An experiment to investigate the effectof Copper (II) Sulphate on the activity of amylase.

An experiment to investigate the effect of Copper (II) Sulphate on the activity of amylase

Introduction

Copper (II) Sulphate is an enzyme inhibitor. An enzyme inhibitor is any substance or chemical which if added to the enzyme substrate complex will decrease the rate of reaction.

Inhibitors come in two forms: competitive and non-competitive inhibitors. A competitive inhibitor is one that competes for the active site of an enzyme with the enzymes’ substrate. It does not affect the enzyme in any way other than occupy the active site making the productivity of the enzyme less efficient. This can be explained clearly in the diagram below:

Competitive inhibitors are reversible, that is the inhibitor does not stay bound with the active site but is released again so the enzyme is still able to form the enzyme-substrate complex. It is summarised in the following equation:

Enzyme + Inhibitor Enzyme-Inhibitor Complex

Competitive inhibitors are always reversible.

Non-competitive inhibition occurs when the inhibitor does not compete with the active site but binds to another part of the enzyme changing its shape, thus denaturing a part of the enzyme. Non-competitive inhibitors can be reversible or irreversible (however irreversible inhibitors are always non-competitive). Irreversible inhibitors act as in the equation above, but do not have a double arrow but one arrow indicating that the reaction occurs one way. This can be shown diagrammatically below.

Copper (II) sulphate is an inhibitor and depending upon what it affects is competitive or non-competitive. For example with trypsin, copper (II) sulphate is a competitive inhibitor.

However, in this experiment we are dealing with amylase, and copper (II) sulphate is in fact a non-competitive inhibitor of this enzyme. The inhibiting component of copper (II) sulphate is the Cu2+ ion (although the SO42- component can also cause inhibition if it is in high enough concentrations). The reason it is a good inhibitor is because of a few reasons. Firstly, copper can displace other metals that are essential for the activity of the enzyme.

Secondly, enzymes contain several amino acid residues, which are negatively charged. The copper ions with its +2 charge are attracted to these negatively charged amino acids and bind onto them (if possible). This could upset the catalytic properties of the enzyme in many ways. For example, if the negatively charged amino acid is a component in the chemical mechanism of catalysis, then having the copper bound onto it may interfere with its activity. This affect could be reversible. The copper could also have an effect further away from the active site affecting the bonding of the enzyme. This would disrupt the tertiary structure of the enzyme. This may be reversible or irreversible depending upon whether the enzyme becomes unfolded or not. If the enzyme becomes unfolded it is likely that it would be irreversible as the enzyme would not be able to fold back together.

Thirdly, copper working with hydrogen peroxide can lead to proteolysis. Proteolysis is the breaking up of the amino acid chains that form the enzyme. Amylase especially is susceptible to this reaction. Although hydrogen peroxide will not be added, trace amounts of hydrogen peroxide are around wherever there are oxygen molecules present. It is worth noting that this way of denaturing the enzyme is minute compared with the first two. There are other ways in which copper could denature an enzyme, however, they would be insignificant in comparison with the stated reason.

Amylase requires calcium ions to facilitate catalysis. Copper (II) Sulphate inhibits amylase mainly by the Cu2+ ion displacing the Ca2+ ion in the active site. The copper ion is unable to facilitate catalysis and thus the enzyme becomes inhibited.

The copper ion could also disrupt the bonding of amylase as stated in the second point above. That is by bonding onto other parts of the amylase molecule, it may change the shape of the molecule and changing the shape of an enzyme can lead to the enzyme becoming denatured.

In this experiment I will be using amylase as the inhibited enzyme because it is easily accessible, it is relatively inexpensive and it is a digester of a very common substrate, namely starch. Starch is a polysaccharide and when mixed with iodine solution stains a blue-black colour, however, the disaccharides and the monosaccharides that make up the starch molecule (alpha glucose) are not stained blue-black by the iodine colour. This makes it very useful to know when the molecule is digested, as the solution would go from a blue-black colour to colourless.

Hypothesis

I predict that with increasing concentrations of Copper (II) sulphate, the rate if reaction for amylase digesting starch will decrease (i.e. the time for the reaction to take place will increase). This is because as you increase the concentration of copper (II) sulphate, the concentration of copper ions increases in the solution too. These ions will begin to inhibit the enzyme amylase and thus the enzyme will not be as efficient as it was before the copper (II) sulphate was added, due to the displacement of the calcium ions and so on.

Now as you double the concentration of Copper (II) sulphate, twice the amount of copper ions will be present for the same given volume. If there are twice the amount of copper ions in the solution that means that the chance of the copper ion colliding with the amylase would increase by a factor of 2. So the chance that the copper ion will collide with the amylase in the correct position in order to inhibit the enzyme will increase by a factor of 2. Although the process of collision is random, the average chance of meeting a copper ion would increase by 2. Having this fact in mind, I predict that as the concentration of copper (II) sulphate doubles the time for the reaction to take place will double. That is the concentration of copper (II) sulphate will be proportional to time of the reaction. In other words I predict that the rate of reaction will be inversely proportional to the concentration of copper (II) sulphate, i.e. as the concentration of copper (II) sulphate doubles, the rate at which amylase digests starch will half.

However, amylase and starch solution on its own will not digest instantaneously. Clearly the starch molecules will need to collide with the amylase enzyme many times for the complete digestion of starch to occur. Hence I predict the graph of increasing concentrations of copper (II) sulphate and time for the reaction to take place should look like the diagram below:

This graph is in the form y = m x + c where c is the y-intercept and is the length of time it takes for starch to be digested by amylase in the presence of no ...

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This graph is in the form y = m x + c where c is the y-intercept and is the length of time it takes for starch to be digested by amylase in the presence of no inhibitor. As the concentration of copper (II) sulphate would increase in a straight line afterwards we can say that the concentration of copper (II) sulphate is proportional to the time taken for the reaction to occur. If we took c to be 0, i.e. subtracted the value c from the other results gained by adding the copper (II) sulphate, then the relationship would be directly proportional.

As time is related to rate, I predict that the graph of rate verses the concentration of copper (II) sulphate will look like the following:

This is not a typical inversely proportional graph. Firstly the line touches the y-axis at the point where the concentration of copper (II) sulphate is equal to 0. This is because the rate cannot be increased any further (with the same concentrations of starch and amylase solution) because there are no inhibitors and that the enzyme would be working at its optimum rate.

Key Variables

Before continuing with the pilot run of the experiment it is important to note the variable being tested in this experiment and the other key variables that may have an affect on the experiment if they are not kept constant.

The variable being tested in this experiment is the concentration of the inhibitor, namely copper (II) sulphate. Discussion about how this will affect the results was discussed already in the hypothesis, but briefly as the concentration of copper (II) sulphate increases, the rate of reaction decreases due the inhibition of the enzyme.

Other variables that will be discussed below will be kept constant as far as humanly possible.

Temperature affects the rate of reaction. This is because as you increase the temperature, you increase the energy supplied to the molecules of the reaction. An increase in energy means the molecules move about more as kinetic energy is increased. This causes an increase in successful collisions and so an increase in the rate of reaction.

The concentration of all other chemicals will have to remain constant in order for this experiment to be a fair test. For example if the concentration of amylase were increased, then the rate of reaction would also increase due to the increase in the number of active sites and so there would be an increase in the number of successful collisions. Similarly if the concentration of starch were to decrease then the rate of reaction would increase, as there would be less starch molecules to digest. The opposite effect would occur if the amylase concentration were decreased as the number of active sites would decrease, i.e. the rate of reaction would decrease. If the concentration of starch increased then the rate of reaction would decrease, as there would be more starch to digest.

Safety and Risk assessment

In all experiments there are hazards and it is important to be fully aware of them in order to conduct the experiment efficiently and safely.

Copper (II) sulphate is a harmful chemical. It could cause irritation if not handled with care. It is very important to transport and handle this chemical in a safe manner, wearing the appropriate equipment, such as safety goggles. After the experiment, it should be disposed of down a fume cupboard sink in order that the chemicals do not react with other substances in the outside environment and so not affect the habitat of any organism or cause any damage to the environment.

Amylase and starch although they are not as harmful as copper (II) sulphate should also be handled with care. After the experiment it too should be disposed of with care.

Sensible handling of equipment is also required.

Hands should be washed immediately in case of any spillage. Hands should be washed after the experiment is completed.

Pilot Method

You will need the following apparatus:

Copper (II) sulphate solution 1.0M 100cm3 (harmful)
Amylase solution 1.0% 50cm3
Starch solution 0.1% 50cm3
Iodine solution (kept in a small bottle) 20cm3
Boiling tubes (10)
Test tube rack
Beakers (5 X 100cm3)
Pipettes (4)
Distilled water
Stopwatch

Wash all apparatus (i.e. boiling tubes, beakers, pipettes, syringes) with distilled water to get rid of any unwanted chemicals that may hinder the experiment.
Add 2cm3 of starch solution 0.1 % to one of the boiling tubes using a pipette (this pipette should be kept with the beaker full of starch solution as it will be only used for starch to prevent mixing chemicals prematurely).
Add one drop of iodine solution to the starch solution in the test tube. Iodine is a good way of showing the presence of starch as it turns the clear starch solution into a dark blue colour. However, when starch is digested, the iodine no longer stains, so when in the presence of amylase, the solution appears to go clearer as the amylase digests the starch.
Add 2cm3 of amylase solution to the same boiling tube using a different pipitte and simultaneously start the stopwatch.
Time how long it takes for the mixture of solution to go colourless from the dark blue colour.
Repeat the steps from 1 to 5 but add 2cm3 of copper (II) sulphate solution using another pipette allocated for the adding of this chemical only before adding the iodine.
Record the results in a table

These steps were followed and the following pilot results were recorded:

Results from pilot

As you can see the above pilot results are far from satisfactory. It is clear that the method and the way the practical is performed are amended to yield better results.

Amendments

Due to the unsuccessful results of the pilot, i.e. the lengthy nature of the results and the results unattained for the higher concentrations of Copper (II) sulphate it is necessary to amend the method of the pilot in order to attain more reliable results. This will be done in two major ways:

Firstly the problem of the long, inaccurate and unattainable results should be addressed. It is clear that these results are not the type one can assess with ease. Therefore the boiling tubes should be placed in a 50oC water bath. This will yield better results due to the optimum temperature theory of enzymes. As you increase the temperature, you will increase the kinetic energy of the molecules and therefore the substrate and enzyme molecules will collide more often and hence the reaction will take place faster.

There is an equation:

Temperature Co-efficient Q10 = Rate of reaction 10oC above temperature X

Rate of reaction at temperature X

For most enzymes between 0oC and its optimum temperature, every 10oC increase the rate of reaction doubles. That is Q10 = 2 (between 0oC and optimum)

For example if the rate was 5 at temperature X then 10oC above the temperature of X the rate would be 10.

However this trend will not continue forever. Eventually as you increase the temperature, the kinetic energy of the molecules increase, but at the same time the bonds of the molecules begin to break. If the bonds (i.e. the covalent bonds holding the molecule together) of the enzyme, (e.g. amylase) begins to break, then the shape of the enzyme changes due to unfolding and therefore the substrate (e.g. starch) will no longer be able to fit in the active site of the molecule and so no digestion occurs. This is the point where the enzyme is said to have denatured

So we find that the rate of reaction keeps increasing up until the point where the bonds of the enzyme begin to break, after this point the rate of reaction decreases again. There must be an optimum point as shown in the diagram below:

If the experiment were conducted at this temperature, which happens to be around 50oC, then the rate of reaction would be significantly increased, to be precise around 6 times as fast. As all of the chemicals, i.e. copper (II) sulphate solutions, amylase and starch will take place in this environment, then 50oC will become a constant. This will have no overall effect on the experiment other than to increase the rate by the same amount each time. The variability would come about only due to the concentrations of copper (II) sulphate.

Another factor that could improve the set of results is when to put the iodine solution into the mixture. The dark blue colour is produced by iodine binding onto the starch. As this could cause a change in the shape of starch, it is likely to cause an interference with the activity of amylase. This affect is known as stearic hindrance, literally iodine would hinder starch from binding at a molecular level, so a clear indication of how well amylase is working will not be seen.

The best thing to do would be to add the iodine to the solution a little while after the amylase has been added. In this way the amylase would be able to work on the starch and the iodine will still be able to show you when the starch ahs been digested.

Method

You will need the following apparatus:

Copper (II) sulphate solution 1.0M 100cm3 (harmful)
Amylase solution 1.0% 50cm3
Starch solution 0.1% 50cm3
Iodine solution (kept in a small bottle) 20cm3
Boiling tubes (10)
Boiling tube rack
Beakers (5 X 100cm3)
Pipettes (4)
Distilled water
Stopwatch
Bunsen Burner
Ceramic Mat
Tripod and gauze
Mercury thermometer
Water Bath

All the apparatus that would come into direct contact with chemicals was washed with distilled water. That is apparatus such as boiling tubes, beakers, and pipettes. A Bunsen burner was set up on top of a ceramic mat with a tripod and gauze. A water bath was place on top and was heated to 50oC and this temperature was maintained. Three boiling tubes were set up in a boiling tube rack. In the first of the boiling tubes 2 cm3 of 1.0% amylase solution was put in using a pipette. This pipette was placed next to the amylase solution, as it would only be used for the purpose of extracting amylase to prevent any premature reactions from taking place. The same was done with the pipettes for the other solutions. In the second boiling tube, 2 cm3 of starch solution was put in using a separate pipette. 2 cm3 of the relevant copper (II) sulphate solution was put into the third boiling tube using a separate pipette. All three of the boiling tubes were placed into the 50oC water bath and a timer was started which lasted for three minutes. This was to ensure that all of the solutions were at 50oC before they were mixed together. After three minutes the solutions were mixed together and a timer was started simultaneously. After 30 seconds, iodine solution was added to the mixture and the mixture turned a dark blue colour. When the solution had reached its original colour, which was determined by eye, the timer was stopped. The results were recorded in a results table, and the experiment was repeated for the remaining concentrations of copper (II) sulphate. The whole experiment was repeated three times for increased accuracy.

N.B. For the result where the concentration of copper (II) sulphate is 0, 2 cm3 of distilled water should be added to make the chances of collision the same. I.e. the distilled water will not inhibit the amylase, but will provide the necessary amount of water molecules to make the chance of amylase and starch colliding the same as if there were copper (II) sulphate solution present.

Results

The method was followed and the following results were achieved:

A results table showing the time and rate at which it took amylase to digest starch in the presence of copper (II) sulphate

The results were taken and tabulated in the above table. The moles if copper (II) sulphate is the amount of moles of copper (II) sulphate used for that sequence of results. It increases in 0.2 moles. The time 1st (S) is the first set of results taken for the varying concentrations of copper (II) sulphate, time 2nd (S) is the second set of results, and time 3rd is the third set of results taken. Average time is the average of these three results of time. Rate 1st, Rate 2nd, and Rate 3rd, are rates calculated from time 1st, time 2nd, and time 3rd respectively. Average rate is the average of these three rates.

Conclusions

Trends

As you can see from the first graph as the concentration of CuSO4 increases the time for digestion increases in a straight line, but towards the end curves slightly. Inconsistencies were minimal in the results, and the results had a fairly small spread, which means the results attained were quite accurate. The y-intercept which was 99.67 seconds was the average time it took for the amylase to digest starch without the presence of an inhibitor. So we can say that the relationship between increasing concentration and time is proportional to a certain extent, but ceases to be after 0.6M of copper (II) sulphate. The time for starch to digest increased by about 35 seconds every 0.2 moles of copper (II) sulphate. Towards the end however, the trend breaks and the time increases by about 45 seconds every 0.2 moles.

The rate of reaction naturally decreases as the concentration of copper (II) sulphate increases. As you can see from the graph as it progresses, the rate of reaction between each result roughly decreases by a factor of 1.2 each time as the concentration of copper (II) sulphate increases by 0.2 moles. The only exception is the difference between the 0.0M and 0.2M solution of copper (II) sulphate where the decrease in rate is larger than with the higher concentrations. This is shown in the table below:

So we could say that the relationship was inversely proportional to at the start was it not for the fact that the line of the graph touches the y-axis at the maximum, but after 0.6 moles the decrease in rate was in a straight line constantly decreasing at 1.2 each time. rate (i.e. when the concentration of copper (II) sulphate is 0).

We can clearly see that the copper (II) sulphate had an affect on the activity of amylase. As the concentration of the copper (II) sulphate increased, the activity of amylase to digest starch decreased. We know that copper (II) sulphate does not affect starch, as starch is chemically stable so the copper ions could not displace any other element present in the starch molecules. So the copper ions must have affected the amylase.

Explanation of results

Clearly the results achieved and the pattern observed was due to the reaction of the copper ions with the amylase molecule. Amylase is an enzyme, and all enzymes are globular proteins. The reason enzymes decrease the activation of the energy of a reaction is due to the effective shape of the enzyme to fit specifically the shape of its substrate in order for it do digest or decompose. As it is made up of many folded polypeptide chains, bonded uniquely, the shape of the protein is vital for the function of it.

Copper (II) sulphate is made up of two ions: a copper 2+ (Cu2+) ion and a sulphate 2- ion (SO42-), the two charges cancel out when together. In solution however, the water molecules being polar, separate the copper ions and the sulphate ions so the ions are free to move around in solution. The key part of the inhibition of amylase, however, is the copper ion. Amylase contains calcium in its structure, as this is bonded to other atoms, it is part of its structure and therefore is vital for the activity of the enzyme. Copper has the ability to displace the calcium, forming calcium ions and copper. The copper could therefore change the shape of the enzyme, thus changing the shape of the active site and therefore rendering the enzyme useless. As we can see in the results, with increasing concentrations of copper (II) sulphate (and therefore increasing concentrations of copper ions) the rate of reaction decreased. As the moles of copper (II) sulphate increased, the time it took for starch to denature increased proportionally at first (increasing by around 35 seconds every 0.2 moles), but after 0.6 moles, it began to increase further, i.e. 40seconds and 45seconds. An explanation for this is as the amylase molecule was becoming inhibited by the copper ions at lower concentrations of copper (II) sulphate, the general shape of the amylase molecule had changed, but not as much as to alter the active site as much. As the concentration of copper (II) sulphate increased after 0.6 moles, the time increased further as the shape of the molecule was beginning to change drastically. The enzyme was becoming more and more inhibited: meaning not only the shape of the active site was changing, but also the tertiary structure of the enzyme itself. This too can explain the late proportionality of the rate of reaction

Secondly enzymes contain negatively charged amino acid residues. Copper with its positive charge can bind onto these negative charged residues, drawing electrons away from their current positions, and therefore could end up denaturing the enzyme through disruption of bonds and so changing the shape. This binding could upset the catalytic properties of amylase as the copper could disrupt the tertiary structure of the protein, as it seemed at higher concentrations of copper (II) sulphate, around 0.6 molars. As some negatively charged residues participate in catalysis, having the copper bound on is going to interfere with activity.

Evaluation

The results achieved in the pilot were unsatisfactory with regards to finding out how copper (II) sulphate affects the activity of amylase. An improvement suggested on the method was to have a water bath at 50oC. We can calculate the temperature coefficient using the equation below and using the 3 attained results verses the same 3 but with the amended method:

Temperature Co-efficient Q10 = Rate of reaction 10oC above temperature X

Rate of reaction at temperature X

As you can see there was a great improvement in rate from the room temperature to the water bath at 50oC.

The spread of data was fairly small and is shown in the below table:

As you can see the range was quite low which indicates a good set of results. The range for rate was in general quite good too. However, the range for 0.0 moles was quite large. This could be due to the fact that the rate was very fast and as the collisions are random, then the faster the rate the more random it is likely to be and so the more spread the data. This experiment went quite well in accordance to drawing a firm conclusion and finding out the affect of copper (II) sulphate on the activity of amylase. It was important to repeat the results in order to get a more accurate overall result. The more times the experiment would be repeated the more accurate the results are likely to be.

The main flaw of this experiment was to judge when the enzyme had finished digesting the starch. It was very hard to make an accurate decision when the end point was. This was due to the fact that it was up to the individual to decide when the reaction was over. Even though a test-tube containing the relevant colour the solution should change back to was placed beside, it never went precisely that colour. It was especially harder in the longer reactions, such as the 1.0molar solution of copper (II) sulphate as the colour change was very long and the solution changed from dark blue to blue (due to the colour of the copper (II) sulphate).

Another limitation was the adding of the iodine. Iodine binds onto starch thus forming the dark blue colour. The reaction needed to have the iodine added near the start, as the change from dark blue to colourless had to be seen. However, as iodine binds onto the starch, there would always be some of the starch molecule left undigested and so a faint dark blue colour would be left.

As the copper (II) sulphate was made to a 1.0 molar solution, all the other concentrations had to be made from the 1.0molar solution. This too would prove an inaccuracy, as there would be a variation between the concentrations made and tested each time.

However, the experiment did yield satisfactory results in respect with what the hypothesis suggested. In this aspect the experiment was successful, even though there were inaccuracies in the method.

Further experimentation could be done by firstly using a colorimeter to detect the colour changes of the solution as the starch is being digested. This would yield increased accurate results. The experiment could be extended to see whether alpha amylase would be inhibited more or less than beta amylase using copper (II) sulphate.