Fluorimeter 6285 Application note: A10-007A

Phosphatase assays using DiFMUP as substrate

Introduction carried out in a final volume of 2.0ml in plastic

Phosphatases are enzymes that remove phosphate
groups from a substrate; this may be another enzyme,
protein, DNA, RNA or an artificial substrate used to
measure phosphatase activity. De-phosphorylation
may either activate or deactivate an enzyme, resulting
in an increase or decrease in activity of a particular
biochemical pathway. Due to this ‘switch’ like activity,
phosphatases are often integral to many signal
transduction pathways; they are also important in
digestion and turnover and mobilisation of inorganic
phosphate.
In this application note we demonstrate use of the
®
EnzChek Phosphatase Assay Kit (E12020) from
Invitrogen. This fluorimetric kit contains the artificial
substrate, 6,8-difluoro-4-methylumbelliferyl phosphate
(DiFMUP), a fluorinated relative of 4-
methylumbelliferyl phosphate (MUP). Unlike MUP
however, which is limited to measuring alkaline
phosphatase activity, DiFMUP can be used at neutral,
acidic and alkaline pH. Using this kit with the Jenway
Model 6285 fluorimeter we demonstrate activity of
both acid phosphatases (potato and prostatic) and
calf intestinal alkaline phosphatase simply by
changing the reaction buffer and hence pH conditions.
Methods
® 80
The EnzChek Phosphatase Assay Kit (E12020) was
purchased from Invitrogen. This kit includes DiFMUP
fluorimeter cuvettes (060 247).
The 6285 fluorimeter was fitted with the UG1 320-
380nm bandpass filter (627 126) for excitation and the
interference filter 460nm (627 167) for emission; the
reaction product of the assay has an
excitation/emission maxima of approximately
358/455nm.
The fluorimeter was connected to a PC and Jenway’s
DataWay software used for data acquisition; the
instrument was set up to take readings every 20
seconds. For experiments where temperature control
was required, the heated cell system (628 200,
comprising heated cell holder, controller and
connection leads) was also fitted to the fluorimeter.
Results
The time course of phosphatase activity was
measured over a period of 2 hours for both POTAP
and CIAP. Both enzymes were diluted to 1U/ml and
10µl added to a cuvette containing 1x reaction buffer
(enzyme specific) and 100µM DiFMUP to give a final
enzyme amount of 0.01U in a 2.0ml volume.
Reactions were performed at room temperature and
the results shown in Figure 1.
70

substrate, N,N-dimethylformamide (DMF) for 60

dissolving the DiFMUP, 5x reaction buffer (0.5M 50
RFU

sodium acetate, pH 5.0) and a sample of potato acid 40

phosphatase (POTAP). Prostatic acid phosphatase 30 POTAP
(PAP) from bovine prostate was purchased from 20 CIAP
Sigma (P6409). Calf intestinal alkaline phosphatase 10 Blank
(CIAP) and buffer was purchased from Promega 0
(M1821). 0 20 40 60 80 100 120
Time (min)
The kit components were prepared according to the
Figure 1: Time course for POTAP and CIAP activity.
manufacturer’s instructions. Briefly, just before use, a
The fluorimeter gain was set to 35%.
vial of DiFMUP was dissolved in 200µl DMF to give a
stock solution of 10mM; this is equivalent to a 100x
Both enzymes gave a steady increase in fluorescence
working solution. The POTAP was diluted to 10U/ml
with CIAP showing a slower activity than POTAP.
using 1x reaction buffer (0.1M sodium acetate, pH
This is possibly due to the incubation temperature:
5.0) and stored in aliquots at -20ºC. PAP was also
CIAP in vivo would normally be most active at 37ºC,
dissolved in 1x reaction buffer to a final concentration
whereas the plant POTAP needs to be active at
of 10mg/ml; this too was stored in aliquots at -20ºC.
cooler, ambient temperature conditions.
Both POTAP and PAP were diluted as required in 1x
reaction buffer. CIAP was supplied in storage buffer at
1U/µl and was diluted as required in 1x CIAP buffer Next, the detection of various amounts of POTAP was
(50mM Tris-HCl (pH 9.3 at 25ºC), 1mM MgCl2, 0.1mM measured over a period of 90 minutes at room
ZnCl2 and 1mM spermidine). All reactions were temperature. The results are shown in Figure 2 and
indicate that as little as 0.0001U of POTAP can be

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clearly detected after only a relatively short incubation temperature approximately two degrees lower than
period, illustrating the high sensitivity of the assay. that set on the heated cell controller (Figure 4).

A similar experiment was repeated with PAP. This For the temperature controlled assay, the cuvette
enzyme was supplied by weight and not in units; containing buffer and DiFMUP was placed in the cell
therefore the amount added per assay is shown in mg holder and allowed to equilibrate for 15 minutes prior
in Figure 3. to adding 0.1mg PAP. Readings were then taken for a
100
further 30 minutes and the results are shown in Figure
90
5. Results show that activity at 45ºC (sample chamber
80
A temperature) was approximately twice that at 25ºC.
0.02

70 0.01
0.005
60 40
0.002
RFU

50 0.001
0.0005 35
40
0.0002

Sample temp ( o C)
30 0.0001
20 0 30

10
25
0
0 20 40 60 80
Time (min) 20

10 15
0 2 4 6 8 10 12 14 16 18 20
9

8
B Time (min)

7
0.001
Figure 4: Sample temperature measured using a
6
0.0005 thermocouple with the cuvette holder set to 37ºC. 2ml
RFU

5 0.0002 of water were placed in the cuvette.

0.0001
4
0 80
3
70
2
60
1
25ºC
0 50
30ºC
0 20 40 60 80
RFU

40 35ºC
Time (min)
40ºC
30
45ºC
Figure 2: Time course for detection of different 20
amounts of POTAP. A: all data (U added per cuvette).
10
B: expanded detail of the low enzyme concentrations.
0
The fluorimeter gain was set to 35%. 0 5 10 15 20 25 30 35 40 45
Time (min)

120
Figure 5: Effect of temperature on PAP activity.
100 0.1mg PAP added to reaction after 15 min
0mg
1mg
temperature equilibration. Temperature shown is that
80
0.5mg set on the heated cell holder.
0.2mg
RFU

60
0.1mg
0.05mg
CIAP is commonly used in molecular biology to
40
0.02mg remove 5’ phosphates from DNA and RNA. Following
20
0.01mg
dephosphorylation, the CIAP activity needs to be
removed to allow downstream processes. This can be
0 done by heat denaturing the enzyme at either 65ºC
0 10 20 30 40 50 60 70 80 90
Time (min)
for 60 min or 75ºC for 10 min in the presence of 5mM
EDTA (1). To check that these conditions completely
inactivate CIAP, phosphatase activity was measured
Figure 3: Time course of PAP reaction with DiFMUP. before and after heat treatment and in the absence
Different amounts of PAP were incubated at room and presence of 5mM EDTA.
temperature. The fluorimeter gain was set to 35%.
Six samples of 0.01U/µl CIAP were prepared, three of
Enzyme activity is affected by temperature. We which contained a final concentration of 5mM EDTA.
therefore looked at the effect of different incubation Two pairs of samples (with and without EDTA) were
temperatures on PAP activity using the heated cell then heated at either 65ºC for 1 hour or 75ºC for 10
holder and controller. Preliminary experiments using a minutes in a dry block heater. 10µl of each sample
thermocouple placed in 2ml water in a cuvette (0.1U CIAP) was then assayed with DiFMUP to check
showed that the sample reached a steady for phosphatase activity. Results are shown in Figure
temperature after about 15 minutes in the cell holder. 6.
Under the conditions used, the sample settled at a

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Adding EDTA to the sample appeared to enhance Conclusions
activity of unheated CIAP, although after heat We have demonstrated here that the Jenway Model
treatment some residual activity remained after 1 h at 6285 fluorimeter in combination with DataWay data
65ºC in the sample where EDTA had not been added. acquisition software can be used for dynamic
Therefore the combination of both EDTA and heat fluorescence assays such as the EnzChek®
treatment is important for complete enzyme Phosphatase Assay Kit. Time course and enzyme
inactivation. concentration determination are readily achieved with
a high level of sensitivity. In addition, the temperature
80 controlled cell holder enables specific assay
70
A temperatures to be maintained.
60
No EDTA, no heat The assay could equally be performed on the Model
50 + EDTA, no heat
No EDTA, 65ºC 60min
6280 fluorimeter which has the same sensitivity
RFU

40
+EDTA, 65ºC 60min specification as the 6285. Both models employ a
30 No EDTA, 75ºC 10min
+EDTA, 75ºC 10min
highly sensitive photomultiplier detector, with red
20 enhancement on the model 6285 allowing detection
10 up to 850nm.
0
0 5
Time (min)
10 15
References
(1) Sambrook, J. and Russell, D.W. Molecular
1.5
Cloning: A Laboratory Manual. Third Edition. Cold
Spring Harbor Laboratory Press. 2001.
B
1.4
No EDTA, 65ºC 60min
+EDTA, 65ºC 60min
RFU

No EDTA, 75ºC 10min

+EDTA, 75ºC 10min
1.3

1.2
0 5 10 15
Time (min)

Figure 6: Heat denaturation of CIAP. Background

measurements were taken for the first 4 minutes
followed by the addition of 0.1U CIAP. A: activity with
and without heat treatment. B: expanded detail of the
heat-treated samples showing some activity in the
sample heated at 65ºC without EDTA.

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Tel: 01785 810433