P i Release Kinetics
1. Prepare 600–800 μl solution containing 20 μM SufBC and
20 μM MDCC-PBP.
2. Prepare 600–800 μl 4 μM ATP.
3. Flush the stopped-flow system thoroughly with buffer, before
loading the reaction solutions.
4. Load the drive syringes with SufBC/MDCC-PBP and ATP
and prime with three pushes of both syringes.
5. Keep all the settings (excitation wavelength, slits, PM voltage)
as in the calibration.
6. Record traces on different time scales (e.g., 1, 10, 100 s) to find
the optimal time range for the measurement.
7. Record at least three traces in the appropriate time scales (here
10 and 100 s).
To convert the fluorescence traces into P i concentration
changes:
8. Subtract the minimum fluorescence value from all other values
in a trace to set the initial fluorescence to zero.
The P i concentration is calculated either by using the result from
the calibration curve (step 9) or by setting the end value of the P i
release trace to the used ATP concentration (step 10) (see Note 18).
9. Divide all offset corrected data points by the slope of the
calibration curve.
or
10. Divide all offset corrected data points by the end point of the
trace (average of the last few data points) and multiply by the
total ATP used (here 2 μM).
11. At the beginning of the trace, an artificial increase or decrease
of the fluorescence signal is often observed due to
re-equilibration of P i binding to PBP due to different concentrations of contaminating P i in the solutions. These data
points are deleted before data analysis.
12. While calibration is necessary for the interpretation and analysis of multi-turnover data, for single-turnover experiments
conversion of fluorescence data into P i concentration is not
required, but rate constants can directly be obtained from
curve fitting to the raw data (arbitrary fluorescence units).
Data Interpretation
and Analysis
P i release kinetics during a single turnover of ATP hydrolysis by
SufBC are shown in Fig. 7. The increase in [P i ] was analyzed using a
single-exponential curve (see Eq. 6), which gives a rate constant of
0.11 s
À1 (see Note 19). A very similar rate constant has been
previously determined for the cleavage of a fluorescent analogue,
mant-ATP (k +2
mATP
¼ 0.088 s
À1
) using the quench-flow technique
310
Simone Kunzelmann
1. Prepare 600–800 μl solution containing 20 μM SufBC and
20 μM MDCC-PBP.
2. Prepare 600–800 μl 4 μM ATP.
3. Flush the stopped-flow system thoroughly with buffer, before
loading the reaction solutions.
4. Load the drive syringes with SufBC/MDCC-PBP and ATP
and prime with three pushes of both syringes.
5. Keep all the settings (excitation wavelength, slits, PM voltage)
as in the calibration.
6. Record traces on different time scales (e.g., 1, 10, 100 s) to find
the optimal time range for the measurement.
7. Record at least three traces in the appropriate time scales (here
10 and 100 s).
To convert the fluorescence traces into P i concentration
changes:
8. Subtract the minimum fluorescence value from all other values
in a trace to set the initial fluorescence to zero.
The P i concentration is calculated either by using the result from
the calibration curve (step 9) or by setting the end value of the P i
release trace to the used ATP concentration (step 10) (see Note 18).
9. Divide all offset corrected data points by the slope of the
calibration curve.
or
10. Divide all offset corrected data points by the end point of the
trace (average of the last few data points) and multiply by the
total ATP used (here 2 μM).
11. At the beginning of the trace, an artificial increase or decrease
of the fluorescence signal is often observed due to
re-equilibration of P i binding to PBP due to different concentrations of contaminating P i in the solutions. These data
points are deleted before data analysis.
12. While calibration is necessary for the interpretation and analysis of multi-turnover data, for single-turnover experiments
conversion of fluorescence data into P i concentration is not
required, but rate constants can directly be obtained from
curve fitting to the raw data (arbitrary fluorescence units).
Data Interpretation
and Analysis
P i release kinetics during a single turnover of ATP hydrolysis by
SufBC are shown in Fig. 7. The increase in [P i ] was analyzed using a
single-exponential curve (see Eq. 6), which gives a rate constant of
0.11 s
À1 (see Note 19). A very similar rate constant has been
previously determined for the cleavage of a fluorescent analogue,
mant-ATP (k +2
mATP
¼ 0.088 s
À1
) using the quench-flow technique
310
Simone Kunzelmann