Supporting Information
A water-soluble selenoxide reagent as a useful probe for the reactivity
and folding of polythiol peptides
Kenta Araia, Masato Noguchia, Beena G. Singhb, K. Indira Priyadarsinib, Katsuhiko Fujioa, Yurika
Kuboc, Kyoko Takayamac, Setsuko Andoc, and Michio Iwaokaa*
a
Department of Chemistry, School of Science, Tokai University, Kitakaname, Hiratsuka-shi,
Kanagawa 259-1292, Japan.
b
Radiation and Photochemistry Division Bhabha Atomic Research Centre, Trombay, Mumbai
400085, India.
c
Department of Chemistry, School of Science, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku,
Fukuoka 814-0180, Japan.
Contents:
Figure S1–S2. Typical HPLC chromatogram obtained by short-term oxidation experiment of Ins-A
and Rlx-A using 1 equivalent of DHSox for 1 min at 25 °C.
Table S1–S3. Observed and expected molecular masses of the folding intermediates fractionated
from HPLC of Ins-A, Rlx-A and Ins-B as determined by ESI(+)-TOF-MS spectrometry.
Figure S3-S35. Relative populations of SS intermediates of CX-397, Ins-A, Rlx-A, and Ins-B as a
function of the reaction time under various conditions.
Figure S36. UV absorbance changes at 310 nm and the plots of the second-order rate constant
against the pH obtained by the short-term oxidation of DTTred with DHSox using a stopped-flow
instrument.
Figure S37. HPLC chromatograms of the peptide fragments obtained from 3Sº and 3S by digestion
with thermolysin.
1
UV absorbance at 280 nm
7
3
1
2
5
4
10
14
6
18
x
8
22
Retention time (min)
Figure S1. Typical HPLC chromatogram obtained by short-term oxidation experiment of Ins.-A
using 1 equivalent of DHSox for 1 min at 25 °C. The fraction number is corresponding to that in the
table S2. The symbols of x is a unknown impurities.
Table S1. Observed and expected molecular masses of the folding intermediates and the native state
of Ins-A as determined by ESI(+)-TOF-MS spectrometry.
Species
Fraction No.
Observed mass (Da)
Expected mass (Da)
R
1
2640.1
2638.7
1S
2
2485.2
2486.7
3
2485.9
2486.7
4
2485.2
2486.7
5
2485.9
2486.7
6
2333.9
2334.7
7
2333.9
2334.7
8
2334.3
2334.7
2S
2
UV absorbance at 275 nm
3
4
1
5
2
6
x
x
x
9
13
17
21
25
Retention time (min)
Figure S2. Typical HPLC chromatogram obtained by short-term oxidation experiment of Rlx-A
using 1 equivalent of DHSox for 1 min at 25 °C. The fraction number is corresponding to that in the
table S2. The symbols of x is a unknown impurities.
Table S2. Observed and expected molecular masses of the folding intermediates and the native state
of Rlx-A as determined by ESI(+)-TOF-MS spectrometry.
Species
Fraction No.
Observed mass (Da)
Expected mass (Da)
R
1
2970.5
2971.3
1S
2
2819.5
2819.3
3
2819.4
2819.3
4
2819.5
2819.3
5
2667.1
2667.3
6
2667.0
2667.3
2S
3
Table S3. Observed and expected molecular masses of the folding intermediates and the native state
of Ins.-B as determined by ESI(+)-TOF-MS spectrometry.
Species
Observed mass (Da)
Expected mass (Da)
Rins-B
3549.8
3549.9
1Sins-B
3397.8
3397.9
100
Relative populations (%)
R
80
60
2S
40
3S
1S
20
Figure S3. Relative populations of SS intermediates
of CX-397 as a function of the reaction time.
Reaction conditions were [R]0=[DHSox]0/2=47.9 μM at
25 °C and pH 4.0.
0
0.1
1
100
10
Reaction time (sec)
100
Relative populations (%)
R
80
3S
60
40
2S
1S
20
Figure S4. Relative populations of SS intermediates
of CX-397 as a function of the reaction time.
Reaction conditions were [R]0=[DHSox]0/3=45.8 μM at
25 °C and pH 4.0.
0
0.1
1
10
100
Reaction time (sec)
4
Relative populations (%)
100
R
80
60
1S
40
20
2S
3S
0
0.1
100
10
1
Figure S5. Relative populations of SS intermediates
of CX-397 as a function of the reaction time.
Reaction conditions were [R]0=[DHSox]0=43.6 μM at
25 °C and pH 8.0.
Reaction time (sec)
100
Relative populations (%)
3S
80
R
60
1S
40
2S
20
0
0.1
100
10
1
Figure S6. Relative populations of SS intermediates
of CX-397 as a function of the reaction time.
Reaction conditions were [R]0=[DHSox]0/3=44.1 μM at
25 °C and pH 8.0.
Reaction time (sec)
Relative populations (%)
100
R
80
60
1S
40
2S
20
3S
0
0.1
1
10
Reaction time (sec)
Figure S7. Relative populations of SS intermediates
of CX-397 as a function of the reaction time.
Reaction conditions were [R]0=[DHSox]0=52.5 μM at
25 °C and pH 10.0.
100
5
Relative populations (%)
100
80
R
60
2S
1S
40
3S
20
0
0.1
1
100
10
Figure S8. Relative populations of SS intermediates
of CX-397 as a function of the reaction time.
Reaction conditions were [R]0=[DHSox]0/2=53.6 μM at
25 °C and pH 10.0.
Reaction time (sec)
100
Relative populations (%)
3S
80
R
60
40
1S
2S
20
0
0.1
1
Figure S9. Relative populations of SS intermediates
of CX-397 as a function of the reaction time.
Reaction conditions were [R]0=[DHSox]0/3=44.2 μM at
25 °C and pH 10.0.
100
10
Reaction time (sec)
100
Relative populations (%)
R
3S
80
60
40
2S
1S
20
0
0.1
1
10
Reaction time (sec)
100
Figure S10. Relative populations of SS intermediates
of CX-397 as a function of the reaction time.
Reaction conditions were [R]0=[DHSox]0/4=61.6 μM at
25 °C and pH 4.0 in the presence of 2 M Gdn-HCl.
6
Relative populations (%)
100
R
80
60
2S
40
20
1S
3S
0
0.1
1
Figure S11. Relative populations of SS intermediates
of CX-397 as a function of the reaction time.
Reaction conditions were [R]0=[DHSox]0/2=41.4 μM at
25 °C and pH 7.0 in the presence of 2 M urea.
100
10
Reaction time (sec)
Relative populations (%)
100
R
80
3S
60
40
2S
1S
of CX-397 as a function of the reaction time.
Reaction conditions were [R]0=[DHSox]0/3=29.7 μM at
25 °C and pH 8.0 in the presence of 2 M urea.
20
0
0.1
Figure S12. Relative populations of SS intermediates
1
100
10
Reaction time (sec)
Relative populations (%)
100
80
R
60
2S
40
3S
1S
20
0
0.1
1
10
Reaction time (sec)
Figure S13. Relative populations of SS intermediates
of CX-397 as a function of the reaction time.
Reaction conditions were [R]0=[DHSox]0/2=32.3 μM at
25 °C and pH 8.0 in the presence of 2 M Gdn-HCl.
100
7
Relative populations (%)
100
R
80
3S
60
1S
40
2S
20
0
0.1
100
10
1
Figure S14. Relative populations of SS intermediates
of CX-397 as a function of the reaction time.
Reaction conditions were [R]0=[DHSox]0/3=26.3 μM at
25 °C and pH 8.0 in the presence of 2 M Gdn-HCl.
Reaction time (sec)
Relative populations (%)
100
R
80
60
40
Figure S15. Relative populations of SS intermediates
1S
20
2S
0
0.1
1
of CX-397 as a function of the reaction time.
Reaction conditions were [R]0=[DHSox]0=23.9 μM at
25 °C and pH 7.0 in the presence of 2 M urea.
100
10
Reaction time (sec)
Relative populations (%)
100
R
80
2S
60
40
20
Figure S16. Relative populations of SS intermediates
of CX-397 as a function of the reaction time.
Reaction conditions were [R]0=[DHSox]0/2=18.7 μM at
25 °C and pH 7.0 in the presence of 2 M urea.
1S
0
0.1
1
10
Reaction time (sec)
100
8
Relative populations (%)
100
80
2S
R
60
40
20
Figure S17. Relative populations of SS intermediates
of CX-397 as a function of the reaction time.
Reaction conditions were [R]0=[DHSox]0/3=18.5 μM at
25 °C and pH 7.0 in the presence of 2 M urea.
1S
0
0.1
1
100
10
Reaction time (sec)
Relative populations (%)
100
80
R
2S
60
40
Figure S18. Relative populations of SS intermediates
1S
20
of CX-397 as a function of the reaction time.
Reaction conditions were [R]0=[DHSox]0/2=14.5 μM at
25 °C and pH 8.0 in the presence of 2 M urea.
0
0.1
1
100
10
Reaction time (sec)
Relative populations (%)
100
80
2S
R
60
40
Figure S19. Relative populations of SS intermediates
of CX-397 as a function of the reaction time.
Reaction conditions were [R]0=[DHSox]0/3=14.8 μM at
25 °C and pH 8.0 in the presence of 2 M urea.
1S
20
0
0.1
1
10
Reaction time (sec)
100
9
Relative populations (%)
100
R
80
60
1S
40
Figure S20. Relative populations of SS intermediates
of Ins-A as a function of the reaction time.
Reaction conditions were [R]0=[DHSox]0=10.5 μM at
25 °C and pH 10.0 in the presence of 2 M urea.
20
2S
0
0.1
10
1
100
Reaction time (sec)
Relative populations (%)
100
80
R
2S
60
40
Figure S21. Relative populations of SS intermediates
1S
20
of Ins-A as a function of the reaction time.
Reaction conditions were [R]0=[DHSox]0/2=16.5 μM at
25 °C and pH 10.0 in the presence of 2 M urea.
0
0.1
10
1
100
Reaction time (sec)
Relative populations (%)
100
R
80
60
40
20
Figure S22. Relative populations of SS intermediates
of Ins-B as a function of the reaction time.
Reaction conditions were [R]0=2[DHSox]0=41.3 μM at
25 °C and pH 4.0 in the presence of 2 M urea.
1S
0
0.1
1
10
Reaction time (sec)
100
10
Relative populations (%)
100
R
80
60
40
Figure S23. Relative populations of SS intermediates
of Ins-B as a function of the reaction time.
Reaction conditions were [R]0=[DHSox]0=42.1 μM at
25 °C and pH 4.0 in the presence of 2 M urea.
1S
20
0
0.1
1
10
100
Reaction time (sec)
Relative populations (%)
100
R
80
60
40
Figure S24. Relative populations of SS intermediates
20
of Ins-B as a function of the reaction time.
Reaction conditions were [R]0=2[DHSox]0=21.5 μM at
25 °C and pH 7.0 in the presence of 2 M urea.
1S
0
0.1
1
10
100
Reaction time (sec)
Relative populations (%)
100
R
80
60
40
20
1S
0
0.1
1
10
Reaction time (sec)
100
Figure S25. Relative populations of SS intermediates
of Ins-B as a function of the reaction time.
Reaction conditions were [R]0=[DHSox]0/0.65=18.7 μM
at 25 °C and pH 7.0 in the presence of 2 M urea.
11
Relative populations (%)
100
R
80
60
40
20
1S
0
0.1
1
10
Figure S26. Relative populations of SS intermediates
of Ins-B as a function of the reaction time.
Reaction conditions were [R]0=[DHSox]0=25.7 μM at
25 °C and pH 7.0 in the presence of 2 M urea.
100 .
Reaction time (sec)
Relative populations (%)
100
R
80
60
40
Figure S27. Relative populations of SS intermediates
20
1S
0
0.1
1
10
of Ins-B as a function of the reaction time.
Reaction conditions were [R]0=2[DHSox]0=25.8 μM at
25 °C and pH 8.0 in the presence of 2 M urea.
100
.
Reaction time (sec)
Relative populations (%)
100
R
80
60
40
20
1S
0
0.1
1
10
Reaction time (sec)
Figure S28. Relative populations of SS intermediates
of Ins-B as a function of the reaction time.
Reaction conditions were [R]0=[DHSox]0/0.45=46.9 μM
at 25°C and pH 8.0 in the presence of 2 M urea.
100 .
12
Relative populations (%)
100
R
80
60
40
20
Figure S29. Relative populations of SS intermediates
of Ins-B as a function of the reaction time.
Reaction conditions were [R]0=2[DHSox]0=40.0 μM at
25 °C and pH 10.0 in the presence of 2 M urea.
.
1S
0
0.1
1
10
100
Reaction time (sec)
Relative populations (%)
100
R
80
60
40
Figure S30. Relative populations of SS intermediates
20
of Ins-B as a function of the reaction time.
Reaction conditions were [R]0=[DHSox]0=40.0 μM at
25 °C and pH 10.0 in the presence of 2 M urea.
1S
0
0.1
1
10
100
Reaction time (sec)
Relative populations (%)
100
R
80
60
40
1S
20
0
0.1
1
10
Reaction time (sec)
100
Figure S31. Relative populations of SS intermediates
of Ins-B as a function of the reaction time.
Reaction conditions were [R]0=[DHSox]0/2=42.6 μM at
25 °C and pH 10.0 in the presence of 2 M urea.
13
Relative populations (%)
100
R
80
60
40
1S
20
0
0.1
1
10
100
Figure S32. Relative populations of SS intermediates
of Ins-B as a function of the reaction time.
Reaction conditions were [R]0=2[DHSox]0=31.6 μM at
25 °C and pH 10.0 in the presence of 0.2 M urea.
Reaction time (sec)
Relative populations (%)
100
R
80
60
40
1S
20
0
0.1
1
10
100
Figure S33. Relative populations of SS intermediates
of Ins-B as a function of the reaction time.
Reaction conditions were [R]0=[DHSox]0=31.1 μM at
25 °C and pH 10.0 in the presence of 0.2 M urea.
Reaction time (sec)
100
Relative populations (%)
R
80
60
40
20
Figure S34. Relative populations of SS intermediates
of Ins-B as a function of the reaction time.
Reaction conditions were [R]0=2[DHSox]0=37.2 μM at
25 °C and pH 10.0 in the presence of 4 M urea.
1S
0
0.1
1
10
Reaction time (sec)
100
14
Relative populations (%)
100
R
80
60
40
20
Figure S35. Relative populations of SS intermediates
of Ins-B as a function of the reaction time.
Reaction conditions were [R]0=[DHSox]0/2=31.0 μM at
25 °C and pH 10.0 in the presence of 4 M urea.
100
1S
0
0.1
10
1
k
DHSox + DTTred
10000
10
8000
8
UV absorbance
･ｓ-1
kDHSox +(mM
,-1M
s)
DTTred
-1 -1
12000
12
milliAbsorbance
at 310 nm (x 10-3)
Reaction time (sec)
1.2
B
A
d
c
0.8
0.4
b
0.0
0.0
6000
6
0.2
0.4
a
0.6
0.8
Time, s
4000
4
2000
2
00
4
4
55
66
7
7
88
9
9
10
10
pH
Figure S36. UV absorbance changes at 310 nm (panel A) and the plots of the second-order rate
constant against the pH obtained at 25 °C by the short-term oxidation of DTTred with DHSox using a
stopped-flow instrument. The reaction conditions of panel A were (a) [DTTred]0 = 2 mM, [DHSox]0 =
0.1 mM in 200 mM acetate buffer at pH 4.0, (b) [DTTred]0 = 2 mM, [DHSox]0 = 0.1 mM in 100 mM
Tris-HCl buffer at pH 7.0, (c) [DTTred]0 = 2 mM, [DHSox]0 = 0.1 mM in 100 mM Tris-HCl buffer at
pH 8.0, and (d) [DTTred]0 = 2 mM, [DHSox]0 = 0.1 mM in 25 mM NaHCO3 buffer at pH 10.0. See
the text for details of determination of the second-order rate constants.
15
UV absorbance at 215 nm
A
0
10
UV absorbance at 215 nm
B
0
20
30
40
50
40
50
Retention time (min)
10
20
30
Retention time (min)
Figure S37. HPLC chromatogramss of the peptide fragments of (A) 3S° and (B) 3S digested by
thermolysin. For detailed digestion and HPLC conditions, see experimental section in the text.
16