Variables:
Variable: The temperatures of the hydrogen peroxide and the yeast.
Controlled Variables:
Volume of water in the measuring cylinder: 100.0ml
Times: 0, 1, 2, 3, 4, 5 minutes
Types of liquid: Water, Hydrogen Peroxide, and Yeast solution
Volumes of substances: 40.00ml yeast, 20.00ml hydrogen peroxide
Room temperature: 25ºC approximately
Temperatures of mixture: 20.0?C, 30.0?C, 40.0?C, 50.0?C, and 60.0?C. These must be kept as exactly as possible as yeast is very receptive to changes in temperature.
If these variables were altered, it would not be fair test.
Results:
20.00 20.00 20.00 Avg 30.00 30.00 30.00 Avg
0 100.00 100.00 100.00 0 100.00 100.00 100.00
1 75.00 58.00 66.50 1 41.00 42.00 41.50
2 61.00 45.00 53.00 2 2.00 10.00 6.00
3 50.00 27.00 38.50 3 0.00 0.00 0.00
4 39.00 13.00 26.00 4 0.00 0.00 0.00
5 30.00 2.00 16.00 5 0.00 0.00 0.00
40.00 40.00 40.00 Avg 50.00 50.00 50.00 Avg
0 100.00 100.00 100.00 0 100.00 100.00 100.00
1 58.00 60.00 59.00 1 25.00 22.00 23.50
2 39.00 27.00 33.00 2 0.00 0.00 0.00
3 18.00 9.00 13.50 3 0.00 0.00 0.00
4 2.00 0.00 1.00 4 0.00 0.00 0.00
5 0.00 0.00 0.00 5 0.00 0.00 0.00
60.00 60.00 60.00 Avg
0 100.00 100.00 100.00
1 69.00 28.00 48.50
2 26.00 2.00 14.00
3 5.00 0.00 2.50
4 0.00 0.00 0.00
5 0.00 0.00 0.00
Graphs on following pages
Conclusion:
I conclude that my prediction was partly correct. I was correct by saying that the reaction would be quicker as the temperature was 30?, but it was slower at 40? and again quicker at 50?. This may be because the catalyst, in this case catalase worked best at that temperature, allowing for more successful collisions between the yeast and hydrogen peroxide molecules.. I cannot explain these results because I can guarantee that I made sure that it remained a fair test throughout the experiment. I didn´t alter any of the other variables. All the other results came out to be what I expected, with the reactions slower at 20? and 60?.
Evaluation:
I was a little surprised at some of the results, and if I were to do this experiment again, I would try to discover what it was that gave these findings. I would also do the experiments for every 5? instead of every 10?. I would also measure other variables, to ensure that there cannot be any more fluke results. I would conduct the experiment more times to get a more accurate average. Although I conducted the experiment as accurately as I could there were many sources of error in the method that I used. Firstly, some help from friends was required to begin the experiment and this lead to a small delay in starting the stopclock. I would have to find a way to be a little more accurate. This would ensure that my results were as accurate and as precise as I could possibly get them.
How temperature affects the breakdown of hydrogen peroxide by the enzyme catalyse
Planning
Aim
My aim is to find out how temperature affects the breakdown of hydrogen peroxide by the enzyme catalyse.
Apparatus & Diagram
Delivery tube Measuring cylinder
Boss & Clamp
Boiling tube Stand
Water basin
Stopwatch Bunsen burner
Thermometer Heatproof mat
Weighing scales Tripod
Beaker Gauze
Measuring cylinder Safety goggles
Scalpel 6 x Boiling tubes
Plastic tile Potato
Ruler Hydrogen Peroxide
Variable to Alter
The variable I will alter in my experiment, is the temperature of the catalyse within the potato. This is because; by doing this I will answer my aim and also supply myself with the evidence needed to find out if the temperature of the enzyme (catalyse) does affect the breakdown of hydrogen peroxide. The temperature I measure will be in a regular scale, which starts at 10°c and goes up in tens to 50°c.
Variable to keep the same
The variables I will keep the same are mainly the amounts of hydrogen peroxide in each of the five boiling tubes, the mass of the potato in each test and that the potato pieces had the same surface area. I have kept these the same, because by doing this I have instantly blocked out any faults in the experiment, which may be due to those variables changing.
Safety Used Within the Experiment
The safety I will have to use is firstly safety goggles through out the experiment and also to have my tie tucked into my shirt, to avoid contact with a naked flame. I will also need a safety tile whilst cutting the potato to ensure that the potato or bench wasn't in any danger of germs. I will also have to consider the fact that other members of the group were to work along side myself, so I will have to be aware of other people's movements. The main term of safety is to be sensible and act quickly if any spillage of Hydrogen Peroxide was to occur.
Method
To begin with, acquire five boiling tubes and deposit in them 2 ml of Hydrogen Peroxide. Once you placed the liquid in all five boiling tubes, label each one of the boiling tubes starting with 10°c and then ending up with the fifth tube saying 50°c on it. There should still be one empty tube. This boiling tube is used to place the cube of potato in so that it can be heated or cooled to the required temperature, in the water bath. To set up the water bath, you will need a Bunsen burner and the general equipment that is required with it, and a beaker ½ full of water. The water bath for 10°c and 20°c is not filled with water and doesn't require the Bunsen burner; instead, it needs to be ½ full of ice.
After you have done this, cut up fifteen pieces of potato 1cm³ and making sure that they have the same properties as each other, for instance the weight, surface area and source of potato must all be the same. Fifteen potato cubes are needed, because the experiment is to be repeated three times for each temperature, to ensure that I get accurate results and able to draw a average of each temperature. Place one potato piece into the spare boiling tube and according to the label on the tube in which you are going to place the potato in after heating/cooling, put the cube of catalyse (potato) into the water bath for exactly 4 minutes. After the potato has been in the water bath for 4 minutes, carefully take it out of the boiling tube and into the boiling tube in which there is 2 ml of hydrogen peroxide, making sure that the label is correct to the temperature you set in the water bath.
The instant you place the potato into the liquid, quickly put the delivery tube that should be placed at one end in a water basin, which leads to a 100 ml measuring cylinder full of water. The water basin should be ½ full of water. The measuring cylinder would collect all the hydrogen being produced. Let the measuring cylinder collect the hydrogen for 4 minutes and the record the amount of water displaced. Repeat this procedure for each temperature three times to insure we have an average amount of hydrogen produced.
Prediction
By looking at my experiment, I predict that the rate of break down of hydrogen peroxide by the enzyme catalyse will increase as the temperature increases, but I also know that this will only increase to a certain point. This point is the denature stage, which usually occurs around 50°c - 60°c. So, I will be expecting to see results that increase and the suddenly decrease.
Explanation
I have predicted the above because from my knowledge, I have studied that in general terms enzyme activity increases as the temperature increases. This is because the molecules have more energy and therefore collide more often. This happens to a point where the enzyme becomes denatured and no longer works as usual. This is all because of how enzymes work, which is shown in the diagram below.
Enzyme
Substrate
This is how the enzyme looks before heating. Notice how the substrate fits exactly in the active site.
Enzyme
Substrate
This is how the enzyme looks after heating of 50°c-60°c. The substrate no longer fits the active site.
This sketch graph below shows how the activity of an enzyme increases as the temperature goes up, and it also shows how it suddenly drops as the enzyme is denatured.
Preliminary Work
I did a brief experiment in order to receive a more knowledgeable view over the final task. The changes that had to be enforced were that the hydrogen peroxide, which I had as 2 ml in the method, had to be increased to 4 ml per boiling tube. This is because the 1cm³ potato didn't fully get coated with the hydrogen peroxide. The usefulness of the preliminary work was that I got the idea of using instruments like a measuring cylinder for example, with more accuracy by measuring below the meniscus. The main advantage of the preliminary experiment, was that in my method I stated that one should use a 100 ml measuring cylinder for the retrieval of oxygen, but this test proved to me that the results were between 0-10 ml.
Hydrogen Peroxide
An investigation into how the amount of oxygen is produced by changing one variable effects the decomposition of Hydrogen Peroxide in the presence of the enzyme.
Hydrogen peroxide Water + Oxygen
2 H2O2 2H2O + O2
This shows us that when the H2O2 has reacted with the catalyst this is what it produces.
There are many ways in which the amount of oxygen produced could be affected. I believe that, surface area of the chip, temperature of liquid that is reacting, the concentration of the liquid or the amount used are going to be the major variables in the experiment.
From these variables I clearly believe that looking at the concentration of the liquid, in this case H2O2, will be the best experiment to do. Also I believe that this will give us a strong set of results with which we will be able to pull good clear conclusions from.
To keep all other variables constant so they do not influence my results, we will do all experiments in the same room hopefully the temperature remains constant in that one room, all potato chips will be cut with a size 4 cork bora and then cut into 3 centimetres, as to keep each test fair. Also the volume of the concentration H2O2 will remain at 10ml3 and the amount water that is placed into the measuring cylinder will also be 10ml3.
To set-up this experiment will be quite tricky, as we have to place the bung into the water and up the measuring cylinder very quickly as to not loose any of the oxygen produced in the first few seconds. We will overcome this problem by paralysing the measuring cylinder with a clamp stand, and another to hold the boiling tube where the H2O2 and potato chip are; which contain the enzymes used to catalyse the H2O2 to help produce the oxygen a lot quicker. We used the water in the measuring cylinder to measure the overall amount of oxygen produced by seeing how much the oxygen had displaced.
This a diagram of how my experiment is set up:
The effect of hydrogen peroxide on catalase if you change the temperature
AIM
Effect of temperature of the action of the Enzyme Catalase.
PLANNING
Background Knowledge
An enzyme is a biological catalyst, it alter the rate of reaction without being changed itself. Enzymes are proteins; they have a very precise three-dimensional shape, which forms a one specific active site on the enzyme. Each enzyme can only convert one kind of substrate molecule in to one kind of product molecule. These are specific.
What affects Enzymes?
· Temperature- Enzymes stop working if the temperature rises above 40ºC. Increasing the temperature alters the 3D shape and so the enzyme can no longer fit the substrate.
· pH- They work best in neutral conditions neither acidic nor alkaline.
What affect does catalase have?
Catalase is a very fast reacting enzyme, it is found in many living cells, it breaks down hydrogen peroxide to water and oxygen. In fact one molecule of it can deal with six million molecules of hydrogen peroxide in 1 minute. Hydrogen peroxide is toxic so needs to be changed into harmless substances.
Catalase
Hydrogen peroxide water + oxygen
2H2O2 2H2O + O2
References to practicals referring to enzymes
· Biology for You Pg 30 - Experiment 3.1
From looking at this I found out that catalase reacts with hydrogen peroxide to give out water and oxygen. Oxygen bubbles produce froth on the surface of the solution. In my forthcoming experiment I will expect to see froth being produced.
· Biology- Nelson Science Pg 25 - Picture 4
From looking at this graph, see below. I have learnt that the affect of temperature does in fact change the rate of reaction. From the graph the reaction reaches 40ºC but then denatures and the rate of the reaction decreases. The rate falls rapidly suggesting denaturing.
Taking this information into account I would expect the enzyme catalase to show a similar pattern with respect to the temperature.
In order to observe the effect of temperature on catalase we will be maintaining in the amount of oxygen released. The oxygen produces a froth which we will then measure in mm and the volume of oxygen given off which will be measure in cm³
Method- measuring the height of froth and volume of oxygen
1. Put work shirt on and goggles on. Carry out the rest of safety precautions.
2. Gather equipment as shown on diagram1.
3. Using a cork borer make 5 cylinders from the large potato.
4. Cut them into all the same length (6cm)
5. Using a pestle and mortar mash up each cylinder separately.
6. Measure 25ml of hydrogen peroxide using a measuring cylinder.
7. Select the temperature you are going to study
0ºC- iced water
25ºC-no extra equipment
37ºC-water bath required
55ºC-water bath required
100ºC-beaker of boiling water
8. Place on mashed cylinder into a boiling tube add the measured hydrogen peroxide and attach the rubber bung connected to the measuring syringe.
9. Start stop watch and record volume of gas collected every 30 seconds. At the same time measure the amount of froth produced at 30 seconds intervals
Apparatus
· 5 beakers
· 5 test tubes
· Thermometers
· Cork borer
· Potato
· Ruler
· Knife
· Tile
· Measuring syringe
· Heat proof mat
· Bunsen burner
· Tri-pod
· Wire gauze
· Pestle and mortar
· Hydrogen peroxide
· Matches
· Spills
· Ice cubes
· Water bath
· Goggles
· Spatula
· Stopwatch
· Measuring cylinder
Fair test
In this investigation I will keep constant the following
· The surface area of the potato. I will use the mashed up form as it will be a faster reaction as there is more area to react on, as we have to consider the time span.
· The same volume of hydrogen peroxide in each part of the investigation.
· The same size equipment e.g. boiling tubes as the readings for the results will be wrong if this is not constant.
· Use the same method for each experiment so that there won´t be any major differences. Only alter the temperature.
· Keep the amount of potato the same amount.
· Measure the temperature with a thermometer.
Accuracy
In order to make my investigation go to plan I will be as accurate as I can be so I will measure to the correct measuring size.
· Measure the volume in cm³ and amount of potato in grams to make sure that they are exactly the same mass before using them in the experiment.
· Do the experiment three times to ensure that there isn´t an odd result. Three is a good number to use as you can see if there is one odd one where if you just done the experiment twice then you wouldn´t know which one odd and which isn´t.
· Also to average out the results.
Safety precautions
· Wear goggles
· Tuck tie in skirt
· Wear work shirt
· Handle the hydrogen peroxide with care as it is corrosive and an irritant
Predictions and Reasons
From my research I think that the enzymes will denature after 40ºC and any other temperature above that. Reason being that enzymes are proteins and their structure is three-dimensional. Increasing the temperature disturbs the intra molecular bonds that hold the 3D shape. Because of this the shape is altered. Enzymes have an active site. This fits into the substrate molecular (see diagram2-lock and key). If the active site is altered the substrate will no longer fit in and so the enzyme doesn´t work properly.
The rise of reaction rate is also due to the increase in temperature, relating to the kinetic theory. The higher the temperature, the faster they move. This happens but only to an optimum of 40ºC. The curve leading up to the optimum point is gradual but as it is reached it falls dramatically. The reason being that the active site is destroyed therefore no reaction can take place as there is only one specific active site per substrate.
Analysing results and Conclusion
From my results it appears that catalase works best at 37ºC, and it is virtually denatured at boiling point.
Looking at the initial part of the reaction (see graph 1) it is clear that the gradient at the beginning gets steeper when looking at the temperatures between 10ºC-55ºC. At each temperature the line levels off towards the end of five minutes. Looking at graph 2, there is a steady rise in height of froth up to 37ºC and then a gradual fall up to 100ºC.
Looking at my background knowledge and prior experiments using enzymes I can explain my results as follows.
Kinetic theory states that particles, which gain heat energy, move more quickly. In our case the reacting particles are the substrate (hydrogen peroxide) and the enzyme catalase. As the temperature is increased the particles of hydrogen peroxide have more energy therefore they collide with the potato more frequently and so increasing the rate at which the product is formed. However at a certain temperature this is no longer the case. This is because enzymes are proteins and proteins can be denatured at high temperatures. This is because proteins have a 3D shape. In our case the catalase has a certain shape that the substrate fits into. At high temperatures the active site on the enzyme is altered, see diagram below.
(Diagram showing active site on the enzyme is altered therefore stopping products being formed)
This stops the substrate from 'fitting´ and so no product is formed.
My results do not totally support or undermine my original prediction. The reason being that on graph 1, my results suit my prediction. It shows that the temperature, 37ºC was the fastest and 100ºC is when the enzyme denatures. But in graph 2, my results undermine my original prediction as at 55ºC the reaction still takes place where as in my prediction I stated that enzymes would denature at 40ºC approximately, I didn´t expect this is happen.
Evaluation
In my investigation I was pleased with my achievements.
In my method, keeping the temperature constant throughout the investigation was hard to maintain, as the temperature of the contents of the tube would change quite quickly and therefore the hydrogen peroxide wouldn´t be at the temperature required. To overcome this problem I could keep the test tubes in a hot water bath for all the temperatures making sure that the water bath was the suitable depth. This would ensure constant temperature throughout the whole 5 mins. Also another problem that I encountered was to keep the height of the froth fair. I measured the height of the froth with a 30cm ruler against the test tube rack, with the support of my hand. As I was measuring, my hand would move from time to time and therefore didn´t know where I should place my ruler afterwards. To over come this I should attach the ruler onto the test tube rack with cello tape, as it is transparent or maybe use a pointer.
With respect to I measured the height of froth in cm, but to be more precise I should have measured it in mm. To over come this I should use a ruler with mm readings. Also another problem that I observed on accuracy was that I didn´t allow the temperature to equilibrate to the right temperature. In this case I wasn´t using the correct temperature that I wanted, this could have led to some anomalous results. Ideally I should have brought the temperature of the hydrogen peroxide up to the needed temperature before adding to the potato.
Looking back at my results I found some anomalous results in my findings. When averaging I used these results, which could of made the average either lower or higher than it should be. To improve this I should have missed these results. Not including some sets of results when making averages may have led to better values.
My results are in line with those I predicted. Graphs indicate rise in temperature up a point leads to an increase in oxygen production. This is in line with kinetic theory. However it is very clear that after a certain temperature is reached the enzyme actually virtually stops. This supports my theory of lock and key fit.
However optimum activity of enzyme is at about 37ºC this is as we expected. But at 55ºC the enzyme is still not denatured according to my results. This is a higher temperature than I would expect. Possible not allowing solutions to reach temperatures selected has led to an inaccuracy. It may be that in fact that many temperatures of solutions were lower than we stated.
Overall, due to reliable repeats and in general predictions being confirmed I feel my results are reliable enough to make a conclusion.
The obvious thing I would improve about the measurements I made would be to increase the range of temperatures used. Especially between 55ºC-100ºC. In this way it may be clearer at the temperature which denaturing took place, and would possibly give a graph that resembled the graph in background knowledge.
Another way of improving this investigation is to change the method. I measure the volume of oxygen that was produced. In order to get pure oxygen without any other gases that are in the air I would use the same equipment but make sure that the gap between the rubber bung and solution was free from any other gases.