The Effect of Temperature on Catalase Enzyme.
The Effect of Temperature on Catalase Enzyme
Aim: Investigate the effect of temperature on the activity of catalase.
Introduction: Enzymes are biological catalysts. They speed up metabolic reactions in the body but remain chemically unchanged themselves. Enzymes contain an active site. This is a region, normally a depression or cleft, to which another molecule may bind. This molecule is known as the substrate, and is usually specific to the active site of the particular enzyme, which breaks it down. Substrates will not usually fit into any other active sites other than that of the enzyme it is specified to. This can be explained as a lock and key model, where the lock and key are specific to each other, only, that there are many of the same kinds of lock and key when it come to the enzymes.
Just as lock and keys have three-dimensional shapes, proteins are also three-dimensional. Usually, there is only one active site on an enzyme; however there can be more. Some energy releasing reactions in cells produce hydrogen peroxide. This is acidic, and can thus, kill cells. Normally, hydrogen peroxide decomposes to form hydrogen and oxygen:
2H2O2 2H2O + O2
However, this process is very lengthy. There is an enzyme known as catalase in cells which dramatically increases the rate of decomposition of hydrogen peroxide.
catalase
2H2O2 2H2O + O2
This type of reaction where a molecule is broken down into smaller pieces is known as a catabolic reaction.
In order to investigate the effect of temperature on the activity of catalase, I will record the amount of oxygen released when hydrogen peroxide is broken down.
Variables: There are quite a few variables which can alter the rate of reaction, and need to be kept constant. They are as follows:
a) PH: at too high PH, the enzyme is denatured due to the loss of H+ ions. The same applies for too low a PH level, where too many H+ ions would attach to the negative regions of the enzyme, changes its shape and causing it to denature.
b) Concentration of enzyme: The higher the concentration, the higher the rate of reaction will be. With a larger number of catalase molecules, the chance of successful collisions between enzyme and substrate will be increased. In order to keep this constant, I will make sure I use the same volume of tissue containing catalase each time I conduct the experiment.
c) Surface area: The previous also applies to this.
d) Mass of tissue: Here it needs to be taken into account that different liver will not give the same mass, even if equally sized pieces are cut. Different pieces liver will not have exactly the same water and catalase content.
The mass will be kept constant in the same way as surface area and concentration of enzyme.
Prediction: The higher the temperature, the higher the rate of reaction up to a certain point. This is due to the fact that the particles gain kinetic energy and subsequently move around more vigorously. Thus, the chance of there being a successful collision between the enzyme and substrate molecule increases as reacting particles with collide more frequently with increased kinetic energy.
Enzymes have a very specific three-dimensional shape, held together by ionic and hydrogen bonds. If the amino acids are too vigorous in their motion, then, these bonds will brake. Once the bonds have been broken, the enzyme is said to have become denatured. As a result of becoming denatured, the enzymes' rate of activity becomes less because the enzyme loses its specific three-dimensional shape. The enzyme will start to become denatured after around 40ºC as enzyme activity is usually at its optimum at this temperature. After this, the rate of reaction will probably deteriorate. After 60ºC, there is likely to be no reaction, as the enzymes would probably be completely denatured by then.
Methodology:
. Wear goggles for protection, and lab coat if available.
2. Arrange apparatus.
3. Get 250cm3 beaker and fill with water to about the 150cm3 mark with water at a specific temperature. This temperature can be reached by either cooling, via the use of ice, or by heating, via the use of a Bunsen burner.
4. While the water is reaching the desired temperature, bore out a cylinder of tissue with cork borer.
5. Cut the cylinder, with a ruler so that it is three centimetres long. Then, cut this three centimetre long cylinder up into a further six equal pieces, each 5 millimetres wide. Make sure to keep eyes level with ruler so as to minimise the chance of parallax error. The diameter of the cylinder remains constant due to the use of the cork borer.
6. Put the pieces into a boiling tube and put aside.
7. ...
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4. While the water is reaching the desired temperature, bore out a cylinder of tissue with cork borer.
5. Cut the cylinder, with a ruler so that it is three centimetres long. Then, cut this three centimetre long cylinder up into a further six equal pieces, each 5 millimetres wide. Make sure to keep eyes level with ruler so as to minimise the chance of parallax error. The diameter of the cylinder remains constant due to the use of the cork borer.
6. Put the pieces into a boiling tube and put aside.
7. Measure out 30ml of hydrogen peroxide, using a 50cm3-measuring cylinder. Pour this into a boiling tube.
8. Get the two boiling tubes containing one containing hydrogen peroxide and the other liver. Put them both into the beaker of water once the water has reached the desired temperature (this can be done by heating or adding ice to water). After this, wait until the temperature of the hydrogen peroxide and the water bath are equal.
9. Once the temperatures of the liver and hydrogen peroxide are equal, clamp the boiling tube containing the hydrogen peroxide to a clamp stand. Then add the liver into the test tube
containing the hydrogen peroxide and put bung on top of the test tube, which now contains both liver and hydrogen peroxide, and make sure it is in the beaker of water.
0. Now, measure the water being displaced from the 100cm3 measuring cylinder every thirty seconds using a stop watch.
1. Take readings every thirty seconds for five minutes.
2. Repeat the above steps for different temperatures.
Risk assessment:
During this investigation, I will use catalase from liver cells to speed up the breakdown of hydrogen peroxide. Hydrogen peroxide is an oxidising agent, as oxygen will be given off during the reaction. This implies that it could help the burning of fires. Due to this, reasonable care must be taken to ensure that the oxygen is not directly exposed to the flame of the Bunsen burner while water is being heated. As well as this, hydrogen peroxide can be very harmful if it enters the eyes. Thus, goggles must be worn. Also, if available lab coats will also be worn.
The cork borer is also a potential hazard as it is quite sharp. When cutting out the piece of liver, it must be ensured that the liver is resting on the tile before it is cut. The methodology explains how it will be cut.
The blade which will be used to cut the liver, is also another hazard. It must be handled with appropriate care as it is sharp.
Glass apparatus must be handled with care so that they do not break. If they do, that is also another hazard, and should be cleared up and disposed of in an appropriate fashion.
Fair testing:
While conducting the experiment, it has to be taken into account that a few variables will have to be controlled so as to ensure that the test is fair.
. The PH of the reactants should be kept constant. This will not be hard to ensure as the PH of the reactants does not vary significantly during the course of the reaction.
2. The temperature of the reactants will have to be kept constant during the reaction. This is a variable which will be harder to control. There will be used, a beaker of 150cm3 of water at the appropriate temperature into which will be immersed, the boiling tube containing the reactants once their temperatures have equalised and stabilised. This will ensure that the temperature of the reactants is stable for longer as the larger volume of water in the beaker will maintain its temperature for longer. Also, the same volume of water will be added to the beaker each time a new test is to be done, so as not have any differences in how long the water maintains its heat for.
3. It must be ensured that when taking readings off the measuring cylinders and thermometers etc, the readings are taken with the graduated markings at eye level, so as to minimise the risk of inaccurate readings.
4. All boiling tubes etc. must be washed thoroughly with water after their use, so as to minimise the chance of contamination.
5. Making sure that all the liver pieces are cut to the same width, and have the same diameter will control the surface area of the liver.
While conducting the experiment, it must be ensured that the utmost attention is paid to accuracy due to the fact that accurate measurements will result in accurate results. Subsequently, the evidence for or against my prediction regarding the enzyme activity in relation to temperature will be more strong, and, more reliable. The above mentioned methods of accurate measurement will be used as they are most appropriate for the situation, with time factors being one of the major reasons for the augmentation of such methods, as well as the availability of equipment.
During the experiment, the following apparatus will be used:
Cork borer (size four)
Liver
Hydrogen peroxide
Water
Blade
Ruler (15cm)
Stop watch
Test tube rack
Boiling tubes
Delivery tube and rubber bung
Water container
Measuring cylinders (100 cm3 and 50cm3 )
Goggles
Cutting tile
Clamp and stand
Beaker (250cm3)
Graduated pipette (5ml)
ice
Thermometer (0 ºC-100 ºC)
During the experiment, the apparatus will be set up in the way shown below:
During preliminary work, I found that the 100 cm3 measuring cylinder was ideal for the measurement of water being displaced by oxygen formed during the reaction. This is because it is graduated in millilitres. Thus, it will be possible to measure the displacement of water to the nearest 0.5 of a millilitre. Also, the measuring cylinder is not so small that the volume of oxygen produced will be greater than the cylinder can hold within the course of the reaction.
The 50cm3 measuring cylinder used to measure the volume of hydrogen peroxide was ideal as the amount of hydrogen peroxide was wasn't too little that it would be inappropriate and also inaccurate to measure with a 50cm3 measuring cylinder, nor was it so large, that it would just about be measured.
Boiling tubes are ideal for the reaction to take place in, as the volume of oxygen produced is quite small. Thus, it will be quicker for the oxygen produced to be able to displace the water in the measuring cylinder. With a conical flask, it would take much longer.
I also found that the breakdown of hydrogen peroxide at room temperature is very slow (Without a catalyst). No oxygen was given off at all over the period of time I observed the hydrogen peroxide for any reaction. However, I did not test whether this was true for higher temperatures. If it was not, then there is the likelihood of major inaccuracies in the conducting of the experiment.
Results:
A Table to show the Volume of Oxygen Produced due to the Catabolic breakdown of Hydrogen Peroxide in Relation to Temperature:
Key:
Time Volume of oxygen produced Temperature
(Seconds) (Cm3) (ºC)
0
20
30
40
50
60
70
0
0
0
0
0
0
0
0
30
0
2
3
2
0.5
60
0.5
2
2.5
3.5
2.5
.5
0.5
90
3
4
4
3
2
20
.5
4.5
5.5
5
4.5
2
50
2
5.5
6
7
5
2
80
2.5
6.5
7
9
5
2
210
3
7
8
0
5
2
240
3.5
7
0
1
5
2.5
270
4
8
1
2
5
3
300
4
8.5
2
3.5
6
3
A Table to show the Volume of Oxygen Produced due to the Catabolic breakdown of Hydrogen Peroxide in Relation to Temperature (repeat tests):
Key:
Time Volume of oxygen produced Temperature
(Seconds) (Cm3) (ºC)
0
20
30
40
50
60
70
0
0
0
0
0
0
0
0
30
0.5
2
0.5
0
60
0.5
2
3
3.5
2
0
90
3
3.5
5
2.5
.5
0.5
20
2
3
5
5
3
2
0.5
50
2
4.5
6
7
3
2.5
0.5
80
3
5.5
7
7.5
3.5
2.5
0.5
210
3
6
8
9
4
2.5
0.5
240
3.5
7
9
1
4
3
0.5
270
4
7.5
1
2
4.5
3
0.5
300
4.5
0
2
3
4.5
3.5
From the graphs, the gradients were taken for the graph of oxygen produced at each temperature. This gave the rate of reaction for the breakdown of hydrogen peroxide, at the different temperatures the experiment was carried out at, in volume of oxygen produced in cm per second. The rate of reaction was then converted to the volume of oxygen produced in minutes, by multiplying each gradient by sixty. The two sets of gradients obtained for the graph of each temperature (one for the initial experiment, and the other set of gradients for the graph drawn from the repeat tests) were matched according to temperature at which the hydrogen peroxide was catabolically broken down at, and their average was taken for a more true picture of what the rate of reaction really is at the different temperatures.
A table Showing the Rates of Reaction for the Initial Tests, the Repeat Test, and also the Average Rate of Reaction
Temperature (ºC)
Rate of reaction for the results obtained in initial test (cm3/min)
Rate of reaction for the results obtained in repeat test (cm3/min)
The average rate of reaction (cm3/min)
0
5.30/270 = 1.06
5.20/294.5 = 1.04
(1.06+1.04)/2 = 1.05
20
9.90/300 = 1.98
9.95/300 = 1.99
(1.98+1.99)/2 = 1.99
30
3.1/300 = 2.62
2.65/300 = 2.53
(2.62+2.53)/2 = 2.58
40
3.4/300 = 2.68
3.5/300 = 2.7
(2.68+2.70)/2 = 2.69
50
5.50/300 = 1.1
4.85/300 = 0.97
(1.10+0.97)/2 = 1.04
60
2.85/300 = 0.57
2.35/300 = 0.47
(0.47+0.57)/2 = 0.52
70
0.70/300 = 0.14
0.6/300 = 0.12
(0.14+0.12)/2 = 0.13
Conclusion: The results obtained during the course of the experiment seem to be quite conclusive. It possible to identify a pattern or trend in the results obtained. From the rate of reaction graph, we can see that the oxygen is produced more and more quickly via the breakdown of hydrogen peroxide (when the reaction is catabolic) with an increase in temperature. This is, however, only up to a certain point. We see that the rate of reaction keeps on increasing until 40ºC, after which it starts to fall. There is still some oxygen being produced after 70ºC, but only very little, almost zero.
With respect to the results obtained, I can now say that the prediction I made earlier on was more or less correct, although not as correct as I had hoped they would be, as I had predicted that the rate of reaction would be zero after 60ºC, and this is more true of enzyme activity at 70ºC. However, the rest of my prediction seems to be in support of my hypothesis. Thus, it would seem that the enzyme activity (in other words, the rate of reaction) increases with temperature up until around 40ºC as the enzyme and substrate molecules gain more and more kinetic energy. As a result, the reactants move around with increased vigour. This results in there being an increased number of effective collisions. Subsequently, the rate of reaction increases. After 40ºC, the rate of reaction deteriorates. Although the kinetic energy increases, and in essence, the rate of reaction should keep on increasing, this is however not true. This due to the fact that after 40ºC, the optimum temperature for catalase enzyme activity, the weak bonds that hold together the enzyme structure, start to break (this is especially true of hydrogen bonds), due to the increased kinetic energy. Resultantly, the rate of reaction deteriorates as the enzyme becomes denatured due to the fact that its active site, and ultimately its whole structure, is lost due to the breaking up of the bonds that hold it together. This means that the substrate molecule can no longer fit into the active site of the enzyme as the shape of the active site changes.
Evaluation: Although the results obtained from the experiment were more or less support in my prediction, I was not totally satisfied with the experiment. There were a lot of errors, both in the conducting of the experiment, and in the results obtained.
First of all the way I conducted the experiment was quite flawed. For example, the measuring cylinder used to measure the volume of oxygen produced, was quite inappropriate, and probably resulted in many of the readings being taken being quite inaccurate as it is easy to make a mistake in reading off the value while trying to hold the measuring cylinder straight, and also trying to keep the bottom of the meniscus of the water at eye level, all at the same time. Also, it was only possible to measure to the nearest 0.5 of a centimetre cubed, as the measuring cylinder was only graduated in 1cm3
The method used to keep the temperature of the reactants constant at certain temperatures was also improper, as water in the beaker which was used for the above purpose, was not in a large enough volume or quantity to retain its heat for more than a minute or two. Thus, the temperature of the reactants was fluctuating during the experiment. Also, the water in the beaker did not cover all the reactants in the boiling tube, and some of the reactants were not immersed by water. Thus, there were regions of unequal temperatures in the reactants.
The controlling of the surface area of the liver was also inaccurate as it was impossible to measure the lengths of liver to exactly 5mm throughout.
Also, it was assumed that the PH of the reactants would remain constant throughout. This may not have been the case
Considering the above, it is feasible to say that the results obtained during the experiment are neither likely to be very reliable nor very accurate.
Considering that the there was so much possibility for inaccuracy, there were not however, any major anomalies in the results obtained, although the results obtained in the repeats at
20ºC and 50ºC, were on the doubtable side, as there was quite a difference when the volumes of oxygen obtained were compared with those obtained during the first test:
Volume of oxygen produced (in cm3) at:
20ºC 50ºC
st test: 8.5 6
2nd test: 10.0 4.5
These slight anomalies may have arisen may have arisen due to a number of reasons:
a) Improper measurement off measuring cylinder
b) Improper surface area of liver, and effectively enzyme
c) Difference in temperature due to loss of heat (kinetic energy)
d) PH level may have altered
If I was to conduct the experiment again, I would make sure that it was more accurate overall. This would ensure that the results obtained were more reliable and accurate. I would do this in the following ways:
) I would use a graduated syringe, instead of a measuring cylinder as this would ensure that it was easier to measure the volume of oxygen produced. It would also be more accurate as there would be a clear line to mark the amount of oxygen produced and there would not be a need to observe where thw meniscus of water touched on the graduation marks.
2) I would use a greater volume of water to ensure that the temperature of the reactants remained constant, due to the fact that water is such that, the greater the volume, the greater its ability to retain heat.
3) I would use a buffer to control the PH of the reactants as it would ensure that the Ph remained constant.
4) I would crush the liver to a paste, using a mortar ant pestle, and drain the filtrate for use as the catalase sample. A measure of the volume would be made.In this way, the exact amounts of enzyme could be calculated. If not, then I would use yeast as my enzyme, as it can be measured out to very precise quantities.
5) I would also use a graduated syringe to measure out the amounts of hydrogen peroxide, and other fluids. The reasons for this are the same as the ones mentioned above.
With the above taken into account, I would say that my conclusion is in fact, not very safe due to the fact that there were too many errors and uncertainties concerning the results obtained. Subsequently, the results do not provide a very stable evidence for support of my hypothesis. Although the percentage error of individual equipment may have been at first glance, small, they add up to large percentage errors which then render useless, the legitimacy of the results obtained.
