126
1. Dissolve 15 mg of adenylic acid (AMP) and 5 mg of LH 2 in
1 mL of dimethyl sulfoxide.
2. Dissolve 100 mg of dicyclohexylcarbodiimide in 1 mL of
dimethyl sulfoxide.
3. Mix the solutions of 1 and 2.
4. Incubate for 10 min at room temperature (see Note 14).
5. Add 5 mL of acetone to stop the reaction.
6. Centrifuge and carefully remove the supernatant.
7. Add 3 mL of acetone.
8. Centrifuge and carefully remove the supernatant.
9. Repeat steps 7 and 8.
10. Dissolve the precipitate in 1.5 mL of the solution of acetic acid
(10 mM) and NaCl (40 mM).
11. Apply to the column containing the resin of Toyopearl HW-40S
(bed volume ~2 mL).
12. Collect the fraction by adding 1 mL of diluted hydrochloric
acid (pH 4.5) (see Notes 15 and 16).
13. Repeat step 12 10–15 times.
14. Measure the fluorescence spectrum of the each fraction in
steps 12 and 13 with excitation wavelength of 327 nm, and
collect the fractions that emit fluorescence peaking at around
535 nm (see Note 17) [7]. When the peak values of LH 2 -AMP
and x nM of LH 2 are y and z, respectively, the concentration of
LH 2 -AMP can be determined as follows (see Note 18).
The concentration of LH 2 -AMP (nM) =
0.45
z
x y
×
×
.
1. Prepare substrate solutions with varied concentrations
1–2000 μM of LH 2 in the MOPS buffer containing 20 mM of
ATP (see Note 20).
2. Dispense 50 μL of the solution to a well of 96-well plate.
3. Measure the bioluminescence intensity after injection of 50 μL
of 50 nM of the probe (BD1/Donor or BD2/Acceptor)
diluted in the MOPS buffer with 0.1-s interval for 10 s.
4. Use the maximal light intensity when each concentration of
LH 2 was applied and perform nonlinear curve fitting to derive
V max and K m using Kaleida Graph 4.1.
1. Prepare solutions with varying concentrations (3–2000 μM)
of ATP in MOPS buffer containing 10 mM of LH 2 (see
Note 20).
2. Follow the steps 2–4 in Subheading 3.6.
3.5 Chemical
Synthesis
and Purification
of LH 2 -AMP
(See Note 13) [5, 6]
3.6 LH 2 Kinetic
Determination of BD1/
Donor and BD2/
Acceptor (See Note 19)
3.7 ATP Kinetic
Determination
of BD1/Donor and
BD2/Acceptor
(See Note 19)
Yuki Ohmuro-Matsuyama and Hiroshi Ueda
1. Dissolve 15 mg of adenylic acid (AMP) and 5 mg of LH 2 in
1 mL of dimethyl sulfoxide.
2. Dissolve 100 mg of dicyclohexylcarbodiimide in 1 mL of
dimethyl sulfoxide.
3. Mix the solutions of 1 and 2.
4. Incubate for 10 min at room temperature (see Note 14).
5. Add 5 mL of acetone to stop the reaction.
6. Centrifuge and carefully remove the supernatant.
7. Add 3 mL of acetone.
8. Centrifuge and carefully remove the supernatant.
9. Repeat steps 7 and 8.
10. Dissolve the precipitate in 1.5 mL of the solution of acetic acid
(10 mM) and NaCl (40 mM).
11. Apply to the column containing the resin of Toyopearl HW-40S
(bed volume ~2 mL).
12. Collect the fraction by adding 1 mL of diluted hydrochloric
acid (pH 4.5) (see Notes 15 and 16).
13. Repeat step 12 10–15 times.
14. Measure the fluorescence spectrum of the each fraction in
steps 12 and 13 with excitation wavelength of 327 nm, and
collect the fractions that emit fluorescence peaking at around
535 nm (see Note 17) [7]. When the peak values of LH 2 -AMP
and x nM of LH 2 are y and z, respectively, the concentration of
LH 2 -AMP can be determined as follows (see Note 18).
The concentration of LH 2 -AMP (nM) =
0.45
z
x y
×
×
.
1. Prepare substrate solutions with varied concentrations
1–2000 μM of LH 2 in the MOPS buffer containing 20 mM of
ATP (see Note 20).
2. Dispense 50 μL of the solution to a well of 96-well plate.
3. Measure the bioluminescence intensity after injection of 50 μL
of 50 nM of the probe (BD1/Donor or BD2/Acceptor)
diluted in the MOPS buffer with 0.1-s interval for 10 s.
4. Use the maximal light intensity when each concentration of
LH 2 was applied and perform nonlinear curve fitting to derive
V max and K m using Kaleida Graph 4.1.
1. Prepare solutions with varying concentrations (3–2000 μM)
of ATP in MOPS buffer containing 10 mM of LH 2 (see
Note 20).
2. Follow the steps 2–4 in Subheading 3.6.
3.5 Chemical
Synthesis
and Purification
of LH 2 -AMP
(See Note 13) [5, 6]
3.6 LH 2 Kinetic
Determination of BD1/
Donor and BD2/
Acceptor (See Note 19)
3.7 ATP Kinetic
Determination
of BD1/Donor and
BD2/Acceptor
(See Note 19)
Yuki Ohmuro-Matsuyama and Hiroshi Ueda