Supplementary Data
Significance of anionic functional group in betaine-type metabolite
analogs on the facilitation of enzyme reactions
Yuichi Nakagawa, Kotomi Takagi, Ryutaro Genjima, Kazuya Koumoto*
Department of Nanobiochemistry, FIRST (Frontiers of Innovative Research in Science and Technology), Konan
University, 7-1-20 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan.
Contents
1.
Influence of sulfobetaines on the facilitation of various enzyme reactions S2
2.
Solution properties of sulfobetaine-containing aqueous solutions
S4
3.
1
S5
4.
13
C NMR spectra of sulfobetaines
S7
5.
HRESI-MS spectra of sulfobetaines
S9
H NMR spectra of sulfobetaines
S1
1. Influence of sulfobetaines on the facilitation of various enzyme reactions
<Materials>
Sulfatase from Helix pomatia, p-nitrophenylphosphate disodium salt hexahydrate and potassium
p-nitrophenylsulfate were purchased from Sigma-Ardrich. -Glucosidase from agrobacterium sp.
was purchased from Megazyme International. Alkaline phosphatase from calf intestine and
horseradish peroxidase were purchased from Wako Pure Chemical Ltd. ABTS and
p-nitrophenyl--D-glucopyranoside were purchased from Tokyo Chemical Industry, Ltd. They were
used without purification unless otherwise noted. The following substrate-enzyme pairs were used:
p-nitrophenyl-β-D-glucopyranoside for -glucosidase, p-nitrophenylphosphate disodium salt
hexahydrate for alkaline phosphatase, potassium p-nitrophenylsulfate for sulfatase, and
2,2′-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) ammonium salt (ABTS) for horseradish
peroxidase.
<Enzyme reaction protocol using -glucosidase,alkaline phosphatase, sulfatase >
The enzyme reactions were performed in a 96-well multi-plate. A 1 µL aliquot of enzyme stock
solution (-glucosidase: 50 µg/mL,alkaline phosphatase: 50 µg/mL, sulfatase: 626 µg/mL) was
placed onto the wall of each well of a 96-well plate containing 199 µL of the substrate, analog and
buffer solutions, which were prepared by mixing the aqueous stock solutions, and incubated at
37 °C for 5 min. The enzyme reaction was started by shaking the plate, thus mixing together the
enzyme stock solution, substrate, analogs and buffer. The final concentration of each reaction is as
follows:
[-glucosidase]=0.25
µg/ml,
[phosphate
buffer
(pH
7.0)]=100
mM,
[p-nitrophenyl-β-D-glucopyranoside]=2.0 mM, [analog]= 0-1000 mM; [alkaline phosphatase]= 0.25
µg/ml, [Tris buffer (pH 7.5)]=100 mM, [p-nitrophenylphosphate disodium salt hexahydrate]=2.0
mM, [analog]= 0-1000 mM; [sulfatase]=3.13 µg/ml, [phosphate buffer (pH 7.0)]=100 mM,
[potassium p-nitrophenylsulfate]=20 mM, [analog]= 0-1000 mM.
The initial slopes of the absorbance against incubation time were converted into the initial velocities
using the molar extinction coefficient (11,200 cm-1M-1 at 405 nm for -glucosidase; 15,100 cm-1M-1
at 405 nm for alkaline phosphatase; 11,200 cm-1M-1 at 405 nm for sulfatase). The initial velocities
were estimated as the average of five measurements.
<Enzyme reaction protocol using horseradish peroxidase>
The enzyme reactions were performed in a 96-well multi-plate. A 1 µL aliquot of horseradish
peroxidase stock solution ( µg/mL) and 1 µL aliquot of H2O2 stock solution were placed onto the
wall of each well of a 96-well plate containing 198 µL of the substrate, analog and buffer solutions,
which were prepared by mixing the aqueous stock solutions, and incubated at 37 °C for 5 min. The
enzyme reaction was started by shaking the plate, thus mixing together the enzyme, H2O2, substrate,
analog and buffer. The final concentration is adjusted to [horseradish peroxidase]=5.0 ng/ml, [citric
S2
acid buffer (pH 4.2)]=100 mM, [H2O2]=20 mM, [ABTS]=1.0 mM, [analog]= 0-1000 mM. The
initial slopes of the absorbance against incubation time were converted into the initial velocities
using the molar extinction coefficient (25,000 cm-1M-1 at 405 nm). The initial velocities were
estimated as the average of five measurements.
2.0
2.0
(a)
(b)
1.5
Activity
Activity
1.5
1.0
0.5
0.5
0.0
1.0
0
200
400
600
800
0.0
1000
0
200
[SB-4 or CB-4] / mM
(c)
800
1000
800
1000
(d)
1.5
Activity
1.5
Activity
600
2.0
2.0
1.0
1.0
0.5
0.5
0.0
400
[SB-4 or CB-4] / mM
0.0
0
200
400
600
800
1000
[SB-4 or CB-4] / mM
0
200
400
600
[SB-4 or CB-4] / mM
Fig. S1 Comparison of enzyme activation behaviors as a function of SB-4 (closed circle) or CB-4
(open circle) concentration for (a) -glucosidase from almond (b) alkaline phosphatase from calf
intestine (c) sulfatase from Helix pomatia (d) horseradish peroxidase. Error bars were represented
as the standard deviation of five measurements.
S3
2. Solution properties of sulfobetaine-containing aqueous solutions
Table S1. Average grain diameter (nm) of sulfobetaines estimated by dynamic light scattering
300 mM
500 mM
700 mM
1000 mM
SB-1
0.87±0.09
0.88±0.06
0.87±0.02
0.85±0.09
SB-2
0.97±0.05
0.98±0.03
0.96±0.02
0.97±0.01
SB-3
1.07±0.05
1.16±0.03
1.23±0.02
1.32±0.05
SB-4
1.42±0.08
1.41±0.03
1.50±0.04
1.66±0.04
Table S2. Osmolarity (mOsm kg-1) of sulfobetaine-containing aqueous solution.
0
100 mM
400 mM
700 mM
1000 mM
SB-1
12.67±0.94
98.60±6.38
457.60±3.72
874.40±1.85
1365.00±1.79
SB-2
12.67±0.94
105.20±3.66
488.60±2.50
955.20±1.72
1525.80±2.48
SB-3
12.67±0.94
100.20±4.40
478.60±2.58
966.20±3.25
1612.00±6.13
SB-4
12.67±0.94
102.20±2.64
498.60±4.22
1064.40±3.61
1877.60±5.24
Table S3. Water density (mg mL-1) of sulfobetaine-containing aqueous solution.
0
100 mM
400 mM
700 mM
1000 mM
SB-1
1005.85±0.93
1013.95±1.35
1025.32±1.09
1040.11±0.83
1055.59±0.88
SB-2
1005.85±0.93
1012.62±1.52
1024.19±0.44
1037.31±0.85
1052.78±1.23
SB-3
1005.85±0.93
1011.04±1.21
1021.84±0.70
1034.03±1.00
1047.58±2.85
SB-4
1005.85±0.93
1010.69±0.98
1021.03±0.64
1032.40±1.34
1043.18±1.10
S4
3. 1H NMR spectra of sulfobetaines
Fig. S2 1H NMR spectrum of SB-1
Fig. S3. 1H NMR spectrum of SB-2
S5
Fig. S4 1H NMR spectrum of SB-3
Fig. S5 1H NMR spectrum of SB-4
S6
4. 13C NMR spectra of sulfobetaines
Fig. S6 13C NMR spectrum of SB-1
Fig. S7 13C NMR spectrum of SB-2
S7
Fig. S8 13C NMR spectrum of SB-3
Fig. S9 13C NMR spectrum of SB-4
S8
5. HRESI-MS spectra of sulfobetaines
Fig. S10 HRESI-MS spectrum of SB-1
Fig. S11 HRESI-MS spectrum of SB-2
S9
Fig. S12 HRESI-MS spectrum of SB-3
Fig. S13 HRESI-MS spectrum of SB-4
S10