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The Effect of Differing pHs on the Action of the Enzyme Pancreatin

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The Effect of Differing pHs on the Action of the Enzyme Pancreatin

I predict that the optimum pH for the enzyme pancreatin is pH 9. I
came to this decision after taking into consideration the following
information.

Catalysts speed up a reaction without being used up and so therefore
they can be used over and over again. Due to them not being used up
they are only required in very small amounts. Enzymes are biological
catalysts and I will be using digestive enzymes for the experiment
that I will be carrying out. The digestive juices contain enzymes,
which break down large insoluble molecules into small soluble
molecules. There are 3 main types of digestive enzymes and a different
one is needed for each category of food. The enzymes which catalyse
the breakdown of carbohydrates is carbohydrase; the enzymes which
catalyse the breakdown of fats is lipase and the enzymes which
catalyse the breakdown of protein is protease. Carbohydrates are
broken down into glucose, fats are broken down into fatty acids
(glycerol) and proteins are broken down into amino acids. Pancreatin
contains a protease enzyme.

Enzymes are all proteins and they are affected by temperature and pH;
they have an optimum. Enzymes work on substances called a substrate.
Enzymes have very specific shapes; each enzyme only works on its own
specific substrates. This can be explained by using the lock and key
theory where the lock is the enzyme and the key is the substrate. Only
the correct shape and sized substrate (key) can fit into the active
site (keyhole) of the enzyme (lock). The substance made at the end is
called the product.

[IMAGE]

Diagram from www.elmhurst.edu/~chm/vchembook/571lockkey.html

The enzyme is held into the very specific 3D shape by different types
of bonds. If these bonds become broken or damaged in any way then the
active site could change shape and so therefore the substrate would no
longer bind as tightly. The conditions that damage the bonds are
temperature and pH. Enzymes have an optimum for both of these factors.
If the optimum pH for an enzyme was pH8 and the pH of the surroundings
was altered slightly this would cause some of the ionic bonds holding
the enzyme in its shape to break. The same also goes for temperature.
However, the shape would only become slightly changed and so therefore
the enzyme would still work but just not as quickly as it would in the
optimum pH and temperature.

On the other hand if the pH was altered a long way from the optimum
this would then cause the shape to become more distorted and
decreasing the chances of the substrate binding to the active site.
This again also applies to temperature. When the substrate no longer
fits into the active site we say that the enzyme has become denatured
and so therefore can no longer be used in a reaction. This happens at
high temperatures and extreme pHs.

[IMAGE]

The aim of my experiment is to find out the optimum pH for pancreatin.
Conditions in the stomach are acidic (approximately pH2/3) because our
stomach contains hydrochloric acid. There are two reasons as to why
the conditions are like this

1. To kill any bacteria

2. It provides the correct conditions for the stomach protease.

Food is the passed down into the duodenum (the 1st part of the small
intestine) which is where the digestion of food occurs. Pancreatin is
secreted into the duodenum to carry on the breakdown of food. Bile
(which is made in the liver and stored in the gall bladder) is also
secreted to neutralise the stomach acid and it also emulsifies fats.
Bile is an alkali due to it containing sodium hydrogen carbonate
NaHCO3 which is around pH10. Pancreatin itself is also slightly
alkali.

41-16-Duodenum-L.gif

Diagram from http://35.9.122.184/images/41-
AnimalNutrition/HTML/source/32.html

I carried out preliminary work to find the optimum temperature for
pancreatin and my results showed me that the optimum temperature was
50°C. As a result of my preliminary work, I will be carrying out my
experiment in water kept at the temperature of 50°C. I will control
this by using a thermostatic water bath.

For this experiment I will be using the following pHs 7, 8,9,10,11
because as stated at the beginning I predict that the optimum pH will
be pH9 and so therefore I am doing a range of two pHs either side of
this. In order to create the correct pH for the enzyme to work I will
add a liquid called a pH buffer to the enzyme. A pH buffer is a liquid
which is added to a liquid to make the solution the correct pH for my
experiment. I will be using test tubes which will contain 7 cm³ of
liquid in total. This will consist of 4 cm³ pancreatin and 3 cm³ of
buffer. I will carry out the experiment three times for each pH so
that I can then work out an average time. To show that the results are
due to the enzyme being at different pHs I will use a control. My
control will be that I will add a piece of film negative to boiled
enzyme and if the negative doesn't go clear then that proves that the
enzyme is needed.

[IMAGE]

I will be using film negatives to find out what the optimum pH for
pancreatin is. The film negatives are made up of gelatine (which is an
insoluble protein), which is sandwiched between celluloid (transparent
plastic) and the pigment. The gelatine acts to stick the pigment to
the celluloid. During the experiment that I am going to carry out, the
pancreatin will digest the gelatine and turn it into soluble amino
acids. This will mean that the pigment will have nothing sticking it
to the celluloid and so therefore the pigment will fall off. This will
just leave the transparent piece of celluloid and so therefore I then
know that the experiment has finished.

Plan

Apparatus

5 test tubes

1 Test tube rack

15 splints

15 Film negatives (35mm, 2 holes wide)

Stop Clock

Weights

Scissors

6 Syringes 5cm³

Pancreatin (4 cm³ in each test tube)

5 pH buffers (3cm³ in each test tube) pH7 pH8 ph9 pH10 pH11

Thermostatic water bath-50°C

Diagram

Method

1. I will collect my apparatus.

2. I will use test tubes so that I can have 1 film negative in each
test tube so that I can get more accurate results because I will
only have to watch 5 film negatives at a time.

3. I will label the 5 test tubes with what pH they will contain (7,
8, 9, 10, and 11).

4. I will set up the 5 test tubes in a test tube rack.

5. In each test tube I will add 4cm³ of pancreatin.

6. In tube 1 I will add 3cm³ of pH buffer 7 using a 5cm³ syringe.

7. In tube 2 I will add 3cm³ of pH buffer 8 using a 5cm³ syringe.

8. In tube 3 I will add 3cm³ of pH buffer 9 using a 5cm³ syringe.

9. In tube 4 I will add 3cm³ of pH buffer 10 using a 5cm³ syringe.

10. In tube 5 I will add 3cm³ of pH buffer 11 using a 5cm³ syringe.

11. I will then put the test tube rack with the 5 test tubes in,
into the thermostatic water bath.

12. I will allow 10 minutes before placing my film negatives into
the test tubes. This is so the enzyme can acclimatise so that when
I put the film negatives into the test tubes the reaction will
always be taking place at the temperature 50°C. I will be using
this temperature because from my preliminary work I found that
50°C was the optimum temperature.

13. I will then prepare the film negatives to be 35mm long and 2
holes wide. Each piece of negative will be cut from the same roll
of film, so that each piece of film will have been exposed the
same amount.

14. I will make a 1cm cut up the splints so that the negatives will
slot in place and stay secure all throughout the experiment so
that I will be able to check if the film has gone clear or not
easily.

15. I will label each splint with the pH that it is going to be
placed into. This means that I will be able to make sure that the
film negatives do not get switched round.

16. After the enzyme has acclimatised for 10 minutes I will add the
film negatives and start the stop clock.

17. I will not check the negatives for the first 2 minutes because
from my preliminary work I found that nothing occurred.

18. After 2 minutes, I will check the film negatives every minute
until they start to go clear. When there are signs of this
happening, I will start to check the film negative every 30
seconds.

19. I will not check every 30 seconds from the very start or until
some signs of a reaction taking place can be seen, because each
time the film negative is taken out of the test tube no reaction
can take place as the negative isn't in contact with the
pancreatin.

20. Once the film negative has gone totally transparent, I will
record down the time that it reads on the stopclock. I will do
this for each negative until all of the film negatives have gone
totally transparent.

21. When this has happened I will replace the film negatives and
repeat the experiment again for another 2 times so that I will
have 3 sets of results for each pH. Therefore, I will be able to
work out an average time.

22. When the optimum pH has been found, I will do the same
experiment but including half values into the experiment.

23. The same method will apply for the half values, making sure that
for each pH there are 3 sets of results so that again I can find
an average time for each pH.

In this experiment I am going to record in the results how long it
takes for the film negative to go totally transparent.


Fair Test Table

Variable

Why does it need to be controlled?

How I will control it

The temperature of the water bath

Every enzyme has an optimum temperature, so therefore if the
temperature changes, this could affect the results by either slowing
or speeding up the reaction.

I will be using a thermostatic water bath at the temperature 50°

The Volume of Pancreatin and Buffer

If there is more pancreatin, then there is more active sites so
therefore the reaction will happen a lot faster. The buffer also needs
to be equal or less than the amount of pancreatin.

I will use a 5cm³ syringe so I get the exact amount of pancreatin and
buffer that is necessary for the experiment that I will be carrying
out.

The Size of the Film Negative

If the film negatives are different sizes then this will affect the
results because there will be either be a greater or smaller quantity
of gelatine. This will mean that either the reaction will occur
quicker or slower affecting the time that it takes for the film
negative to go totally transparent.

I will be using the same size of film which will be 35mm long and 2
holes wide. As well as all the pieces of film negatives coming from
the same roll of film so that they all have been exposed the same
amount.

Concentration of pancreatin

If the concentration of pancreatin is greater then there will be more
active sites and so therefore this will speed up the reaction. As well
as if the concentration is smaller then there will be less active site
and so therefore the reaction will be slower.

I will use a 2% pancreatin solution throughout my experiment.

The size of the splint

If the split in the splint is larger then this will prevent the
pancreatin react with the gelatine because the splint will be in the
way.

I will measure the splits in the splint with a ruler so that are as
accurate as possible.

Method

I did exactly what I planned to do.

Results

Time taken for the negative to go clear/secs

pH

Trial 1

Trial 2

Trial 3

Average

7

499

536

579

538

8

478

525

274

525

9

454

494

542

496

10

457

505

554

505

11

20+

20+

20+

20+

Time taken for the negative to go clear/secs

pH

Trial 1

Trial 2

Trial 3

Average

7.5

917

981

1024

974

8

543

628

546

572

8.5

870

907

966

1371

9

502

607

508

539

9.5

561

694

552

602

Conclusion

From my results I can conclude that the optimum pH was pH9. I have
concluded this because in both experiments pH9 took the lowest time
for the negative to go clear. In my first experiment pH8 took 525
seconds, pH 9 took 496 seconds and pH10 took 505. From this data it
can be said that pH10 on average didn't take much longer than pH9 but
this can also be said for pH8 however it did take slightly longer. In
my second experiment where I investigated half values, I can again
from my results conclude that pH9 took the lowest time for the
negative to go clear. However from my results of my second experiment
it can be seen that pH8.5 took the longest on average but this could
be an anomalous result. It was expected that pH9 would be the optimum
because of the conditions in the duodenum. Bile and pancreatin juices
are secreted into the duodenum. Bile is an alkali due to it containing
sodium hydrogen carbonate NaHCO3which is around pH10. Pancreatin
itself is also slightly alkali. However acid from the stomach is also
passed down into the duodenum causing the pH to be lowered slightly. I
can also conclude from my results that pH took the longest. This could
be because pH11 denatured the enzyme. When an enzyme is denatured
bonds (what holds the enzymes specific shape) become broken or damaged
causing the active site to change shape and so therefore the substrate
would no longer bind as tightly slowing down the reaction. I agree
with my prediction because I predicted that pH9 would be the optimum
pH and my results confirm this.

Evaluation

Overall I feel that both experiments went well and I didn't occur many
problems. After analysing my results I feel that I can rely on them
except for pH8.5 which I have regarded as an anomalous result because
it took 1371 seconds for the negative to go clear which was even
longer than pH8 which isn't expected. During the experiments there
were many elements that may not have been accurate. The splits in the
splints were not all the same because even though I measured them some
split even more when I cut them. If I was to carry out the experiment
again I would use a Stanley knife instead of scissors because I could
control the cut more easily. The size of the negative may not have
been accurate because I cut them by freehand and so therefore I can't
be 100% sure that they were exactly the same size. To improve this I
would not only measure it using the holes already on the negative but
I would also use a ruler. Another factor is also that when I kept
taking the negative out to see if it had gone clear I would have been
preventing a reaction from taking place. As a results the overall time
would be longer however I can't do anything about this really because
I have to check otherwise I wouldn't know when the negative had gone
clear. Another problem that I occurred is judging exactly when the
experiment had ending because I couldn't be a 100% sure that the
negative had gone completely transparent and so I have taken this into
consideration when looking over my results.

How to Cite this Page

MLA Citation:
"The Effect of Differing pHs on the Action of the Enzyme Pancreatin." 123HelpMe.com. 16 Apr 2014
    <http://www.123HelpMe.com/view.asp?id=149179>.




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