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Investigating the Effect of Copper Sulphate on Amylase Activity

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Investigating the Effect of Copper Sulphate on Amylase Activity


Aim

The aim of my experiment is to observe the affect on amylase when
adding copper sulphate to a starch solution.

Introduction

Enzymes are that act as catalysts, in other words they increase the
rate of chemical reactions. Consider the following general reaction
between two substances, A and B, which react together to form a
product, substance C:

A + B = C

In biological systems, this reaction might occur very slowly, or not
at all, in the absence of an enzyme. Enzymes will greatly increase the
rate of formation of the product. They can increase the rate of
reactions by a factor of at least one million. Most enzymes are large
protein molecules, with complex three-dimensional shapes.

Enzymes increase the rates of reactions by reducing the free energy of
activation, so that the barrier to a reaction occurring is lower in
the presence of an enzyme. The combination of enzyme and substrate
creates a new energy profile, for the reaction, with a lower free
energy of activation.

Once the products have been formed, they leave the active site of the
enzyme, which is left free to combine with a new substrate molecule.
Enzymes, like chemical catalysts, are not used up in the reaction they
catalyse so they can be used over and over again. The overall reaction
between an enzyme and its substrate can be represented by the
following equation:

ENZYME+SUBSTRATE à ENZYME-SUBSTRATE complex àENZYME + PRODUCTS

Amylase is an enzyme that is found in saliva and pancreatic juice and
aids the digestion of starch and other polysaccharides, which it
breaks down into glucose, maltose and dextrins. It is required to
digest carbohydrates (polysaccharides) into smaller units
(disaccharides), and eventually converting them into even smaller
units (monosaccharides) such as glucose. So Starch being a
carbohydrate, Amylase hydrolyses the Starch.

Inhibitors are substances that reduce the activity of enzymes. They
act by interfering with the activity of the active site, either
directly or indirectly. There are several types of inhibitors, which
can be either reversible or (if they bind reversibly with the enzyme)
or irreversible (if they bind permanently to the enzyme).

Copper sulphate is known to be an Irreversible inhibitor. Irreversible
inhibitors attach tightly to the enzyme molecule so that it loses its
catalytic properties. Irreversibly inhibitors include ions of heavy
metals, such as mercury and certain nerve gases.

Theories that will help explain my reasoning

Lock and key theory

The basic mechanism by which enzymes catalyze chemical reactions
begins with the binding of the substrate (or substrates) to the active
site on the enzyme. The active site is the specific region of the
enzyme which combines with the substrate. The binding of the substrate
to the enzyme causes changes in the distribution of electrons in the
chemical bonds of the substrate and ultimately causes the reactions
that lead to the formation of products. The products are released from
the enzyme surface to regenerate the enzyme for another reaction
cycle.

Substrate (key)

The active site has a unique geometric shape that is complementary to
the geometric shape of a substrate molecule, similar to the fit of
puzzle pieces. This means that enzymes specifically react with only
one or a very few similar compounds. This diagram will aid your
understanding:

[IMAGE]

[IMAGE]

[IMAGE]

[IMAGE]

[IMAGE]


Smaller keys, larger keys, or incorrectly positioned teeth on keys
(incorrectly shaped or sized substrate molecules) do not fit into the
lock (enzyme). Only the correctly shaped key opens a particular lock.
This is illustrated in graphic on the left.

The specific action of an enzyme with a single substrate can be
explained using a Lock and Key analogy.

In this analogy, the lock is the enzyme and the key is the substrate.
Only the correctly sized key (substrate) fits into the key hole
(active site) of the lock (enzyme).

An enzyme inhibitor is a substance that slows down the rate at which
an enzyme-catalysed reaction take place.

Copper Sulphate is a non-competitive inhibitor which means that the
copper sulphate molecules bind to a part of the enzymes tertiary
structure disfiguring the enzyme and most importantly disfiguring the
active site.

Therefore the substrate is now unable to fit into the active site of
the enzyme. Thererfore there will be a less concentration of working
enzymes that could catalyse the reaction.

Collision theory

Theory that explains how chemical reactions take place and why rates
of reaction alter. For a reaction to occur the reactant particles must
collide. Only a certain fraction of the total collisions cause
chemical change; these are called successful collisions. The
successful collisions have sufficient energy (activation energy) at
the moment of impact to break the existing bonds and form new bonds,
resulting in the products of the reaction. Increasing the
concentration of the reactants and raising the temperature bring about
more collisions and therefore more successful collisions, increasing
the rate of reaction. As copper sulphate is a non-competitive
inhibitor it binds to the enzymes tertiary structure breaking peptide
bonds as enzymes are proteins therefore disfiguring the enzyme and its
active site. Less successful collisions will occur as there is a lower
concentration of working enzymes. Therefore slowing down the rate of
reaction.

[IMAGE]

The Variables

My experiment has range of different variables, here are a few and a
brief description of how they are going to be kept constant:

Concentration of copper sulphate:- I will make sure I use 1% copper
sulphate by reading the bottle.

Concentration of amylase:- I will make sure I use 1% copper sulphate
by reading the bottle.

Volume of amylase:- I will measure out the chosen volumes of Amylase
carefully.

Volume of copper Sulphate:- I will measure out the chosen volumes of
copper sulphate carefully.

Temperature:- Temperature will be kept constant by using an electronic
water bath.

Ph:- We cannot use a ph buffer solution as it may be interfere with
our experiment making our results unreliable.

Pressure:- we are going to maintain the normal atmospheric pressure of
1.

My dependant variable is the concentration of copper sulphate.

My independent variable is the time taken to reach achromatic point.



Hypothesis
==========

I predict that copper sulphate will inhibit amylase activity.


Reasons
-------

I predict this because copper is known to inhibit the activity in Zinc
in alcohol dehydrogenase and because amylase contains calcium which is
also a metal, so it is reasonable to say that the copper will displace
the calcium slowing down amylase activity. I also think that the
copper sulphate will inhibit the amylase according to the lock and key
and collision theorys. The lock and key theory tells us that enzymes
break up bonds in a substrate by using an active site, the substrate
being the starch in the food and the substrate has to have the shape
to fit into the active site. The collision theory tells us that in
order for something to react or to break a bond there has to be a
collision of 2 particles, in this case it would be the enzyme and the
starch. So when copper sulphate is added the CuSO4, which has almost
the same shape as starch, the active sites are taken up by the CuSO4
particles leaving the amylase 1 less active site to use until the
copper sulphate is released therefore the amylase having less
successful collisions, therefore slowing down the rate of activity.
Another factor to add to my reasons is this, Enzymes contain several
amino acids with negatively charged residues. Copper, with it's
positive charge, would like to bind to these negative charges if it
can gain access to them. That can upset the catalytic properties of
an enzyme in a number of ways. For example, if the negatively charged
residue participates in the chemical mechanism of catalysis, then
having Cu bound is going to interfere with activity. This effect
should be reversible. Cu can also have an effect at sites more
distant from the active site of the enzyme if it disrupts the tertiary
structure (the folded structure of the protein) of the enzyme. Again,
these effects are most likely to be reversible, unless the Cu leads to
unfolding of the protein. Very often, proteins cannot refold properly
when they are unfolded.

Equipment


Test Tubes:- test tubes are needed to carry the substrate, the copper
sulphate and the amylase, they are also used to mix the solutes.

Thermometer:- Thermometers are used to measure the temperature of the
solutes so it could b analysed as a fair test.

Water bath:- Water baths are used to increase the temperature to a
specific degree, so as it could not be a variable.


Stop watch:- the stop watch will be used to time the intervals in
which a sample of the mix should be taken out and tested with iodine.

Pippette fillers:- will be used to fill up the graduated pipettes.

Pasteur pipettes:- will be used to take out samples of the mixed
solution to be tested.

Graduated Pipettes:- These will be needed to accurately extract the
chemicals and place them in the glass wear. I als selected the
graduated pipette to use in my experiment due its low tolerance level
of +0.06ml giving a tolerance level of 2 decimal places and
percentage error of 0.24%.

Iodine:- will be used to test the solution.

1% Copper Sulphate:- the substance we are testing if it is an
inhibitor or not.


1% Stach Solution:- this is the substance we are using to test amylase
activity after adding copper sulphate.

1% Amylase:- amylase Is used to break down the starch.

White Dimple trays:- These will be used to add the iodine then the
mixed solution to check if the starch has been broken down or not.

Beakers:- These are needed for any dilutions that will have to be
made.

Pilot study

For my pilot study I shall conduct a test to see what concentrations
and volumes should be used to conduct a reasonable experiment. In
light of the results of this experiment I will change the amount of
concentration accordingly. To judge I would see what changes occur
during the experiment, for instance, if the experiment takes too long
I would have to decrease the concentration of copper sulphate.

I would add different volumes of copper sulphate into the solution of
amylase and starch and time how long it takes for me to see a change.

I had to change the volume and concentrations of copper sulphate a
number of times before I had a concentration/volume that was
reasonable to use in my experiment. The 1st time I tested what
concentrations I should use, I started with 0.1% copper sulphate with
volume of 5cm3 and the temperature at 350. The result was inconclusive
because my limiting factor was 12 minutes meaning any result after 12
minutes I would regard it as an inconclusive result.

Inconclusive means that there was no result as my limiting factor was
12 minutes.


CONCENTRATION

VOLUME

TEMP

Time to reach achromatic point

0.1% Copper sulphate

5 cm3

35oC

INCONCLUSIVE

0.5% Amylase

5 cm3

1% Starch

5 cm3

The 2nd time I tested what concentrations to use, I used 0.01% copper
sulphate with the volume of 5cm3 and the temperature of 350.

CONCENTRATION

VOLUME

TEMP

Time to reach achromatic point

0.01% Copper sulphate

5 cm3

35oC

INCONCLUSIVE

0.5% Amylase

5 cm3

1% Starch

5 cm3

The result was inconclusive so I conducted another test, with the
concentration of 0.01% copper sulphate, the volume of copper sulphate
as 2.5cm3, and the temperature as 400C. The result was that the Iodine
didn’t detect any starch at the time of 500-520 seconds. Therefore I
used these concentrations/volumes/temperature:-

CONCENTRATION

VOLUME

TEMP

Time to reach achromatic point

0.01% Copper sulphate

2.5 cm3

40oC

500-520 seconds

0.5% Amylase

5 cm3

1% Starch

5 cm3

Method

1. Prepare 4 different concentrations of the copper sulphate
solution:

* 0.01

* 0.02

* 0.03

* 0.04

Volume of copper sulphate solution/cm 3_

Volume of distilled water/cm3

Final copper sulphate concentration

1

99

0.01

2

98

0.02

3

97

0.03

4

96

0.04

2. Set up a water bath at 40 0C

3. Pipette 2.5 cm3 of the diluted copper sulphate solution into one
test tube and 5cm3 of starch into another test tube, Pipette 5 cm3
of amylase also and stand all three test tubes in the water bath
and leave for several minutes to reach the temperature of the
water bath.

4. Mix the copper sulphate and starch solutions together, replace
the mixture in the water bath. Then add the Amylase to the
solution and immediately start the stop watch.

5. At intervals of 20 seconds, remove a drop of the mixture and test
it with iodine solution on a white dimple tray.

6. Continue the experiment until the mixture fails to give a
blue-black colour with iodine solution. Record this as the
achromatic point.

7. Repeat this procedure with the other concentrations of amylase.
Use exactly 2.5cm3 of copper sulphate solution, 5cm3 of starch
solution 5cm3 of amylase each time.

8. Repeat the whole process three times to gather averages.


Risk assessment
---------------

In my risk assessment I will be explaining how to make my experiment
safe for me and the people that are around me. For example, Im going
to use reasonable time intervals. This will enable me to handle my
glassware with more care, for instance if the time intervals were very
short there is more chance of either doing something wrong or dropping
the glassware. I will make sure I am wearing goggles while I conduct
this experiment so no harmful substances such could go onto the eyes.
This is especially important in this experiment because I will b using
enzymes which could digest the lining of the eye.

Enzyme name

Hazards

Precautions to reduce risks

Emergency procedures

Storage and disposal

Bacterial amylase

All enzymes are potential allergies therefore should avoid contact and
inhalation because it can cause asthma or irritate the membranes of
the eyes and nose.

bacterial enzyme in its powder form is harmful because it can be
easily inhaled. So take precautions such as stay a metre away from the
powdered enzyme. Also wear goggles to avoid contact with the eye as it
could digest the carbohydrates that the eye is made up of.

If swallowed: wash out mouth and give a glass or 2 of water. Seek
medical attention as soon as possible.

Solids can be mixed with 1kg of sand and placed in the refuse. Aqueous
solutions (100ml) should be diluted in 10 litres of water and run to
waste down the foul-water drain. As a general organic chemical [CG]
unless kept in a refrigerator

If solid or solution gets in eyes: flood the eye with gently running
tap water for 10 minutes. Seek medical attention.

If spilt on skin or clothes: remove contaminated clothing. Flood area
with water and wash thoroughly with soap and cold water.

If spilt in laboratory: scoop up as much solid as possible wash area
thoroughly with detergent and water.


Enzyme name

Hazards

Precautions to reduce risks

Emergency procedures

Storage and disposal

Copper sulphate and Iodine

This substance is harmful if swallowed and it has known to be an
irritant if in contact with the eyes. It also irritates sensitive
skin. It is also very toxic to the aquatic environment and may cause
long-term adverse effects.

The solutions that is equal to and greater than 1M should be labelled
‘HARMFUL’. Wear goggles and you should handle the substance with care.
Once finished using the substance pour down the drain and leave the
tap on for about 2 minutes to reduce the environmental hazards.

If swallowed: wash out mouth and give a glass or 2 of water. Seek
medical attention as soon as possible.

Solids can be mixed with 1kg of sand and placed in the refuse. Aqueous
solutions (100ml) should be diluted in 10 litres of water and run to
waste down the foul-water drain. As a general organic chemical [CG]
unless kept in a refrigerator

If solid or solution gets in eyes: flood the eye with gently running
tap water for 10 minutes. Seek medical attention.

If spilt on skin or clothes: remove contaminated clothing. Flood area
with water and wash thoroughly with soap and cold water.

If dusted inhaled then I would leave the lab to get fresh air. If
breathing is slightly affected then should seek medical attention

If spilt in laboratory: scoop up as much solid as possible wash area
thoroughly with detergent and water.

Results

Inconclusive means that there was no result because the iodine didn’t
stay brown after 12 minutes which is my limiting factor.

Trial 1

CONCENTRATION

VOLUME

TEMP

Time to reach achromatic point

0.01% Copper sulphate

2.5 cm3

40oC

500-520 seconds (510 secs)

0.5% Amylase

5 cm3

1% Starch

5 cm3

Trial 2

CONCENTRATION

VOLUME

TEMP

Time to reach achromatic point

0.01% Copper sulphate

2.5 cm3

40oC

480-500 seconds (490 secs)

0.5% Amylase

5 cm3

1% Starch

5 cm3

Trial 3

CONCENTRATION

VOLUME

TEMP

Time to reach achromatic point

0.01% Copper sulphate

2.5 cm3

40oC

500-520 seconds (510 secs)

0.5% Amylase

5 cm3

1% Starch

5 cm3

Trial 1

CONCENTRATION

VOLUME

TEMP

Time to reach achromatic point

0.02% Copper sulphate

2.5 cm3

40oC

80-100 seconds (90 secs)

0.5% Amylase

5 cm3

1% Starch

5 cm3

Trial 2

CONCENTRATION

VOLUME

TEMP

Time to reach achromatic point

0.02% Copper sulphate

2.5 cm3

40oC

580-600 seconds (590 secs)

0.5% Amylase

5 cm3

1% Starch

5 cm3

Trial 3

CONCENTRATION

VOLUME

TEMP

Time to reach achromatic point

0.02% Copper sulphate

2.5 cm3

40oC

560-580 seconds (570 secs)

0.5% Amylase

5 cm3

1% Starch

5 cm3

Trial 1

CONCENTRATION

VOLUME

TEMP

Time to reach achromatic point

0.03% Copper sulphate

2.5 cm3

40oC

620-640 seconds (630 secs)

0.5% Amylase

5 cm3

1% Starch

5 cm3

Trial 2

CONCENTRATION

VOLUME

TEMP

Time to reach achromatic point

0.03% Copper sulphate

2.5 cm3

40oC

640-660 seconds (650 secs)

0.5% Amylase

5 cm3

1% Starch

5 cm3

Trial 3

CONCENTRATION

VOLUME

TEMP

Time to reach achromatic point

0.03% Copper sulphate

2.5 cm3

40oC

620-640 seconds (630 secs)

0.5% Amylase

5 cm3

1% Starch

5 cm3

1

CONCENTRATION

VOLUME

TEMP

Time to reach achromatic point

0.04% Copper sulphate

2.5 cm3

40oC

INCONCLUSIVE

0.5% Amylase

5 cm3

1% Starch

5 cm3

2

CONCENTRATION

VOLUME

TEMP

Time to reach achromatic point

0.04% Copper sulphate

2.5 cm3

40oC

700-720 seconds (710 secs)

0.5% Amylase

5 cm3

1% Starch

5 cm3

3

CONCENTRATION

VOLUME

TEMP

Time to reach achromatic point

0.04% Copper sulphate

2.5 cm3

40oC

INCONCLUSIVE

0.5% Amylase

5 cm3

1% Starch

5 cm3

CONCENTRATION %

AVERAGES

0.01

503 seconds

0.02

416 seconds

0.03

636 seconds

0.04

1

Time taken to reach achromatic point X 103

710 seconds

[IMAGE]

[IMAGE]The formula I used to calculate the rate of reaction was:

CONCENTRATION %

RATE OF REACTION

0.01%

1.988

0.02%

2.4

0.03%

1.5723

0.04%

1.408

Discussions

For my 1st graph there was a general trend and correlation between the
concentration of copper sulphate and the time taken for the solution
to meet the achromatic point. This positive correlation was that the
higher the concentration of copper sulphate that was added to the
mixed solution, the higher the time taken for the amylase to break
down the starch. The time taken for the solution to meet achromatic
point was certainly higher by looking at these figures you could
decipher that there is a high range between the 1st concentration
tested and the last concentration tested:

CONCENTRATION %

AVERAGES

0.01

503 seconds

0.04

710 seconds

This evidence supports the idea that Copper sulphate inhibits the rate
of reaction of Amylase.

However I did get one anomalous result, on the concentration of 0.02%
copper sulphate I got a higher rate of reaction than my 1st tested
concentration. I think this is because I might of made up the dilution
incorrectly, allowing the amylase to break down the starch at a faster
rate. This brought down the average rate of reaction figure for that
concentration, if I used just the two reasonable results I would have
obtained the average figure of 580. This figure would have
corresponded with all the other figures and with my hypothesis.

For my Rate of Reaction graph there was a general trend and
correlation of my results. The negative correlation means that the
higher the concentration of copper sulphate the lower the rate of
reaction will be. There is a basic trend of a negative correlation
because copper sulphate is an inhibitor. More precisely Copper
sulphate is a non – active site directed inhibitor. Non-active site
directed inhibitors bind strongly with enzymes tertiary structure
breaking peptide bonds, as all enzymes are proteins. This alters the
shape of the enzyme and its active site disabling the enzyme to
function properly as the substrate is no longer able to fit into the
enzymes active site. This means that copper sulphate probably bonded
strongly with the amylase altering its tertiary structure which also
alters its active site. As the active site has most likely changed
(according to the lock and key theory) the starch may no longer fit in
to the amylases active site as they are no longer geometrically
compliant. Therefore there will be a lower concentration of working
amylase enzymes so less successful collisions will take place.
Therefore slowing down the rate of reaction.

These two graphs suggest that my hypothesis of “copper sulphate
inhibiting amylase activity” is correct.

I think that there were some limitations in my experiment that made my
experiment less accurate and helpful to use. For example I was limited
in the time I had to conduct my experiment, if I had more time I would
have taken more readings for different concentrations to gain further
results that may suggest my hypothesis was right. Another thing I
would do if I had more time is that I would test other types of
amylase such as fungal amylase and observe if copper sulphate had the
same effect. I would also try thing such as test beyond the maximum
concentration of copper sulphate to see if there is a peak in its
inhibiting effect. Also I could have done in hind sight is to increase
the range of the concentrations I used, for example instead of using
0.001, 0.002, 0.003 and 0.004, I could have used 0.001 0.003, 0.006
and 0.009 to receive a greater range of results. Another thing I would
do if I had more time is that I would test other types of amylase such
as fungal amylase and observe if copper sulphate had the same effect.
The in accuracies that I’m worried that affected my results was that
it was hard to detect the colour change in the iodine so my results I
could’ve been carrying on with my experiment not knowing it had gone
past the achromatic point.

Now thinking about it I should have used a colourimeter to detect the
colour change to make my results accurate. A colourimeter is more
accurate as it shines a light through the solution detecting colour
change.

Luckily I didn’t get a lot of wide error bars showing that my results
are still quite accurate. Error bars show the variability of all your
repetitions and as my error bars were quite short u can see that most
of my repetitions are quite close together. However the fact that
there are error bars shows that there are flaws in my experiment and
that is mostly due to the fact of trying to pin point the achromatic
point. There is 1 very wide error bar in the 0.02% concentration,
because the error bar is so big, this result cannot be used as it is
inaccurate.

References

Nelson Advanced Science:

Molecules and Cell

Collins Guide to As Edexcel Biology:

Unit 1

www.bbc.co.uk/asguru

www.elmhurst.edu/~chm/vchembook/

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