcare, phastsytem) and the number of chemical
modification on lysine residues was determined by
trinitrobenzosulfonate reaction.
10.
11.
12.
13.
14.
15.
16.
Determination of the distribution ratio into [P4444][TMBS] phase
(DIL) of cyt.c
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DOI: 10.1039/C9OB00900K
G100.
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Surface charge and hydrophobicity affected the
distribution behavior of chemically modified cyt.c in the
[P4,4,4,4][TMBS]/PKB biphasic system.
●
◆
▲
■
native cyt.c
acetylated cyt.c
methyl-TEG cyt.c
succinylated cyt.c