contrast, chemical cleavage is often reversible (e.g., for myosin
[63, 64]), and this should be considered when interpreting
transient kinetic data of a novel enzyme.
15. Using cold solutions can lead to the formation of gas bubbles
when warmed up in the stopped-flow instrument (in particular
at temperatures above 25
C), which causes disturbances in the
signal. For this reason, it is best to prepare all solutions with
buffer at room temperature and not keep premade solutions
on ice.
16. Ideally, MDCC-PBP is included in both solutions, enzyme and
ATP, at the final concentration to minimize artifacts, e.g., a
small increase or decrease in fluorescence at the beginning of
the reaction caused by re-equilibration of P i binding due to
different P i concentrations in the two solutions. However, to
save material, for example, in the P i calibration or when an ATP
concentration series is used, it is often preferable to use
MDCC-PBP only in the enzyme solution.
17. The slit width and photomultiplier voltage setting depend on
the instrument, lamp age, and concentration of PBP used.
18. Using the calibration data to calculate the P i concentration
changes can sometimes deviate significantly from the expected
concentration of released P i that is the initial nucleotide concentration. Therefore, if there is a defined end value, like in
single-turnover kinetics, the final P i concentration is often set
to the ATP concentration used, assuming that all ATP has been
converted to free P i at the end of the reaction. This is a valid
assumption for most systems. If the reaction is not observed
until the complete substrate turnover, e.g., in multi-turnover
experiments, one has to rely on the calibration data. One
problem with calibration in the stopped-flow is that absolute
fluorescence values (as opposed to amplitudes of fluorescence
changes) are often not very reproducible and due to the fast
binding of P i to MDCC-PBP only end values can be analyzed.
One way around this problem is to measure the calibration by
mixing MDCC-PBP plus different [P i ] with the P i mop and
follow the slow kinetics of P i removal [48]. Other sources of
errors include errors in nucleotide concentration, concentration of the P i solutions, or the fact that the calibration is
normally not measured in exactly the same conditions (in the
presence of enzyme and ATP) as the actual P i release trace.
19. It has to be noted that the P i release data in Fig. 7a are
described better with a double-exponential fit than a single
exponential, suggesting that the mechanism is more complex,
for example, including a conformation change after ATP binding [54, 55].
Phosphate Biosensor Assays
315
[63, 64]), and this should be considered when interpreting
transient kinetic data of a novel enzyme.
15. Using cold solutions can lead to the formation of gas bubbles
when warmed up in the stopped-flow instrument (in particular
at temperatures above 25
C), which causes disturbances in the
signal. For this reason, it is best to prepare all solutions with
buffer at room temperature and not keep premade solutions
on ice.
16. Ideally, MDCC-PBP is included in both solutions, enzyme and
ATP, at the final concentration to minimize artifacts, e.g., a
small increase or decrease in fluorescence at the beginning of
the reaction caused by re-equilibration of P i binding due to
different P i concentrations in the two solutions. However, to
save material, for example, in the P i calibration or when an ATP
concentration series is used, it is often preferable to use
MDCC-PBP only in the enzyme solution.
17. The slit width and photomultiplier voltage setting depend on
the instrument, lamp age, and concentration of PBP used.
18. Using the calibration data to calculate the P i concentration
changes can sometimes deviate significantly from the expected
concentration of released P i that is the initial nucleotide concentration. Therefore, if there is a defined end value, like in
single-turnover kinetics, the final P i concentration is often set
to the ATP concentration used, assuming that all ATP has been
converted to free P i at the end of the reaction. This is a valid
assumption for most systems. If the reaction is not observed
until the complete substrate turnover, e.g., in multi-turnover
experiments, one has to rely on the calibration data. One
problem with calibration in the stopped-flow is that absolute
fluorescence values (as opposed to amplitudes of fluorescence
changes) are often not very reproducible and due to the fast
binding of P i to MDCC-PBP only end values can be analyzed.
One way around this problem is to measure the calibration by
mixing MDCC-PBP plus different [P i ] with the P i mop and
follow the slow kinetics of P i removal [48]. Other sources of
errors include errors in nucleotide concentration, concentration of the P i solutions, or the fact that the calibration is
normally not measured in exactly the same conditions (in the
presence of enzyme and ATP) as the actual P i release trace.
19. It has to be noted that the P i release data in Fig. 7a are
described better with a double-exponential fit than a single
exponential, suggesting that the mechanism is more complex,
for example, including a conformation change after ATP binding [54, 55].
Phosphate Biosensor Assays
315