Extraction and stability of bovine serum albumin (BSA) using
cholinium-based Good’s buffers ionic liquids
Mohamed Taha1, Maria V. Quental1, Isabel Correia2, Mara G. Freire1, and João A. P. Coutinho1*
1
CICECO-Aveiro Institute of Materials, Department of Chemistry, University of Aveiro, 3810-193 Aveiro,
Portugal
2
Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049001 Lisboa, Portugal
Corresponding Author
* Tel.: +351 234 401 507; fax: +351 234 370 084.
E-mail address: [email protected] (J.A.P. Coutinho)
1
Table S1. Characterization of Good’s buffer ionic liquids.
[Ch][Tricine]: 1H NMR (300 MHz, D2O/TSP); δ [Ch], 3.08 (9H, s, C1-C3’s
H), 3.38 (2H, m, C4’s H ), 3.93 (2H, m, C4’s H ); δ [Tricine], 3.42 (6H, s,
OH
H
N
4
1
C1-C3’s H), 3.17 (2H, s, C5’s H). C NMR (75.47 MHz, D2O/TSP); δ [Ch],
13
HO
2
O
3
6
O
5
2
OH
1
4
OH
3
56.73 (C1-C3), 58.47 (C4), 70.28 (C5); δ [Tricine], 63.30 (C4), 63.03 (C1-
N+
-
5
[Ch][Tricine]
C3), 182.19 (C6), 47.62 (C5); melting point = 68 °C.
[Ch][TES]: 1H NMR (300 MHz, D2O/TSP); δ [Ch], 3.08(9H, s, C1-C3’s
H), 3.39 (2H, m, C4’s H ), 3.93 (2H, m, C4’s H ); δ [TES], 3.48 (6H, s, C1C3’s H), 2.96 (2H, t, C6’s H), 2.71 (2H, t, C5’s H). 13C NMR (75.47 MHz,
1
HO
OH
H
4 N
2
2
O
S O
OH O
3
D2O/TSP); δ [Ch], 56.76 (C1-C3), 58.51 (C4), 70.29 (C5); δ [TES], 63.47
3
6
5
N+
OH
1
4
5
[Ch][TES]
(C4), 63.24 (C1-C3), 53.44 (C6), 39.62 (C5); viscous liquid at room
temperature.
[Ch][MES]: 1H NMR (300 MHz, D2O/TSP); δ [Ch], 3.08 (9H, s, C1-C3’s
H), 3.40 (2H, m, C4’s H ), 3.94 (2H, m, C4’s H ); δ [MES], 2.48 (4H, t,
3
4
2
2
C4C6’s H), 2.70 (2H, t, C2’s H), 3.01 (2H, t, C1’s H), 3.64 (4H, t, C3C5’s
O
5
H).
13
6
C NMR (75.47 MHz, D2O/TSP); δ [Ch], 56.76 (C1-C3), 58.50 (C4),
3
O
N
S
1
N+
-
O
OH
1
4
O
5
[Ch][MES]
70.29 (C5); δ [MES], 50.23 (C4C6), 55.17 (C2), 55.49 (C1), 68.86 (C3C5);
melting point = 87 °C.
[Ch][HEPES]: 1H NMR (300 MHz, D2O/TSP); δ [Ch], 3.07 (9H, s, C1C3’s H), 3.39 (2H, m, C4’s H ), 3.93 (2H, m, C4’s H ); δ [HEPES], 2.53
(8H, t, C3C4C5C6’s H), 2.70 (2H, m, C1C8’s H), 2.99 (2H, t, C7’s H), 3.62
(4H, t, C2’s H).
13
2
HO
1
3
4
N
N
7
5
6
C NMR (75.47 MHz, D2O/TSP); δ [Ch], 56.74 (C1-C3),
2
8
O
S OO
3
N+
OH
1
4
5
[Ch][HEPES]
58.48 (C4), 70.28 (C5); δ [HEPES], 50.40 (C8), 54.06 (C4C5), 54.74
(C3C6), 55.00 (C1), 60.69 (C7), 61.57 (C2); melting point = 103 °C.
[Ch][CHES]: 1H NMR (300 MHz, D2O/TSP); δ [Ch], 3.09 (9H, s, C1-C3’s
H), 3.40 (2H, m, C4’s H ), 3.95 (2H, m, C4’s H ); δ [CHES], 0.94-1.79 (10H,
m, C2-C6’s H), 2.39-2.49 (H, m, C1’s H), 2.94 (2H, m, C8’s H), 2.92 (2H, t,
C7’s H).
13
C NMR (75.47 MHz, D2O/TSP); δ [Ch], 56.71 (C1-C3), 58.50
(C4), 70.30 (C5); δ [CHES], 27.32 (C3C5), 28.42 (C4), 34.58 (C2C6), 43.41
4
3
2
6
7
1
HN
3
O
2
5
8
S O-
N+
OH
1
O
4
5
[Ch][CHES]
(C8), 52.80 (C7), 58.44 (C1); melting point = 72 °C.
2
Table S2. Experimental weight fraction data for the binodal curves of the systems composed of
GB/[Ch]Cl/sucrose (1) + PPG 400 (2) at (25 ± 1)°C.
sucrose
TES
100 w1
100 w2
100 w1
100 w2
74.53
3.09
45.61
11.53
73.37
3.30
44.52
11.94
71.79
3.68
41.28
13.00
71.30
3.82
39.76
13.33
70.64
4.00
38.37
14.24
69.71
4.20
30.72
15.54
68.90
4.55
29.33
16.05
67.97
4.81
28.07
16.69
66.62
5.08
27.84
16.76
66.36
5.17
24.46
19.44
65.93
5.28
22.22
20.93
64.62
5.69
20.65
21.53
63.81
5.95
17.59
24.24
62.72
6.28
17.48
24.39
59.33
7.47
15.60
25.94
56.83
8.18
14.84
26.75
51.82
10.07
13.19
28.88
32.51
19.51
10.63
30.81
24.99
23.08
9.75
31.86
13.12
30.87
9.75
31.86
10.22
35.12
9.72
32.42
10.22
35.12
9.72
32.42
7.91
38.47
8.37
35.55
6.95
36.98
6.95
36.98
6.11
38.90
5.19
44.09
3
100 w1
25.13
24.87
27.43
33.74
34.96
38.35
40.86
43.68
44.84
46.43
48.48
48.78
50.67
52.03
53.08
54.35
55.17
56.44
56.53
57.68
58.17
59.60
60.28
60.38
60.72
61.10
61.62
100 w2
25.55
20.61
18.11
13.03
11.83
10.33
9.29
8.45
7.82
6.96
6.49
6.25
5.70
5.19
4.85
4.47
4.23
3.98
3.79
3.46
3.32
3.10
2.92
2.88
2.78
2.69
2.56
HEPES
100 w1
61.79
62.00
62.20
62.20
62.73
63.17
63.81
64.36
65.09
65.61
66.18
66.83
66.88
[Ch]Cl
100 w2
2.44
2.37
2.33
2.27
2.21
2.10
2.02
1.91
1.79
1.73
1.62
1.56
1.53
100 w1
48.71
43.07
39.12
36.28
33.56
31.32
29.62
26.22
25.46
24.24
23.57
20.50
19.97
19.33
18.89
18.46
17.95
17.22
16.79
16.46
15.98
15.48
6.92
5.74
100 w2
6.28
6.95
7.46
8.05
8.44
8.92
9.24
10.62
11.22
11.56
12.01
14.02
14.34
14.57
14.90
15.24
15.43
16.01
16.68
16.88
17.55
17.53
39.23
44.67
4
Table S3. Experimental weight fraction data for the binodal curve of the systems composed of [Ch][GB] (1)
+ PPG 400 (2) at (25 ± 1)°C.
[Ch][Tricine]
100 w1
97.87
69.49
60.19
56.78
54.16
51.55
49.26
47.60
45.85
43.78
42.23
40.60
39.15
37.48
36.35
35.38
34.55
33.51
32.67
31.59
30.46
29.60
29.10
28.25
27.15
26.59
25.94
25.14
23.95
23.04
22.41
21.74
20.96
20.32
19.60
19.00
100 w2
1.30
3.13
3.63
4.28
4.70
5.45
5.76
6.23
6.66
6.95
7.26
7.66
8.11
8.28
8.55
8.81
9.06
9.29
9.49
10.00
10.58
10.74
10.97
11.51
11.73
12.23
12.52
12.63
13.00
13.54
13.85
14.31
14.78
15.21
15.76
16.35
100 w1
18.46
17.79
17.26
16.70
16.07
15.46
14.77
14.23
13.68
12.93
12.40
11.77
10.96
[Ch][HEPES]
100 w2
16.66
17.26
17.62
18.07
18.58
19.03
19.53
20.03
20.60
21.38
21.90
22.68
24.44
100 w1
86.73
65.64
40.58
38.56
37.15
35.58
34.04
32.69
31.83
30.65
29.80
28.92
28.11
26.36
25.45
24.60
24.00
23.34
22.90
22.34
21.76
21.40
20.71
20.17
20.01
19.18
18.53
17.69
17.17
16.56
15.91
15.26
14.63
14.16
13.78
13.45
100 w2
2.30
3.68
8.07
8.67
9.29
9.97
10.47
10.90
11.53
11.99
12.37
12.74
13.36
13.52
14.38
15.01
15.26
15.57
15.99
16.66
16.77
17.07
17.69
17.81
17.65
17.91
19.07
19.14
19.60
20.32
20.48
21.64
22.83
23.41
23.87
24.09
100 w1
13.15
12.76
12.45
12.15
11.68
11.42
11.08
10.81
10.47
10.15
9.89
10.81
10.47
10.15
9.89
100 w2
24.44
25.03
25.35
25.81
26.14
26.48
26.97
27.45
27.87
28.30
28.78
27.45
27.87
28.30
28.78
5
Table S3. Continued
100 w1
69.88
64.79
60.13
55.20
48.50
46.80
43.80
42.43
38.27
36.55
35.08
34.13
32.82
31.70
30.22
28.40
27.84
27.27
26.49
25.49
24.69
23.73
23.28
22.63
22.03
21.35
20.81
20.46
19.99
19.50
18.86
18.40
17.55
17.19
16.17
15.77
[Ch][MES]
100 w2
100 w1
4.23
15.33
5.32
14.76
6.29
13.95
7.18
13.32
8.77
12.63
9.55
12.22
10.90
11.73
11.36
12.78
13.09
13.44
13.98
14.25
14.55
15.33
16.19
16.47
16.73
17.50
18.05
18.74
19.23
19.42
20.02
20.52
20.87
21.40
21.59
22.12
22.67
23.00
23.47
24.17
24.60
24.53
24.83
100 w2
25.34
26.07
26.92
27.87
28.81
29.31
30.03
100 w1
96.95
70.16
62.07
58.09
55.88
52.86
49.21
46.57
44.15
42.22
41.00
39.81
38.36
37.08
35.31
34.03
33.26
32.54
31.28
29.92
29.02
27.98
27.49
26.66
25.81
24.93
24.20
23.97
23.09
22.10
21.35
20.22
19.54
18.70
17.49
16.86
[Ch][TES]
100 w2
100 w1
1.72
16.00
3.71
15.30
4.31
14.67
5.00
13.59
5.64
6.37
7.81
8.29
8.83
9.18
9.67
10.09
10.44
10.75
11.42
11.49
11.88
12.20
12.96
13.47
14.08
14.58
14.82
15.40
15.88
16.31
16.86
16.69
17.25
17.92
18.29
19.23
19.73
20.05
21.21
22.05
100 w2
22.98
23.72
24.42
24.85
6
Table S4. Correlation parameters used to describe the experimental binodal data by Eq. 1a and respective
standard deviations (σ) and correlation coefficients.
cosolute
A±σ
B±σ
105 (C ± σ)
R2
[Sucrose]
115.7 ± 1.8
-0.24 ± 0.01
2.4 ± 0.1
0.9991
[HEPES]
94.2 ± 1.1
0.27 ± 0.01
0.5 ± 0.3
0.9964
[TES]
400.4 ± 3.6
0.64 ± 0.03
2.3 ± 0.3
0.9949
[Ch]Cl
265.4 ± 16.1
-0.70 ± 0.002
1.1 ± 0.2
0.9902
[Ch][Tricine]
180.5 ± 2.4
-0.52 ± 0.01
1.2 ± 0.3
0.9977
[Ch][HEPES]
187.1 ± 9.9
-0.54 ± 0.02
1.0 ± 0.3
0.9938
[Ch][TES]
175.2 ± 3.4
-0.47 ±
0.0
1.2 ± 0.3
0.9960
[Ch][MES]
169.2 ± 4.7
0.42 ± 0.01
1.2 ± 0.3
0.9987
a[𝐏𝐏𝐆
𝟒𝟎𝟎] = 𝑨 × 𝐞𝐱𝐩(𝑩[𝐬𝐨𝐥𝐮𝐭𝐞]𝟎.𝟓 − [𝐬𝐨𝐥𝐮𝐭𝐞]𝟑 ) (𝟏); where [PPG] and [solute] are the PPG 400 and
the second phase-forming component weight percentages, respectively. The coefficients A, B, and C are
adjustable parameters obtained by the regression.
7
Table S5. Data for the tie-lines (TLs) and tie-line lengths (TLLs). Initial mixture compositions are
represented as [PPG 400]M and [solute]M whereas [PPG 400]PPG 400 and [PPG 400]solute are the concentration
of PPG 400 in the polymer-rich phase and in the second phase-forming component or vice-versa.
Weight fraction composition / wt %
solute
[Ch][Tricine]
[Ch][HEPES]
[Ch][TES]
[Ch][MES]
HEPES
TES
[PPG 400]PPG 400 [solute]PPG 400 [PPG 400]M [solute]M [PPG 400]solute
[solute]solute
TLL
66.70
3.24
50.01
7.08
26.25
12.24
42.42
80.08
2.22
49.90
10.17
15.05
19.35
67.24
70.38
3.60
50.16
8.09
29.16
12.74
42.22
77.52
2.93
49.73
9.82
21.64
16.79
57.57
66.69
4.20
49.94
8.53
29.38
13.86
38.54
72.16
3.54
49.73
10.06
21.94
18.13
52.29
63.57
5.48
50.62
9.88
25.45
18.41
40.25
77.01
3.56
56.11
22.24
20.43
57.30
66.38
1.70
49.87
20.06
25.09
22.41
46.20
89.07
0.04
29.99
30.31
22.48
25.49
89.07
94.92
5.13
29.59
29.47
6.98
37.89
93.84
97.71
4.92
30.03
29.80
6.79
38.34
96.87
69.82
4.22
50.32
11.92
37.42
17.01
34.84
86.44
1.42
50.11
14.99
23.23
25.04
67.48
10.00
sucrose
8
Table S6. Estimated -helical content, determined from []222.a
-helical content
PBSb
0.65
HEPES
0.50
TES
0.60
sucrose
0.46
[Ch][Tricine]
0.45
[Ch]][HEPES]
-
[Ch][TES]
0.52
[Ch][MES]
-
[Ch]Cl
0.45
a
J. Morriset, J.S.K. David, H.J. Pownall, A.M. Gotto, Biochem. 12 (1973) 1290–1299.
b
BSA in PBS solution at pH 7.4.
9
[PPG 400] / (mol.kg-1)
6
1.5
1.3
5
1.1
0.9
4
0.7
3
0.5
0.3
0.5
0.7
2
1
0
0.0
0.5
1.0
[Solute] /
1.5
(mol.kg-1)
Figure S1. Ternary phase diagrams for the systems composed of IL+ PPG 400 + water at 25°C and
atmospheric pressure: (▲) [Ch]Cl, () [Ch][Tricine], () [Ch][HEPES], ()[Ch][TES], () [Ch][MES].
10
[PPG 400] / (mol.kg-1)
8
7
6
5
4
3
2
1
0
0
1
2
3
[Solute] / (mol.kg-1)
Figure S2. Ternary phase diagrams for the systems composed of PPG400 + other phase-forming component
+ water at 25°C and atmospheric pressure: (▬) [HEPES], (■) TES and (♦) sucrose.
11
12
11
10
pH
9
buffer region
8
7
6
5
4
3
2
-12-11-10 -9 -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4
ml of titrant
Figure S3. The pH profiles of the investigated GB-ILs in water at (20 ± 1) °C. 10 mL of 0.05 M GB-ILs
titrated with 0.05 M HCl/NaOH; (■) [Ch][Tricine], (●) [Ch][HEPES], (▲) [Ch][TES], and () [Ch][MES].
The (-1 mL) entries correspond to the volumes of 0.05 M HCl that are needed to lower the pH. The addition
of acid is called as a reverse titration.
12
20
20
(a)
(b)
15
RH/nm
RH/nm
15
10
10
5
5
0.05 M TES
0.5 M TES
0
25
30
35
40
45
50 55
o
T/ C
60
65
70
75
0.05 M Tricine
0.5 M Tricine
0
25
80
20
30
35
40
45
50 55
o
T/ C
60
65
70
75
80
20
(c)
(d)
15
RH/nm
RH/nm
15
10
10
5
5
0.05 M HEPES
0.5 M HEPES
0
25
30
35
40
45
50 o 55
T/ C
60
65
70
75
80
0
25
0.05 M Sucrose
0.5 M Sucrose
30
35
40
45
50 o 55
T/ C
60
65
70
75
80
Fig. S4. RH of BSA in (0.05 M and 0.5 M) TES (a), Tricine (b), HEPES (c), and sucrose (d), as a function of
temperature at pH 7.4.
13
20
20
(b)
(a)
15
RH/nm
RH/nm
15
10
10
5
5
0.05 M [Choline]Cl
0.5 M [Choline]Cl
0
25
20
30
35
40
45
50 o 55
T/ C
60
65
70
75
0
25
80
30
35
40
45
60
65
70
75
80
(d)
RH/nm
15
10
10
5
5
0.05 M [Choline][HEPES]
0.5 M [Choline][HEPES]
0
25
50 55
o
T/ C
20
(c)
15
RH/nm
0.05 M [Choline][TES]
0.5 M [Choline][TES]
30
35
40
45
50 55
T / oC
60
65
70
75
80
0
25
0.05 M [Choline][Tricine]
0.5 M [Choline][Tricine]
30
35
40
45
50 o 55
T/ C
60
65
70
75
80
Fig. S5. RH of BSA in (0.05 M and 0.5 M) [Ch]Cl (a), [Ch][TES] (b), [Ch][HEPES] (c), and [Ch][Tricine]
(d), as a function of temperature at pH 7.4.
14
1.2
(a)
Normalised Intensity
1.0
0.8
0.6
0.4
0.2
0.0
1720
1680
1640
1600
1560
1520
1480
-1
Wavenumber(cm )
0.025
(b)
Absorption
0.020
0.015
0.010
0.005
0.000
1720
1700
1680
1660
1640
1620
1600
1580
-1
Wavenumber(cm )
Figure S6. (a) IR spectra of the amide I and II regions of 30 mg·mL-1 BSA in water and in 0.5 M
[Ch][TES] at pH 7.4. (b) Gaussian curve-fitting analysis of amide I spectra in 0.5 M [Ch][TES] at pH 7.4.
15
[] / (deg·cm2/dmol)
5
4
3
2
1
0
200
210
220
230
-1
240
250
260
l / nm
-2
-3
[Ch][Cl]
sucrose
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-5
Figure S7. Circular Dichroism spectra (in []) measured for the top polymeric-rich phase in the systems
containing sucrose and [Ch]Cl, after subtraction of the background spectra.
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