To Investigate how temperature affects the rate at which catalase catalyses hydrogen peroxide.
An Investigation Into How Temperature Affects The Rate At Which Yeast Catalase Catalyses The Decomposition Of H O
Aim:
To Investigate how temperature affects the rate at which catalase catalyses hydrogen peroxide.
Apparatus And Materials:
20cm 5 % yeast solution
2gs yeast
40cm warm water
1 small (50ml ) beaker
Metal spatula
50cm hydrogen peroxide (H O ) 1mo/dm
Stopwatch
Gas syringe
00cm conical flask
2 x 5cm syringe
2 test tubes
Thermometer
Clamp
500cm glass beaker
Measuring cylinder
Balance
Bunsen Burner
Tripod
Gauze
Heat Proof Mat
Method:
) Set up the apparatus as shown above, put on apron and safety goggles.
2) Take the 2gs of yeast and add to the 40cm of warmed water in the small beaker, stir using the metal spatula and leave to stand for a minute.
3) 2cm of the yeast solution should be measured out using a 5ml syringe and placed in 1 of the test tubes, 5cm of the H O should be measured out using the other 5cm syringe and placed in the other test tube.
4) Then place both test tubes and the empty conical flask in the water bath, and heat over the Bunsen burner, checking the temperature on the thermometer until the right temperature has been reached
5) Keep the temperature steady for 2 minutes by removing the heat (if it drops dramatically, heat again) to allow the test tubes to remain at a constant heat
6) Take out the warmed conical flask, pour the yeast solution from the test tube into the warmed conical flask, then add the H O and quickly place the bung into the neck of the conical flask and start the stopwatch
7) After 30 seconds, record the amount of oxygen given off shown by the gas syringe.
8) Repeat from step 3, using the same method but changing the temperature.
I am investigating the differences in the results for 5 temperatures, 20 C, 30 C, 40 C, 50 C and 60 C. I will repeat the experiment twice for each temperature to try and eliminate errors and inaccuracies; I will then calculate a mean value and use that to plot a graph of results.
By warming the conical flask to the appropriate temperature (placing it in the water bath) it means that the temperature remains constant as the two solutions move from the test tubes to the flask, which means more accuracy as to how much O is given off in relation to the temperature of the solution. The balance has two decimal places, that means that the yeast can be measured out to an accurate 2gs. The more accurate the amount of yeast used, the more accurate the percentage of the yeast solution will be, and the more controlled the investigation. I using suitable size syringes to measure out each solution, helping to keep the amounts used accurate. A gas syringe will be used to collect the O which has millimeter divisions, so the amount of O given off can be measured very accurately. To make the investigation more reliable, I will be working with a partner so we will be able to manage the investigation better, eg: putting the bung in the flask and starting the stopwatch at the same time.
Variables
* Volume of hydrogen peroxide
* Concentration of H O
* Temperature
* Volume of yeast solution
* Mass of yeast
* pH
I am going to vary the temperature during the investigation and control all of the other variables. I shall alter the temperature by increasing the temperature of the water bath to heat the hydrogen peroxide and yeast. I will therefore keep the other variables the same in order to make it a fair test, and a comprehensive study without many variables. I will keep my variables the same by making sure that I have implied the same strategy for each test taken. The amount of hydrogen peroxide and yeast solution ...
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* Mass of yeast
* pH
I am going to vary the temperature during the investigation and control all of the other variables. I shall alter the temperature by increasing the temperature of the water bath to heat the hydrogen peroxide and yeast. I will therefore keep the other variables the same in order to make it a fair test, and a comprehensive study without many variables. I will keep my variables the same by making sure that I have implied the same strategy for each test taken. The amount of hydrogen peroxide and yeast solution used is carefully measured out using a syringe; enabling me to make sure they are kept constant throughout the experiment. The human factors in the test are the starting of the stopwatch, the pouring of the solutions and the speed at which the bung is placed in the conical flask once the solutions have been mixed. Since I will be working with a partner, they should be managed adequately but they are factors to evaluate. I am going to use a glass beaker to provide water baths for the solutions instead of a tim bath, in the hope that the fact that glass is a better insulator than tin that it will keep the heat in more effectively, and help keep the temperature more constant.
I will have to be careful when using the hydrogen peroxide, as it is a corrosive chemical, and blistering may occur if it touches skin, so I will be wearing safety glasses to protect my eyes. Hydrogen peroxide is a bleaching agent, so I will be wearing a lab coat so that it does not damage my clothes. I will use tongs to retrieve the test tubes from the hot water bath. The surfaces that I will be working on will be kept free of books, and I will tie my hair back when using the bunsen burner.
I conducted pilot tests to determine what concentration of hydrogen peroxide and yeast solution to use. I decided to used a lower percentage of H O so that that O was given off at a slower rate and was easier to measure.
Prediction:
Catalase reacts with hydrogen peroxide to give out water and oxygen. This can be written as:
2H O 2H O + O
My pilot test showed that in 30 seconds, at a temperature of 40 C, 34cm of O was produced. I predict that between 20 and 40 C, the rate of decomposition will increase, so the volume of O produced will also increase. At 40 C the rate of H O will reach an optimum point, and so the volume of O collected will be the largest result. Above 40 C I predict that the rate of decomposition will drop so the volume of O collected will also drop. I predict that every 10 C the volume of O given off will double, because there is more kinetic energy and this will increase the activity of the hydrogen peroxide and catalse.
I think that the enzymes will denature after 40°C and any other temperature above that.
I predict that my graph of results will look like the one shown here on the right, it shows an exponential shape. The red arrow shows the rising arm of the curve, it peaks at 40 C and then the blue arrow shows the falling arm. The curve steadily rises until it reaches just past 40 C, after
which it drops steeply. I think that the graph will show and exponential shape, like the one shown above.
All enzymes work in a similar way. They are catalysts, changing the rate of a chemical reaction (speeding it up) but not themselves being changed or used up. The lock and key hypothesis describes how they work; each enzyme has a place somewhere on the molecule called an active site. The reacting chemicals (substrates) "fit" the site and it is the binding of the chemical (or chemicals) that causes the reaction. Each enzyme is specific for one particular reaction.
Most enzymes are proteins, they have optimum temperatures, for warm-blooded mammals like ourselves this is our body temperature (37 C). High temperatures cause enzymes to denature. They cause the large complicated protein chains to unravel and change shape permanently. The substrates will no longer fit the active site and the enzyme will not work.
Enzymes are proteins and their structure is three-dimensional. Increasing the temperature beyond a certain point disturbs the hydrogen bonds that hold the 3D shape, and alters the shape. This alters the active site so the substrate will no longer fit in and so the enzyme will not work properly.
Yeast is used most commonly in breweries, because they are unique to all living organisms in that they contain the extra enzymes that allow for fermentation. One enzyme that it contains is catalase. Catalase is used for applications where hydrogen peroxide has to be removed, such as in contact lens cleaning systems, for bleaching of textiles and hair, and in industrial processes that generate hydrogen peroxide. One molecule of catalase can break 40 million molecules of hydrogen peroxide each second.
My predicted graph shows a rising arm, which means there is increased molecular activity within the solution, producing larger amounts of O . This is due to the increase in temperature, relating to the kinetic theory. The higher the temperature, the faster the particles move. The graph shows this happening to an optimum of 40°C. The curve leading up to the optimum point is gradual but as it is reached it falls dramatically. I think this is because the active site is destroyed therefore no reaction can take place.
Results:
Table To Show The Amount Of Oxygen Given Off At The Different Temperatures
Temperature ( C)
Repeat 1 (cm of Oxygen )
Repeat 2 (cm of Oxygen )
20
5
4
30
22
24
40
27
28
50
25
24
60
2
3
Table To Show The Mean Values Of The Amount Of Oxygen Given Off At Different Temperatures
Temperature ( C)
Mean Value (cm of Oxygen)
20
4.5
30
23
40
27.5
50
24.5
60
2.5
Analysis & Conclusion:
In this investigation I have looked at how temperature affects the rate that catalase decomposes hydrogen peroxide, by investigating the volumes of O given off.
From my results it appears that catalase works best at 40 C and starts to denature after this. This shows that the optimum temperature for catalase is 40 C. the steepest part of line on the average graph is between 50 and 60 C where the enzymes have been denatured. The other steep part of the curve is between 20 and 40 C, where the volume of oxygen being given off is increasing.
The graph clearly shows that the volume of oxygen given off increases between 20 and 40 C, as I predicted. It then reaches a peak or optimum, shown more clearly on the Average Graph, at 40 C, also as I predicted. After this it appears that my prediction was correct again, the enzymes denature and the arm of the curve drops from 40 C onwards. The graph shows an exponential shape between 20 and 40 C where the amount of oxygen given off increases, my prediction suggests this and the results support it, overall my prediction is mostly correct, my results support each part of my hypothesis.
The quantitive prediction I made was incorrect, the volume of O given off did not double every 10 C. Although there was an increase in kinetic activity, which resulted in the catalase catalysing at a higher rate, it did not double.
I conclude that altering the temperature of catalase and yeast changes the way the solutions react in terms of the volume of oxygen given off. Up to 40 C increasing the temperature causes a higher rate of decomposition resulting in a higher volume of oxygen being given off. After 40 C the volume of oxygen given off drops because the catalase has passed its optimum temperature and the hydrogen bonds within the molecules have begun to change, causing the catalase to denature and change the shape of the active site. This denaturing affects the catalase's ability to catalyse the substrate, hydrogen peroxide, by destroying the enzymes active site, which is why at 60 C there is almost no catalyzing, as shown on the graph and in my results table.
Evaluation:
All of the points on my graph lie on or near the line of best fit, however there is one I have decided that is anomalous, repeat 1's 30 C result. It is 2cm out from the line of best fit. The rest of the points which do not lie on the line of best fit are within 1cm of it.
There were some limitations to the methods I used to collect my results. There was a limited time in which we had to set up the apparatus, collect the results and clear away, my partner and I finished within this time, but when measuring out the solutions we could have paid more attention to the exact amount, to ensure more accurate results we could have taken more time over the measuring. The yeast solution and the H O were measured out using syringes, and we had basically the right amount each time, but perhaps not as accurately as possible. This may affect the accuracy and in turn the reliability of our results. There was also the possibility of air bubbles occurring in the syringes, which would mean that the amount of solution taken up would be incorrect. There were also a number of jobs to do at once, just as the solution was mixed, such as pouring the solution, starting the stop watch and putting the bung in the flask. It would have been more practical to have a third person to help, so as to ensure each job was done properly. If the bung was not added quickly enough, oxygen could escape and so effect the result recorded. Reading off the volume of oxygen was checked and double checked by my partner and myself, but because we were standing at slightly different angles, and the only way of reading it was by looking at the mm marks, there may be slight inaccuracies in our results. Reading off the volume inaccurately would affect the reliability of our results, although our repeat helps to even out this problem. If the solutions were warmed for too long, the catalase would have more kinetic energy and so would catalyze quicker, affecting the results because there would be more O given off in the time period. There is also no real way of controlling the amount of catalase in 2g of yeast, this varies according to the yeast itself, and we did not try to control it.
I believe that my results are reliable and are substantial enough to support my conclusion. The graph shows 4 anomalous points, but none of them are substantially wrong, and the limitations, as mentioned before, did not heavily affect the reliability of the results. There is no great variability in the results collected; the repeats show close results with the greatest difference being 2, on 30 C.
The method that I used to test my prediction was suitable and fair. There were limitations to the procedure, but not so many that they meant that the method was unsuitable. There are limitations to any method, I feel that I tried to control as many as possible, and that the ones that were not controlled did not heavily affect the reliability of the results.
To over come the limitation of not putting the bung in quick enough, I would work with another partner, so that there were 3 of us, to allow each job to be done as accurately as humanly possible. There is always human error to account for when it comes to starting stop watches, putting bung sin etc. because of differing reflex times, ideally it would be done with a machine which could be programmed exactly, but this is not feasible for a secondary school experiment. To improve accuracy in the amount of solution used, more care should be taken when measuring it out, to ensure that no air bubbles appear. Recording the amount of O given off sometimes causes inaccuracies if the number is not read correctly from the gas syringe, the divisions (cm ) are suitable for this experiment, but to be especially sure, a photograph could be taken at exactly 30seconds, to record where exactly the gas syringe was.
Two repeats were taken, ideally there would be 3, to ensure the most accurate line of best fit. The range of readings taken is also something I would change, I would do more temperatures, I feel that the ones used were not accurate enough, for example I would also record 25, 35, 45 C etc, this would give more accurate and reliable results, and eliminate big gaps in the graph. I believe the apparatus we used was appropriate and suitable for the experiment.
To further support my conclusion, I would look into the amount of froth given off when the H O was mixed with the yeast, I hypothesize that this would increase as the catalase reaches its optimum temperature, and then decrease as it denatures. This could be done with larger amounts of solutions, and perhaps video recording equipment, to record the experiment, as it seems to occur fairly quickly.
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