2 μL of 100 U/mL PNP and 2 μL of the 3 U/mL inorganic
pyrophosphatase (see Note 8). Add enough water to reach a
final total volume of 200 μL minus the volumes of the enzyme,
IPP and DMAPP that will be added later (Table 1).
2. Add 5 μg of the purified enzyme (see Notes 9 and 10) to the
reaction mixture and mix well by up and down pipetting.
3. Preincubate at room temperature for 10 min (see Note 11).
4. Add the enzyme substrates IPP and DMAPP as shown in
Table 1 for a standard reaction (see Note 12) and mix well by
up and down pipetting. It is very important to always perform a
control assay in parallel in which solvent buffer will be added
instead of the experimental substrates (no-substrate control, see
Note 13, Fig. 2).
5. Immediately introduce the plate in the spectrophotometer,
shake it and start measuring the absorbance at 360 nm as a
function of time during 1 h at 25
C. Reading the absorbance
every 2 min should be enough to build the activity curve (see
Note 14).
6. When analyzing the data, remember subtracting the no-substrate control values from the corresponding experimental samples (see Note 15).
7. Calculate the enzyme activity obtained for each condition as
the increase of PPi production per minute and μg of enzyme
(μM PPi min
À1
μg
À1 ) (see Note 16). Use them to build a pH
curve (see Note 17) or to obtain the kinetic parameters of the
enzyme (V max and K m ) from the Michaelis–Menten curve
using the GraphPad Prism software (see Note 18).
3.2 Standard Curve
for PPi Quantification
The linear range for the quantification of PPi using the EnzChek
Kit is from 1 μM to 75 μM.
1. Follow the standard reaction (Table 1) adding increasing
amounts of the 500 μM PPi standard solution (see Note 19)
and omitting the volumes of the substrates (IPP and DMAPP)
and the experimental enzyme.
2. Prepare a no-PPi control without PPi (0 μM point, see Note
19). Also, include a no-pyrophosphatase control as a blank with
no PPi and no inorganic pyrophosphatase (see Note 11).
3. After mixing all the reagents, incubate the plate at 25
C for
60 min (see Note 20).
4. Measure the absorbance at 360 nm.
5. Subtract the no-pyrophosphatase control absorbance value from
each experimental reaction and build the PPi standard curve
plotting the absorbance at 360 nm as a function of PPi
concentration.
32
M. Victoria Barja and Manuel Rodrı ´guez-Concepcio ´ n
pyrophosphatase (see Note 8). Add enough water to reach a
final total volume of 200 μL minus the volumes of the enzyme,
IPP and DMAPP that will be added later (Table 1).
2. Add 5 μg of the purified enzyme (see Notes 9 and 10) to the
reaction mixture and mix well by up and down pipetting.
3. Preincubate at room temperature for 10 min (see Note 11).
4. Add the enzyme substrates IPP and DMAPP as shown in
Table 1 for a standard reaction (see Note 12) and mix well by
up and down pipetting. It is very important to always perform a
control assay in parallel in which solvent buffer will be added
instead of the experimental substrates (no-substrate control, see
Note 13, Fig. 2).
5. Immediately introduce the plate in the spectrophotometer,
shake it and start measuring the absorbance at 360 nm as a
function of time during 1 h at 25
C. Reading the absorbance
every 2 min should be enough to build the activity curve (see
Note 14).
6. When analyzing the data, remember subtracting the no-substrate control values from the corresponding experimental samples (see Note 15).
7. Calculate the enzyme activity obtained for each condition as
the increase of PPi production per minute and μg of enzyme
(μM PPi min
À1
μg
À1 ) (see Note 16). Use them to build a pH
curve (see Note 17) or to obtain the kinetic parameters of the
enzyme (V max and K m ) from the Michaelis–Menten curve
using the GraphPad Prism software (see Note 18).
3.2 Standard Curve
for PPi Quantification
The linear range for the quantification of PPi using the EnzChek
Kit is from 1 μM to 75 μM.
1. Follow the standard reaction (Table 1) adding increasing
amounts of the 500 μM PPi standard solution (see Note 19)
and omitting the volumes of the substrates (IPP and DMAPP)
and the experimental enzyme.
2. Prepare a no-PPi control without PPi (0 μM point, see Note
19). Also, include a no-pyrophosphatase control as a blank with
no PPi and no inorganic pyrophosphatase (see Note 11).
3. After mixing all the reagents, incubate the plate at 25
C for
60 min (see Note 20).
4. Measure the absorbance at 360 nm.
5. Subtract the no-pyrophosphatase control absorbance value from
each experimental reaction and build the PPi standard curve
plotting the absorbance at 360 nm as a function of PPi
concentration.
32
M. Victoria Barja and Manuel Rodrı ´guez-Concepcio ´ n