Investigating an enzyme-controlled reaction: catalase and hydrogen peroxide concentration

2. Identify any anomalies or inconsistencies in your results.

Students may identify some anomalies or inconsistencies in their results. They should be able to explain what to do with them.

3. Describe the shape of the graph.

At low concentrations, the graph will show an increasing rate of reaction as concentration increases, levelling off at higher concentrations.

4. Explain the shape of the graph in relevant biological terms.

The shape is explained by the concentration of substrate directly affecting the rate of reaction until another limiting factor becomes more important.

5. Describe any technical difficulties you had with this apparatus and explain how these could be overcome.

Students should be able to describe technical difficulties and suggest how these could be overcome.

6. Describe how you would extend this investigation to provide more evidence/ data to support your understanding of enzyme-controlled reactions.

Further investigations could investigate other possible limiting factors, such as temperature, pH, or concentration of enzyme.

To help make this experiment more accurate, I repeated it three times and then used the average of all the results to plot a graph with a line of best fit. I tried to keep all the variables except for the concentration of Hydrogen Peroxide the same for all the experiments. However, in reality it is impossible to keep all the variables precisely the same. For example:

a) There is a slight delay between pouring the Hydrogen Peroxide into the yeast, putting the bung on and starting the stopwatch. This will slightly affect all the results but as I carried out all the three steps in the same way for all the experiments it should not make any difference to the overall result.

b) It is also impossible to precisely measure out the amounts of Hydrogen Peroxide, Yeast and Water each time. As the scale on the pipettes shows the volume to the nearest mm3 the volume of the solutions that I used should be correct to the nearest mm3. The volume of gas in the test tube to start with is slightly affected by the amount which the bung is pushed down each time, if the bung is pushed down further then the volume in the tube will be less so the 30cm3 of gas is reached faster.

c) Due to the fairly slow speed of our reactions it is only possible to measure the time of the reaction to the nearest 0.1 second even though the stopwatch shows the measurements to the nearest 0.01 second.

Conclusion

-The conclusion is stated and answers the aim

-The conclusion has been explained using scientific theory

-A comparison is made with a literature value. If no literature value then state attempts made.

-The source of the literature value is fully referenced.

-The results have been referred to as supporting the conclusion.

-Sub-heading ‘error analysis’ has been included.

I predict that as the concentration of hydrogen peroxide increases, the rate of reaction will increase until the pureed potato becomes saturated with the hydrogen peroxide. If the reaction reaches saturation point, no more reaction will be occurred.

The reason that the rate of reaction increases is that more subtracts can react with more active sites of the enzyme when the concentration of hydrogen peroxide increases. As the results, the more oxygen will be produced in the high concentration of hydrogen peroxide.

10% of hydrogen peroxide will produce 10cm3 of oxygen from every cm3 that decomposes. In this procedure, 2 cm3 of 10% hydrogen peroxide will release 20cm3 of oxygen if the reaction goes to completion.

2H2O2   ➔   2H2O + O2

My hypothesis was right. As the concentration of hydrogen peroxide increased, the rate of reaction also increased. This is because more substrates could react with more active sites of the enzyme when the concentration of hydrogen peroxide increases. As a result, the more oxygen was produced in the high concentration of hydrogen peroxide.

There was no oxygen produced in the distilled water. However, as the concentration of hydrogen peroxide increased from 5% to 25%, the amount of oxygen produced increased from 1.80±0.25mL to 6.02±0.25mL.

When the concentration of Hydrogen Peroxide is increased, the rate of reaction increases at a directly proportional rate until the concentration of Hydrogen Peroxide reaches about 16%. If you double the concentration of Hydrogen Peroxide then the rate of reaction doubles as well. When the concentration is doubled from 8-16% the rate goes up from 1.65-2.97 Cm3 Oxygen produced per second, which is an increase of 1.8 times. I would expect the rate to increase two times if the Hydrogen Peroxide concentration is increased two times because there are twice as many substrate molecules which can join onto the enzymes active sites. The reason that the number is less than two times could be put down to the fact that at 16% the Enzyme's active sites may already be close to being saturated with Hydrogen Peroxide. There may also be some experimental error which causes the inaccuracies.

After 16% the increase in the rate of reaction slows down. This is shown by the gradient of the graph going down. At this point virtually all the active sites are occupied so the active sites are said to be saturated with Hydrogen Peroxide. Increasing the Hydrogen Peroxide Concentration after the point of saturation has been reached will not cause the rate of reaction to go up any more. All the active sites are being used so any extra Hydrogen Peroxide molecules will have to wait until an active site becomes available.

The theoretical maximum rate of reaction is when all the sites are being used but in reality this theoretical maximum is never reached due to the fact that not all the active sites are being used all the time. The substrate molecules need time to join onto the enzyme and to leave it so the maximum rate achieved is always slightly below the theoretical maximum. The time taken to fit into and leave the active site is the limiting factor in the rate of reaction.

The diagram below shows what happens

V.  EVALUATION

POST-LAB SURVEY OF STUDENTS' CONCEPTIONS

  Have students retake the Pre-Lab Exercise.  Compare pre-lab and post-lab responses.

TRADITIONAL

1. Describe the general structure and function of an enzyme?

Answer:  An enzyme is a 3-dimensional molecule composed of long chains of amino acids, held together by peptide bonds. They are catalysts that make biochemical reactions possible.

2. Describe what happens in an enzyme catalyzed reaction.  Include diagrams in your explanation.

Answer:  During an enzyme catalyzed reaction the enzyme bonds with a specific substrate at the active site.  This is called an enzyme-substrate complex.  The substrate is converted into a specific product, but the enzyme remains unchanged.  Enzymes accelerate reactions by factors of at least a million.

3. Describe what will happen to the rate of an enzyme controlled reaction if the initial concentration of substrate is increased.

Answer:  As the initial substrate is increased, the initial reaction rate will increase up to the point where all the enzyme molecules are engaging at their maximum rate. Beyond this point the rate of reaction will remain constant (level off).

Initial

Reaction

Rate

                        Concentration of

                        Substrate

4. Describe what will happen to the rate of an enzyme-controlled reaction if higher concentrations of the enzyme are used.

Answer:  As more enzyme is added initially to the substrate, the reaction rate will increase up to the point where all there are too few substrate molecules to allow all of the enzyme molecules to collide with them and catalyze the reaction at the maximum rate. At that point no additional increase in the initial rate of reaction will occur.

Graph for explanation

Initial

Reaction

Rate

                        Concentration of

                        Enzyme

Observations and Measurements – In the boiling tubes it was clear that a reaction was taking place by the observation of bubbles of oxygen gas being released creating a ‘fizzing’ in the boiling tubes.

In order to decide how varying the enzyme concentration affected the decomposition of hydrogen peroxide, the rate of reaction was measured.  To do this accurately, the time taken for a specific quantity of oxygen gas (a product of the reaction) to be released was determined.  This was achieved by observing the time taken for the manometer fluid to travel between the two marked fixed points as it was forced through the manometer by the rising gas.  This was an accurate measure of how the enzyme concentration influenced the breakdown of hydrogen peroxide, as the quantity and speed of gas produced is dependant on the rate of reaction.  The marked points remained the same distance apart for each reading for different enzyme concentrations so that they could be accurately compared and the trend observed.
All measurements were taken so that the stopwatch was started once the rubber tubing was sealed and the stopwatch stopped once the manometer fluid had reached the base of the highest marked point.  To judge  accurately, the point at which the fluid reached the marked line, it was examined at eye level and the measurement taken when the bottom of the meniscus was lined up to the mark.  This was  the same for every reading.

Data handling – The data obtained from this investigation has been recorded in a table showing the time, enzyme concentration and rate of reaction.  This means that the results of the experiment are presented in a clear and orderly fashion that allows patterns in the results to become more obvious.
The rate of reaction was calculated by dividing 1000 by the time taken for the quantity of gas to be produced from the reaction.  By calculating the rate of reaction instead of merely using the time readings, the quicker reactions will be represented as a greater value for the rate of reaction rather than a small time value.  This makes the graph more clear and easier to analyse.
Patterns within the results collected from the experiment, are best shown on a graph.  This is because overall trends between the enzyme concentration and rate of reaction can be portrayed more effectively and become more obvious.

Limitations and Precautions – In this investigation, I measured the rate of reaction with enzyme concentrations of between 0 and 35 units (potato discs).  At 0, there should be no reaction as there will be no substrate, however, I included it to act as a control.  This will show that it is the variable, enzyme concentration that is being measured.
I decided to vary the enzyme concentration by varying the number of potato discs.  However, although the enzyme, Catalase, occurs in the potato tissue, I did not know the exact quantity and certain discs might have more Catalase than others.  This could be a major limitation in this investigation.  I have tried to compensate for this, however, by taking multiple readings for each enzyme concentration so that inaccuracies are minimised once averaged.
As a precaution, I have limited my contact with the boiling tubes, as my body heat will raise the temperature, increasing the rate of reaction or expanding the gas inside the test tube moving the manometer fluid.
I also monitored the temperature using a thermometer to ensure that it remained constant and not disrupt the results of the experiment by affecting the activity of the Catalase.
A pH buffer was used to maintain a consistent pH level in the boiling tubes. This way there was no variation in pH that might have resulted in an increase or decrease in the rate of reaction.
A major limitation of this investigation was the time. It meant that only 8 different enzyme concentrations could be measured at intervals of 5 units or potato discs.  This means that only very general, overall trends can be identified across the results.  Patterns between these values can only be approximated and are not necessarily accurate. 

 
Results – The rate at which hydrogen peroxide was broken down to water and oxygen in the presence of Catalase:
The graph “The decomposition of hydrogen peroxide in the presence of potato catalase Chart 2” shows the rate of reaction up to an enzyme concentration of 25.  Up to this point the line of best fit is a straight line through the origin. This shows that without the enzyme, catalase, present no reaction takes place.  It also indicates that the enzyme concentration is directly proportional to the rate of reaction for the decomposition of hydrogen peroxide in the presence of catalase (the rate of reaction increases with increasing enzyme concentration).         
The other graph, “The activity of potato catalase with differing enzyme concentrations Chart 1”, shows how the rate of reaction varies with differing enzyme concentrations over the whole range that I experimented with.  After an enzyme concentration of 25 potato discs, the line of best fit is no longer a straight line and begins to level off.  The enzyme concentration is no longer proportional to the rate of reaction, and the increases in the rate of reaction reduce dramatically. 

Conclusion – The reaction was fastest at an enzyme concentration of 35 potato discs.  At this enzyme concentration there were the greatest number of free active sites available to the substrate molecules so that they could be broken down. 
The rate increased steadily from 0 up to a concentration of 25 and slowed beyond this point to give a “maximum level”.  It appears that at this “maximum level”, increasing the enzyme concentration had little effect and other factors such as substrate concentration were limiting the reaction and prevented any further increases in the rate of reaction.

Discussion – The results of this investigation are as I predicted in the hypothesis:  “The reaction will increase with increasing enzyme concentration when molecules of hydrogen peroxide are freely available.  However, when molecules of the substrate are in short supply, the increase in rate of reaction is limited and will have little effect”.  The reasons for this are that there are number of variables that influence the decomposition of hydrogen peroxide in the presence of Catalase.  Some of which can be classified as limiting factors i.e. the reaction is dependant or “limited” by their availability, to be able to function effectively; these include enzyme concentration, temperature and substrate concentration. All of these factors are required for an efficient reaction to take place, even when one is freely available the reaction can still be limited by the availability of the others.  When I increased the enzyme concentration, it meant that there were more free active sites for the substrate molecules so that a greater quantity of substrate molecules could be broken down into products.  However past a certain point, which in my investigation was at an enzyme concentration of 25 potato discs, there were many free active sites but insufficient substrate molecules to occupy them.  Increasing the enzyme concentration further without increasing the substrate concentration has no effect on the rate of reaction which eventually will remain constant.
From the line of best fit on the graph “Chart 1”, it is clear that some of the points do not exactly fit.  They are anomalies.  Although they have only slight inaccuracies, they are an indicator of possible errors in the investigation.  These may have occurred in either the measurement of the quantities of the enzyme and substrate or the measurement of the time taken for the manometer fluid to rise five centimetres up the manometer tube.  Another possibility was that fluctuations in temperature caused the rate of reaction to increase or the gas inside the boiling tube to expand, forcing the fluid to rise up the manometer tube.  Although minimal contact was made with the apparatus during the investigation, slight undetected variations in the room temperature may have led to inaccuracies.
The precision of this experiment, generally, was very limited since insufficient readings were taken.  Although the range of enzyme concentrations taken was large, the difference in enzyme concentration between each reading was too great to distinguish a value between them.  For example, the rate of reaction at an enzyme concentration of 15 potato discs was 35 + or – 4.  This results in an error of uncertainty of 11%
The shape of the graph is as I predicted showing that as enzyme concentration increases so does the rate of reaction.  This is because at a greater enzyme concentration, there are more free active sites available for the substrate and so more products can be made in a shorter length of time.  However, it is not possible to take precise readings from the graph between the plotted points since insufficient readings were taken.  To be able to do this, intermediate enzyme concentrations would have to be measured so that the shape of the graph would be more exact.

Suggestions and Improvements - To create a more accurate experiment in the future, several precautions or alterations can be made:
 
· Instead of using potato discs that have slight variations in size, and volume of catalase, as a source for the enzyme, a 1 molar solution of the enzyme could have been diluted to create different concentrations.  This way the concentrations can be measured far more accurately reducing the chances of errors in the investigation. 
· 

In this experiment 8 enzyme concentrations were considered.  However, although there was a large range, insufficient intermediate measurements were made creating gaps between the measurements where guess work is needed to predict the rate of reaction at these points e.g. point A on graph “Chart 2”. In a future investigation, a far greater number of enzyme concentrations between those already recorded should be tested reducing the extent of any anomalies on a graph where the line of best fit must be drawn.
· In this investigation each reading was repeated so that an average rate of reaction for each enzyme concentration could be calculated.  This could be improved by repeating the reading more frequently thus reducing the extent of any anomalies further, once averaged.

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