Effects of Copper Sulphate on the Activity of Catalase

Abdus Salam

A/S Biology Coursework

Introduction

Enzymes are biological catalysts and their contribution to our existence is pivotal. Without them, human and animal digestion may take days. Enzymes therefore, help catalyse reactions at an increased metabolic rate. Each type of enzyme would normally act upon only one type of substrate molecule. This is due to the specific shape of the active site. Sometimes, enzyme activity is disrupted not because of temperature or other environmental factor but by alien molecules, which attach themselves to the enzyme molecules just as easily as the intended substrate. This is called inhibition.

Aim

I will be testing the effects of Copper Sulphate (inhibitor) on a reaction between catalase and hydrogen peroxide. I predict that the greater the concentration of the copper sulphate, the slower the reaction. Reactions will be measured at the rate of which oxygen is released from the H2O2. I will use yeast as a source of the enzyme catalase. If it is an inhibitor, I will go further and try to prove if it is a competitive or non-competitive by increasing substrate concentrations. Fig 1 shows word equation of the reaction.

2H2O2 2H2O + O2

Figure 1

Scientific Involvement

At a molecule level, it is thought that the substrate molecules fit precisely into the enzyme molecules in the same way that a key fits a lock (see Fig 2). The part of the enzyme molecule into which the substrate fits is called the active site.

Figure 2

The configuration of the enzyme molecule is due to ionic bonding, hydrogen bonding, disulphide bridges and hydrophobic interactions

I know that enzymes have optimum temperatures and optimum pH levels. The enzyme is said to denature at temperatures exceeding its optimum or at the wrong pH level resulting in an ineffective enzyme molecule (Fig 3a). Inhibitors that compete for the active site are called competitive inhibitors.

a b

Figure 3

The greater the concentration of substrate, therefore the more likely it is to occupy the active sites and the less likely the effect of the inhibitor. It is said to be competitive because of this reason. Non-competitive inhibitors (Fig 3b) tend to attach themselves to a sight other than the active site. In doing so, they alter the shape of the active site in such a way that the substrate is unable to occupy the enzyme. Similarly, prior binding of the substrate to the active site causes a change in the inhibitor site, which prevents the inhibitor from binding. An increase in substrate does not diminish the affect because neither substrate nor inhibitors are competing for the same site. On the other hand, we have competitive inhibitors, which compete with the substrate for the active site. Increasing the concentration of the substrate reduces their affects.

Enzymes do not alter the equilibrium of a reaction, only the speed at which it is reached. They therefore catalyse the forward and reverse reactions equally.

Provided there is an excess of enzyme molecules, an increase in the substrate concentration produces a corresponding increase in the rate of reaction. At the point where there are sufficient substrate molecules to occupy all active sites on the available enzyme molecules, meaning that the reaction is unaffected by further increases in enzyme concentrations.

Figure 4

At this point the enzymes are working their hardest

A classical non-competitive inhibitor has absolutely no effect on substrate binding. In fact, a change to the shape of the active site is almost certain to alter the ability of the substrate to bind. It won't stop it altogether but the affinity will be reduced. Inhibitors like this are often called mixed inhibitors, as they appear to have some of the properties of competitive and non-competitive types. In fact, classical non-competitive inhibitors are very rare, if they exist at all.

Catalase contains Fe in its active site. Cu++ may inhibit catalase by displacing the Fe from the enzyme. If the Cu is removed and Fe added back, activity is likely to be restored. This suggests that Cu is a non-competitive inhibitor and it alters the enzyme such that it no longer functions. This alteration often involves a change in the catalytic site of the enzyme. In contrast to competitive inhibitors, when the Cu is removed, the enzyme remains inactive.

Hypothesis

I predict that copper sulphate will retard the rate of reaction between the catalase - hydrogen peroxide complex ...

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Hypothesis

I predict that copper sulphate will retard the rate of reaction between the catalase - hydrogen peroxide complex because the molecules of CuSO4 and H2O2 have different structures. I picked up this information during discussions with teachers and students. This implies that the effects of copper sulphate will not be reduced when the substrate concentration is increased, and that the reaction will not reach the v max. This trend can be displayed better in the graph (Fig 5) where the substrate concentration is increased gradually with a fixed amount of inhibitor in the solution.

Figure 6

The reason behind my prediction is that I believe the CuSO4 is a non-competitive inhibitor and will slow down the reaction. However, as the substrate concentration is increased, its activities will stay the same therefore having a lower rate of reaction. This is because I believe CuSO4 is a non-competitive inhibitor and this is what is known to happen to them even when the substrate concentration is increased. If I do achieve similar results to my prediction then it will prove that CuSO4 is a non-competitive inhibitor because the inhibitor still had an effect after the concentration was increased.

Variables involved:

Temperature (Independent)
pH level (Independent)
Yeast concentration (Independent)
CuSO4 (Independent)
H2O2 concentration (Dependent)

As with all enzyme activities, temperatures play important parts in the rates of reactions. They will affect my results if not kept constant, especially when I don't intend to test it. It will be kept constant at room temperature ( ≅ 21°C). I do not expect there to be any drastic fluctuations in the temperature, this makes the use of a water bath unnecessary.

The pH level is another factor which will be kept constant as it is also irrelevant to the investigation. This is because enzymes also have an optimum pH level which can also effect my results. Varying pH levels will make it difficult for me to identify whether the CuSO4 or the pH of the solution is affecting the reaction, thus making my results unreliable.

The yeast and CuSO4 concentrations have to be kept constant as they affect the rate of reaction, since I am trying to find out how CuSO4 interferes with an enzyme-substrate complex. Otherwise, the cause of particular trends will be indistinguishable.

This means that the only variable in this experiment will be the H2O2.

To ensure that it will be a fair test, I will have to carry out all experiments under the same conditions, i.e. same concentrations (except for the variable) and same volumes. I will also repeat experiments three times to give me an average and to help me to identify anomalous results if any.

Method

Procedure:

I will dilute 5ml of H2O2 as required and put it in the boiling tube. After sealing the boiling tube correctly, I will collect 2ml of catalase in the syringe and put it through the bung with the needle end to fitting tightly in the bung. With the measuring cylinder in position, I will squeeze all the catalase into the tube and start the timing simultaneously. From then on the amount of O2 (ml) released will be recorded every 10 seconds.

The same procedure will be used when experimenting with the CuSO4 itself, but this time one ml of it will be added to the H2O2 in the boiling tube. This should produce results showing how it differs from the normal reaction.

Another important point is that I clean the boiling tube thoroughly if I choose to reuse it or use a clean one each time. This is because any remnants could effect the results.

Apparatus:

Figure 7

Diagram of apparatus

Oxygen bubbles

I have chosen this particular apparatus out of a choice of three alternative sets. Out of the other two one used a manometer tube. The problem with this was that if oxygen was produced in large volumes, it will push out all the water from the tube, thus making it very difficult to observe and record amounts of O2 released. This is why I immediately eliminated this from the selection. The second set had a gas syringe for the gas to collect in. in theory it was the soundest method and as I was already familiar with the apparatus I intended to use it. This is because it can be used to easily measure the amount of oxygen produced very accurately and is also very easy to use.

But my plan was thwarted when during preparations I discovered that the syringe was sometimes faulty and would not move smoothly. I could not afford to continue further with my intentions because I could not risk such a disaster during experimenting.

This left me with the apparatus displayed above as my only choice. Although very fiddly, it is probably just as good as the gas syringe apparatus. The idea is that a measuring cylinder full of water is placed upside down in the bowl of water. NB the water does not pour out because there is nothing to fill the empty space it will produce. However, the coming of oxygen encourages the water to pour out. The rate at which the water level falls can be taken as the rate of oxygen produced. This method guarantees that no oxygen will be lost at any stage.

I will be doing two tests. One to show the normal rate of reaction between 2%catalase and various concentrations of 3%hydrogen peroxide and the second will be the same but with One ml of 0.5M copper sulphate added to it. The two different results will allow me to make a comparison and will also allow me to conclude whether copper sulphate is an inhibitor of catalase or not. And testing it with various concentrations will show me if it is a competitive or non-competitive inhibitor.

Dilution Table:

Safety

Enzyme inhibitors are metabolic poisons harmful to all forms of life. They must be handled with care.
Any small volumes of the inhibitor must be measured out using a syringe; safety spectacles must be worn.
Aprons or some other form of protective clothing must be worn because the H2O2 is known to bleach most things it comes in contact with.
Enzyme preparations containing added inhibitors should be in labelled tubes
When the experiment is completed, dispose of mixtures with excess water and rinse out the tubes before setting them aside for washing.
Otherwise good laboratory practice is sufficient to take account of any hazards and avoid significant risks

Bibliography

Toole, G. & Toole, S. ‘A Level Biology’ Letts Educational (1999)
Jones, M., Fosberry, R. & Taylor, D. ‘Biology 1’ Cambridge University Press (2001)

Results

The amount of oxygen produced (ml) in the reaction with no addition of CuSO4.

Table of average results for amount of O2 (ml) released:

The graph (see graph 1) generally shows the trend which occurs when there is no CuSO4 present in the solution. It shows that there is a positive correlation between the amount of oxygen produced and the concentration of the substrate, and as time goes on, more oxygen is produced. By comparing the initial rate of reaction graph of this reaction to that with the CuSO4 in it I will be able to deduce whether CuSO4 is an inhibitor and secondly, if it is a competitive or non-competitive inhibitor.

There is quite a difference between the production of oxygen between the 8vol (40%) and 4vol (20%) concentrations the reason why cannot be identified but must be because of a possible equipment malfunction. This, however, should not effect the trend for the rate of reaction because the result still shows a similar trend.

Table showing how much oxygen was produced (ml) after One ml of 0.5M Copper Sulphate was added

Table of average results for amount of O2 (ml) released:

The graph (see graph 2) shows similar trends to that of graph one but the fact that it does not produce as much oxygen is clearly evident. This goes to conclude that copper sulphate is an inhibitor of this reaction. This time I cannot identify a whole set of results as being anomalous but only one, this the amount of oxygen produced in a 16vol solution at 30 seconds (5.1 ml). This result clearly stands out from its set when observed from the graph. The reason behind the error could be that I misread the value.

Initial rate of reaction was worked out as follows

For example, if I recorded that 20ml of oxygen was produced in 30 seconds, then in one minute (2*30) an estimated 40ml (2*20) of oxygen will be produced. The units of the rate of reaction are therefore in ml/min. The following table shows the expected rates of reaction in ml/min

Table showing the amount of oxygen produced for initial rates of reaction:

The graph (see graph 3) of the results satisfies my prediction because the two lines are how they were expected. The graph goes on to prove that CuSO4 is a non-competitive inhibitor because the activities of the inhibitor aren’t affected even after several concentration increases. The reasons behind this is because all metals tend to be non-competitive inhibitors and a displacement reaction often occurs with the metal found in the active site.

Summary of Results

From the results, I can tell that copper sulphate is a non-competitive inhibitor. This conclusion has been deduced from the fact that the solution with CuSO4 in it did not recover from the effects of the inhibition to something near to that of solution without CuSO4.

The CuSO4 binds to an inhibitor site on the enzyme, which is remote from the active site and brings about a conformational change in the active site. In this sense, it is very similar to one of the competitive inhibitor types. The difference is that this time the change in the active site is such that it does not prevent substrate binding but, rather, prevents the enzyme from converting the bound substrate to product. A classical non-competitive inhibitor has absolutely no effect on substrate binding. In fact a change to the shape of the active site is almost certain to alter the ability of the substrate to bind. It did not stop it altogether but the affinity was reduced.

Evaluation

The procedure I chose was appropriate for the experiment as it allowed me to produce quite reliable and accurate results. The procedure involved making recordings at identical intervals, this was crucial in producing results but the difficulty was that it was difficult to read off values exactly on time when the reaction was still taking place. This was probably why it led to one or two anomalous results. The method must have been the correct method because it produced the correct results. The only thing I have against the method was the refilling of the measuring cylinder with water after each trial. This required a dextrous set of fingers and patience at the same time, both of which deteriorated as the experiment went on. This is why I would have preferred to use the gas syringe apparatus because it is much easier to reset.

From the results, I can only identify a set of anomalous results and an anomalous value. The value, which was mentioned earlier, could have been due to my fault in reading it off accurately. But I wouldn’t believe myself to have read a whole set of results inaccurately. The set of results for 20% H2O2 with no CuSO4 present seems to be less productive of oxygen than expected. I have to attribute this result on faulty apparatus; after all it was the last test for that part of the experiment. Something might have happened to the apparatus during tests done with the other concentrations.

My planned method was followed word for word and at the end of the day I produced some very reliable results, therefore my method must have been very suitable for the task. It was actually my ability to read values and faulty apparatus that lead to errors rather than my method. In my opinion it was ideal for producing such reliable results.

My method have limitations because I didn’t try for a continue to record values until the reaction had stopped completely. I also should have done some experimenting with solutions of more than a 100% concentration to further support that CuSO4 is non-competitive inhibitor. Testing with more concentrations may even surprise me with a shock increase in the rate of reaction, if this does not occur then eventually excess substrate inhibition should take place and reduce the rate of reaction to zero.

The results are satisfying because they represent my prediction and most importantly they represent what is known to happen when CuSO4 is introduced to a catalase and H2O2 reaction. This makes my results very reliable and it poses as firm evidence that proves CuSO4 is a non-competitive inhibitor of catalase.

The use of a gas syringe would help make the results more reliable because I will be less concerned with resetting the equipment, meaning more time would be spent on attempting to acquire more accurate results. Thus avoiding any anomalous results at all. The gas syringe is designed for collecting gas and it will always give you the correct reading no matter what position you hold the syringe in. With a measuring cylinder, you have to keep it straight because the slight tilt will make the meniscus appear to be at one value on one side and a different value on the other.

I don’t have enough anomalous results to make my conclusion invalid, the few that I do have are very minor anyway. All the anomalous results are eliminated when a line of best fit is drawn on the rate of reaction graph, which shows a trend of the completely reliable results.

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