Biology Coursework

How does phosphate concentration inhibit the activity of bean sprout

phosphatase?

Phosphatase is an enzyme found in many organisms which hydrolyzes

molecules containing phosphate groups to release a phosphate ion. It is useful
in the human body to produce phosphate for many important biological
molecules such as ATP and phospholipids. Some phosphatase's work better at
different pH's. Alkaline phosphatase, found in places such as growing bones and
teeth, is so named as the optimum pH is high. Acid phosphatase is has a low
optimum pH and is found in the human kidney and the prostate gland.
Phenolphthalein phosphate is a molecule containing phosphate thats reaction
into phenolphthalein and a phosphate ion is catalysed by phosphatase. In an
alkali solution the free phenolphthalein solution turns pink making a good
indicator of phosphatase activity.

Here is an equation from the Science and Plants for Schools website to show the
reaction:

phenolphthalein ---------------------------------------> free phenolphthalein

phosphate (phosphatase enzyme) + phosphate

Many enzymes are less active when the product they catalyze the
formation of is present. This is called product inhibition. For example the
presence of galactose inhibits the activity of lactase. This is due to competitive
inhibition. Competitive inhibition occurs due to similar structure of the product
and substrate. The substrate and the inhibitor therefore compete for binding to
the active site of the enzyme. As phenolphthalein phosphate is relatively similar
in structure to phosphate it could be deduced that phosphate may act as a
competitive product inhibitor. Therefore my experiment will test this theory.

I will carry out a trial experiment to find a suitable range of phosphate

concentration. The trial experiment will be almost identical to my experiment
however I will use a large range of phosphate concentrations to give an idea of
the scale appropriate for the main experiment. I will choose the concentrations
based on the range that has effect on the end result. Because the trial
experiment will be very similar to my main experiment I will be able to identify
any procedural errors which could be eliminated. I will use phenolphthalein
phospate as the substrate for the phosphatase enzyme as the product,
phenolphthalein, and causes alkaline solutions to turn pink therefore coloration
of the solution will give an indication of enzyme activity. My independent variable
is phosphate concentration. My dependent variable is coloration of my solution
which indicates phosphatase activity. Variables which may affect beansprout
phosphatase activity need to be controlled, such as pH and tempurature. I will
control tempurature by placing my solution in a water bath whilst reaction is
taking place. pH may also be a variable that could affect enzyme activity and so I
will control pH using a buffer solution. It is important to control these variables as
they can affect reaction rate so will affect my results. If these variables are not
controlled it cannot identified clearly how phosphate concentration inhibits the
activity of beansprout phosphatase as other factors will influence the results.

Alternative Hypothesis: The higher the concentration of phosphate present, the

less the coloration of the end solution, indicating more enzyme inhibition. This is
a one-tailed hypothesis.

Null Hypothesis: Phosphate concentration will have no significant effect on

enzyme activity so coloration of end solution will not vary significantly.

Equipment List

1 Measuring cylinder
5 Teat pipettes
7 Test tubes
1 250ml beaker
Muslin Filter
1 Colorimeter
1 Cuvette
30 C Water bath
1 Mortar and Pestle
Distilled water
150 ml Phenolphthalein phosphate
About 150 Beansprouts

Safety

Sodium carbonate is an alkali and therefore is an irritant to the skin and eyes.
Contact should be avoided by wearing a lab coat and safety goggles. Free
phenolphthalein is a suspected carcinogen and therefore contact should be
avoided, especially ingested. However the small amounts present do not
surmount a large risk.

Pilot Procedure

For my test experiment I will use a large range (0-100%) of 0.3 phosphate
solution concentrations so as to get a sense of scale for the main experiment. I
will prepare solutions of 0% 20% 33.3% 40% 66.6% 80% and 100% phosphate
solutions by using appropriate parts 0.3M phosphate solution and distilled water.
I will extract phosphatase enzyme from the bean sprouts. Using a mortar and
pestle and water and filter through muslin filter. I will then add phenolphthalein
phosphate to 7 test tubes and the phosphatase solution prepared. The test tubes
must then be placed in a 30 C water bath controlled and left to allow the reaction
to take place. After 20 minutes I will take the test tubes out of the water bath,
add 5cm of sodium carbonate solution to each test tube and mix. This solution
will turn pink if free phenolpthalein is present as it is alkali. I will then take
readings with the colourimeter to ensure readings are valid.

In my pilot experiment the range of concentrations gave a good range of

intensity of colourations and so I will use the same concentrations in my main
experiment. However one issue raised by the pilot experiment was inaccurate
readings on the colorimeter due to the solution being too cloudy. This meant that
the results were not valid. To rectify this problem in the main experiment I will
use the centrifuge to get a clearer solution. This should allow the colorimeter to
take more valid measurements. I will also repeat the experiment twice more to
improve the reliability of the results and therefore the validity of any conclusions
that can be drawn from them.

Plan

1. Prepare solutions of 0% 20% 33.3% 40% 66.6% 80% and 100% phosphate
solutions by using appropriate parts 0.3M phosphate solution and distilled water.
The solution with 0% phosphate present acts as a control for my experiment.
This is necessary both to test the null hypothesis and to compare my results
against to provide context.

2. Extract the phosphatase enzyme from the bean sprouts. To do this mix about
150 bean sprouts with distilled water in a blender to create about 150cm of
paste.

3.Filter this through a double layer of muslin filter into a clean beaker. This will
leave phosphatase solution ready to be centrifuged

4. Prepare the phosphatase solution to go into the centrifuge by putting it in

centrifuge tubes. Centrifuge for about 5 minutes. Remove from centrifuge.

5. Prepare 7 test tubes by adding exactly 5cm of buffer solution to each test
tube.

6. Write 0%, 20%, 33.3%, 40%, 66.6%, 80% and 100% on 6 stickers.

7. Label each test tube with a sticker.

8. Accurately add 1cm of the corresponding phosphate solution to each test

tube.

9. Add 1cm of phenolphthalein phosphate to each test tube.

10. Next add exactly 1cm of the phosphatase solution prepared in steps 2 and 3.
Mix thoroughly.
11. Place the test tubes in a water bath controlled at 30 C and leave for 20
minutes to allow the reaction to take place.

12. After 20 minutes take the test tubes out of the water bath. Add 5cm of
sodium carbonate solution to each test tube and mix. This solution will turn pink
if free phenolpthalein is present as it is alkali. The more free phenolpthalein
present the faster the reaction rate has been and the pinker the solution will turn.

13. Use distilled water to calibrate the colorimeter. This gives a reference point
for the colourimeter to take readings against. Place a sample of each solution
into a curette. Measure and record readings on colorimeter. The more intense
the coloration of the solution the more phenolphthalein present indicating more
enzyme activity.

I will use Spearman's Rank to identify definite correlation between phosphate

concentration and activity of phosphatase enzyme. This statistical test shows
how strong the correlative relationship is and can be used because I have more
than 5 pairs of values. Although this is sufficient it is far from ideal and
spearman's rank would provide a much more conclusive coefficient if more
concentrations were used.

Results

It is sufficient to leave the results as percentage 0.3M phosphate solution and

absorbance rather than concentrations of phosphate and free phenolpthalein as
absorbance is proportional to phenolphthalein concentration due to the
colouration of free phenolphthalein in the presence of a strong base. The
percentage of 0.3M phosphate solution present in the solution used is
proportional to concentration of phosphate as an appropriate amount of water
was added to each solution to keep the volume constant.

Percentage 0.3M Phosphate Solution/%02033.3405066.6100

A
b

n c e


 

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

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





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 

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

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

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
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The standard deviation is 0.06 which is quite large given that the range of my
data is 0.20. This means that about 68% of the data is 0.06 from the mean, a
relatively large standard deviation is expected as the data has a linear negative
correlation. Due to the nature of the variables being measured results are not
expected to be bunched close to the mean.

The correlation coeffecient shows strong negative correlation meaning that the
less phosphate in the solution the higher the activity of the enzyme, supporting
my null hypothesis. With a 0.05 confidence limit the critical value for 7 pairs of
data is 0.786 my value has a greater absolute vaule than this confirming
correlation for this set of data.

Conclusion

As absorbance indicates the amount of free phenolpthalein produced in

the reaction, it also indicates rate of reaction as the reaction were all carried out
in a time limit. The results clearly show a negative correlation between
absorbance and phosphate concentration.

While the result for 33.3% 0.3M phosphate solution does not fit this trend the
standard error suggests that the actual value could be in line with the trend, this
may have been due to the fact that the phosphatase solution was still quite
cloudy after centrifuging and this will have effected the precision of the results.
This is a systematic error. However it is more likely caused by random error as
the cloudy solution would have effected all results. These results support my
alternative hypothesis. The more phosphate present in the solution the less free
phenolphthalein produced indicating inhibition of the phosphatase enzyme in the
beansprouts. The standard error bars however, do indicate that there may be no
significant difference between each increase in phosphate concentration as the
bars overlap however they do show an overall significant increase in inhibition.
The presence of any phosphate at all in the solution has a clear inhibitory effect
as can be seen from the difference in absorbance at between 0% and 20%
phosphate solution. From these results conclusions the fact the phosphate
concentration affects phosphatase activity can definatley be drawn given the
causal support that the science behind competitive inhibition offers. However it
must be noted that more detailed conclusions about phosphatase inhibition and
how this changes as phosphate concentrate is varied are limited due to the lack
of reliability of my results. Much further repetition of this experiment would
greatly improve this. I feel that while further repetition would give much more
reliable results they would be similar to my results due to product inhibition and
the large difference in reaction rate between reactions in no phosphate and with
100% 0.3M phosphate solution. A large improvement to the experiment would
have to have taken absorbance readings every couple of minutes, this would
have allowed me to calculate values for reaction rate. It is also important to point
out that conclusions drawn from this experiment may not apply to phosphatase
found in different organisms as enzyme optimum conditions vary incredibly from
species to species. Further experiments could be carried out to extrapolate
results to other forms of phosphatase.

References

SCIENCE AND PLANTS FOR SCHOOLS 'Practical Activites' section http://www-

saps.plantsci.cam.ac.uk/worksheets/ssheets/ssheet14.htm [Accessed on 03 July
2009]
LONDON SOUTH BANK UNIVERSITY 'Faculty of Engineering, Science and The
Built Environment' section http://www.lsbu.ac.uk/biology/enztech/inhibition.html
[Accessed on 03 July 2009]
Huggins C and Talalay P (1945) Sodium Phenolphthalein phosphate as a
substrate for phosphatase tests 'The Journal of Biological Chemistry' 159 (2):
399.