Role of Phosphatase Enzymes in Metabolic Reactions
Background
Phosphatase enzymes are involved in a range of
metabolic reactions. A key function of these
enzymes is to release phosphate groups into the
metabolic pool thereby increasing their availability
for use in a range of processes including ATP
synthesis and membrane construction.
Background
Phosphatase is one of the enzyme
systems mentioned into the CfE Higher
and Higher Human course specification.
Background
Acid phosphatases (those with an optimum pH <7.0) can
be extracted from a range of plant tissues – germinating
mung beans or bean sprouts are a cheap and reliable
source.
The substrate is phenolphthalein bisphosphate (PPP).
Under suitable conditions, phosphatase catalyses the
breakdown of PPP to form phenolphthalein (PP).
At neutral or acidic pH, the products of the above
reaction (PP and phosphate) are both colourless – so
their presence is difficult to detect.
This can be overcome through the addition of sodium
carbonate which has 2 effects:
1) raising the pH of the solution >pH10 with cessation of
enzyme activity
2) converts PP to its anionic form, which is pink.
Brief protocol outline
1.
Add 1 cm
3
sodium carbonate solution to 7
cuvettes.
2.
Prepare enzyme extract and substrate
3.
Mix enzyme extract with buffer
4.
Add 1 cm
3
enzyme/buffer mixture to cuvette 1 –
Blank
5.
Add substrate solution to the mixture of
enzyme/buffer.
6.
At 2 minute intervals, remove 1cm
3
of the
substrate/enzyme/buffer mixture and add to one
of the cuvettes.
7.
Measure the absorbance at 550 nm.
Colorimeter – Biochrome (550nm)
To measure absorbance, ensure the colorimeter
has “Abs” on the display for absorbance. If not,
press the Abs / %T button (T – transmission).
The colorimeter has a range of LED light sources
emitting light of specific wavelengths. Turn the
wheel on the right hand side until 550nm is
displayed above the cuvette holder.
When reading the colorimetric blank, press and
hold R (Reference) – should produce a reading of
0.00
When reading a sample, press T (Test).
Part A – Preparing the enzyme extract
Materials
•
20g Bean sprouts
•
Distilled water
•
Pestle and mortar
•
Scissors
•
plastic pipette
•
6x microfuge tubes
•
Microcentrifuge
•
Marker pen
•
Bijou labelled “Enzyme extract”
Germination – if using mung beans (would use
about 30 per extraction)
Take ~20g bean sprouts and place them into a
mortar. Remove and discard the green seed
case (testa) if it is still attached.
Add 5 cm
3
distilled water and grind the bean
sprouts with a pestle to achieve a smooth paste.
Cut the end of a 3 cm
3
plastic pipette. Divide
the bean sprout mixture equally between 6
microcentrifuge tubes. Should be
approximately equal volume to ensure the
centrifuge rotor is balanced. Close the lids and
label with your initials.
Centrifuge your samples for 5 minutes to
produce a pellet.
Carefully remove the supernatant from the
microfuge tubes (using a clean disposable
pipette) and pool their contents in a clean bijou
marked “enzyme extract”.
To carry out the assay at pH5 and pH7, at least
5 cm
3
enzyme extract is required. If the
supernatant is less than this volume, add some
distilled water. A bijou holds 7 cm
3
.
Part B – the phosphatase assay
Materials
•
Enzyme extract (at least 5 cm
3
)
•
10 cm
3
citric acid/phosphate buffer, pH 5.0
•
10 cm
3
citric acid/phosphate buffer, pH 7.0
•
6 cm
3
0.2% phenolphthalein phosphate
•
25 cm
3
10% (w/v) sodium carbonate
•
Stopclock
•
Waterbath (30 ºC)
•
Paper towels
•
14x absorption cuvettes
•
Cuvette rack
•
Colorimeter (we are using a Biochrome colorimeter – 550 nm)
•
Automatic pipette and clean tips
•
Polystyrene cup (or similar)
Pipettes
:
The volumes outlined in the following steps
should be dispensed using the automatic 1 cm
3
pipette with a clean tip.
Temperature
:
All solutions should be kept at 30 ºC
throughout the assay.
Transfer water from the waterbath (30 ºC) to
the polystyrene cup. Stand the universal of
buffer (pH 5.0), bijou of enzyme extract and
bijou of substrate (PPP) in the cup.
Using the 1 cm
3
automatic pipette, add
1 cm
3
sodium carbonate into 7
cuvettes. Discard the tip.
Transfer 2 cm
3
enzyme extract to the
buffer solution in the universal bottle.
For the colorimetric blank: transfer 1 cm
3
of
the mixture from the universal (buffer +
enzyme) to the first cuvette.
Transfer 2 cm
3
PPP to the enzyme/buffer
mixture. Start the stopwatch. Mix the contents
thoroughly but without creating too many
bubbles.
At
2 minute
intervals, transfer 1 cm
3
of the
PPP/enzyme/buffer mixture to a cuvette. The
sodium carbonate will stop the reaction.
Use the colorimetric blank to zero the
colorimeter (contains buffer and enzyme –
cuvette 1). Measure the absorbance of the
remaining solutions at 550 nm.
Repeat the assay using the pH 7.0 buffer.
Results
Results – our results
Your Results – Excel file
Results
Other independent variables?
o
Effect of temperature
o
Phosphatase levels from different
species
o
Test a range of pH values at a fixed
time point (~10-12 minutes)
o
Effect of phosphate concentration
to look at end-product inhibition.
Phosphatase enzymes play a crucial role in various metabolic processes by releasing phosphate groups, increasing their availability for energy synthesis and cell structure formation. Acid phosphatases, with an optimum pH below 7.0, can be extracted from plant tissues like germinating mung beans. A protocol involving the use of sodium carbonate and a colorimeter for measuring absorbance in enzyme reactions is outlined. The process includes preparing enzyme extracts and substrates, conducting the enzyme reaction, and utilizing a colorimeter to measure absorbance at specific wavelengths.
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Presentation Transcript
Background Phosphatase enzymes are involved in a range of metabolic reactions. A key function of these enzymes is to release phosphate groups into the metabolic pool thereby increasing their availability for use in a range of processes including ATP synthesis and membrane construction.
Background Phosphatase is one of the enzyme systems mentioned into the CfE Higher and Higher Human course specification.
Background Acid phosphatases (those with an optimum pH <7.0) can be extracted from a range of plant tissues germinating mung beans or bean sprouts are a cheap and reliable source. The substrate is phenolphthalein bisphosphate (PPP). Under suitable conditions, phosphatase catalyses the breakdown of PPP to form phenolphthalein (PP).
At neutral or acidic pH, the products of the above reaction (PP and phosphate) are both colourless so their presence is difficult to detect. This can be overcome through the addition of sodium carbonate which has 2 effects: 1) raising the pH of the solution >pH10 with cessation of enzyme activity 2) converts PP to its anionic form, which is pink.
Brief protocol outline 1. Add 1 cm3 sodium carbonate solution to 7 cuvettes. 2. Prepare enzyme extract and substrate 3. Mix enzyme extract with buffer 4. Add 1 cm3 enzyme/buffer mixture to cuvette 1 Blank 5. Add substrate solution to the mixture of enzyme/buffer. 6. At 2 minute intervals, remove 1cm3 of the substrate/enzyme/buffer mixture and add to one of the cuvettes. 7. Measure the absorbance at 550 nm.
Colorimeter Biochrome (550nm) To measure absorbance, ensure the colorimeter has Abs on the display for absorbance. If not, press the Abs / %T button (T transmission). The colorimeter has a range of LED light sources emitting light of specific wavelengths. Turn the wheel on the right hand side until 550nm is displayed above the cuvette holder. When reading the colorimetric blank, press and hold R (Reference) should produce a reading of 0.00 When reading a sample, press T (Test).
Part A Preparing the enzyme extract Materials 20g Bean sprouts Distilled water Pestle and mortar Scissors plastic pipette 6x microfuge tubes Microcentrifuge Marker pen Bijou labelled Enzyme extract
Germination if using mung beans (would use about 30 per extraction)
Take ~20g bean sprouts and place them into a mortar. Remove and discard the green seed case (testa) if it is still attached.
Add 5 cm3 distilled water and grind the bean sprouts with a pestle to achieve a smooth paste.
Cut the end of a 3 cm3 plastic pipette. Divide the bean sprout mixture equally between 6 microcentrifuge tubes. Should be approximately equal volume to ensure the centrifuge rotor is balanced. Close the lids and label with your initials.
Centrifuge your samples for 5 minutes to produce a pellet.
Carefully remove the supernatant from the microfuge tubes (using a clean disposable pipette) and pool their contents in a clean bijou marked enzyme extract .
To carry out the assay at pH5 and pH7, at least 5 cm3 enzyme extract is required. If the supernatant is less than this volume, add some distilled water. A bijou holds 7 cm3.
Part B the phosphatase assay Materials Enzyme extract (at least 5 cm3) 10 cm3 citric acid/phosphate buffer, pH 5.0 10 cm3 citric acid/phosphate buffer, pH 7.0 6 cm3 0.2% phenolphthalein phosphate 25 cm3 10% (w/v) sodium carbonate Stopclock Waterbath (30 C) Paper towels 14x absorption cuvettes Cuvette rack Colorimeter (we are using a Biochrome colorimeter 550 nm) Automatic pipette and clean tips Polystyrene cup (or similar)
Pipettes: The volumes outlined in the following steps should be dispensed using the automatic 1 cm3 pipette with a clean tip. Temperature: All solutions should be kept at 30 C throughout the assay.
Transfer water from the waterbath (30 C) to the polystyrene cup. Stand the universal of buffer (pH 5.0), bijou of enzyme extract and bijou of substrate (PPP) in the cup.
Using the 1 cm3 automatic pipette, add 1 cm3 sodium carbonate into 7 cuvettes. Discard the tip.
Transfer 2 cm3 enzyme extract to the buffer solution in the universal bottle.
For the colorimetric blank: transfer 1 cm3 of the mixture from the universal (buffer + enzyme) to the first cuvette.
Transfer 2 cm3 PPP to the enzyme/buffer mixture. Start the stopwatch. Mix the contents thoroughly but without creating too many bubbles.
At 2 minute intervals, transfer 1 cm3 of the PPP/enzyme/buffer mixture to a cuvette. The sodium carbonate will stop the reaction.
Use the colorimetric blank to zero the colorimeter (contains buffer and enzyme cuvette 1). Measure the absorbance of the remaining solutions at 550 nm.
Results Time (min) Time (min) Absorbance Absorbance pH 5.0 pH 7.0 0 2 4 6 8 10 12
Results our results Time (min) Time (min) Absorbance Absorbance pH 5.0 0.00 0.09 0.18 0.22 0.31 0.42 0.56 pH 7.0 0.00 0.02 0.04 0.07 0.12 0.19 0.26 0 2 4 6 8 10 12
Effect of pH on phosphatase activity in bean sprouts 0.6 0.5 0.4 Absorbance 0.3 pH 5.0 pH 7.0 0.2 0.1 0 0 2 4 6 8 10 12 Time (min)
Results Effect of pH on mung bean phosphatase activity 0.25 Relative Activity 0.2 0.15 0.1 0.05 0 2 3 4 5 6 7 8 9 pH
Other independent variables? oEffect of temperature oPhosphatase levels from different species oTest a range of pH values at a fixed time point (~10-12 minutes) oEffect of phosphate concentration to look at end-product inhibition.
