An Investigation Into a Reaction Catalysed by a Protease

An Investigation Into a Reaction Catalysed by a Protease

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An Investigation Into a Reaction Catalysed by a Protease

Aim.

To investigate the effect of temperature and the effectiveness of this
enzyme in breaking down the gelatine (protein) on the back of
photographic film.

Prediction

I predict that the effect of the temperature will be that the higher
the temperature the quicker the enzyme will break down the gelatine,
but the temperature will have to be an optimum temperature because if
it is too high, the enzyme will be denatured. If the temperature is
too low, the enzyme will not work and stay stable.

Increasing the temperature provides more heat energy. This increases
the kinetic energy and makes the enzyme molecules move faster. There
will be an increase in the number of collisions and this will increase
the rates of reaction.

If the temperature continues to increase beyond 40°c, the molecules
move even faster, but the structure of the enzyme molecules has so
much energy that the bonds break. The enzyme begins to lose its
globular shape, which effects the active site, and the enzyme becomes
denatured. Also, for every enzyme there is an optimum PH at which the
reaction it catalyses proceeds most rapidly. Many enzymes work within
a PH range of an about 5-9 and works most efficiently at neutral of PH
7, that is why I will be using water.

Equipment

*10 pieces of photographic film

*20ml of protease enzyme in each test tube

*Water baths (23°c, 40°c, 50°c, 60°c, and 70°c)

*Buffer (5ml in each test tube)

*Stop watch

*Distilled water

*10 boiling tubes

*Goggles

Safety

Wear goggles, wear a lab coat, wash your hands after experiment, make
sure cotton is thread through the photographic film to avoid
contamination.

Variables

*Temperature is kept constant and controlled

* The film size is the same throughout

*Range of temperatures is used

*Use of a buffer (chemical to maintain a constant PH)

Method

Firstly set up the water baths at the specific temperatures and leave

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one at room temperature.

Put 5ml of buffer into each boiling tube using a syringe, the buffer
is used to maintain a constant PH.

Then add 20ml of distilled water, this is used as a control for each
temperature, so that it is to prove that it is the protease that
breaks down the gelatine.

Shake all the boiling tubes so that they are mixed. Then place the
boiling tubes into each water bath, plus the control and leave for 5
minutes so that it reaches the appropriate temperature.

Then add the photographic film (which is attached to cotton) and time
it to see how long it takes for the photographic film to go
transparent. Do this to all the temperatures.

Results

Results 1

TEMPERATURE

TIME IT TOOK FOR THE PROTEASE TO BREAK DOWN THE SILVER NITRATE

TIME IT TOOK FOR THE CONTROL EXPERIMENT

30°c

25.13

-

35°c

17.30

-

40°c

13.12

-

45°c

9.40

-

50°c

8.19

-

Result 2

TEMPERATURE

TIME IT TOOK FOR PROTEASE TO BREAK DOWN THE SILVER NITRATE

TIME IT TOOK FOR THE CONTROL EXPERIMENT

30°c

26.14

-

35°c

18.21

-

40°c

12.17

-

45°c

10.33

-

50°c

8.24

-

‌

Temperature

TEMPERATURE

AVERAGE TIME IT TOOK FOR THE PROTEASE TO BREAK DOWN THE SILVER NITRATE

30°c

25.13

35°c

17.75

40°c

12.64

45°c

9.86

50°c

8.21

Conclusion

I have noticed that my prediction is correct. The effect of
temperature is that the higher the temperature the quicker the enzyme
will break down the gelatine, and the photographic film turns
transparent.

As the graph shows, the higher the temperature the quicker the enzyme
works at breaking down the gelatine.

The higher the temperature, the ore kinetic energy is produced, and
this makes the enzyme molecules and the substrate molecules move
faster. There will be an increase in the number of collisions and this
will increase the rate of reaction. If the temperature continues to
increase beyond 40°c, the molecules move even faster but the structure
of the enzyme has so much kinetic energy that bonds break. The enzyme
begins to lose its globular shape, which effects the active site.

The enzyme and the substrate molecules (protease and the gelatine),
they combine to form an enzyme-substrate complex before the products
of the reaction are released.

This "lock and key" theory increases the probability that they will
react. The chances of molecular collision taking place are thus
increased at higher temperatures so it is more likely that enzyme-
substrate complex will be formed.

If the temperature gets too high, the vibrations become too violent,
chemical bonds in the enzyme break; therefore the enzymes become
denatured.

Also extremes of PH cause the bonds to break resulting in the enzyme
becoming denatured. For every enzyme there is an optimum PH at which
the reaction it catalyses proceeds most rapidly. Many enzymes work
within a PH range of about 5-9 and works most efficiently at neutral
of PH 7.

As my graph shows the enzyme worked at the lowest temperature which
was 30°c, but it took longer because the molecules hadn't gained much
kinetic energy, as the temperature wasn't high enough, that is why it
took about 25-26 minutes to become transparent.

My graph does not show when the enzyme becomes denatured. I predict
that it would become denatured around 70°c or 80°c.

The majority of enzymes become denatured around 45°c.

The enzyme (protease) worked faster around 50°c. it took less time for
the photographic film to become transparent.

Evaluation

I have found that I would need a wider range of temperatures for
observation, as the temperatures I used weren't high enough to
denature the enzymes, so I would need to use temperatures up to about
80°c for more accurate results.

The size of the photographic film wasn't exact; it was cut to an
approximate size so it wasn't exact. To make sure it was the exact
measurements as all the rest I would need to use a template.

The volumes were pretty accurate but they could of been more precise
as I only used a syringe to measure the solution with, but if I used a
volumetric pipette all my volumes would of been the precise
measurement.

There was about 8 test tubes in the water bath that I used, as other
members in my class was using it so the temperature wasn't kept
totally constant.

The temperature was also not kept constant when I kept taking the test
tube out to check the "clearing", so the temperature was not
maintained. To improve this I would need a deep enough water bath so
that I could keep the test tube underwater whilst checking the
"clearing".

I also found that when I removed the photographic film for observation
it was difficult to replace, to stop this from happening, I could use
a beaker, so that it gives me more space to put the film back in.

I also found that I might not of timed the correct time of clearing as
I struggled to see the photographic film in the test tube. To prevent
this I could use a beaker instead of a test tube so that I could
monitor the photographic film at all times.
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