As the temperature increases, the more the atoms which make up the enzyme molecules vibrate. If the temperature continues to rise, the hydrogen bonds and other forces (i.e. ionic bonds) that hold the molecules in their precise shape are broken. If this three-dimensional globular structure is disrupted, the enzyme ceases to function as the active site no longer accommodates the substrate. The enzyme is denatured and loses its catalytic properties.
Graph
Apparatus
150cm3 gas syringe – this is where the oxygen will be collected and measured in cm³.
Delivery tube – this is needed to connect the conical flask to the gas syringe in order for the oxygen gas to flow through.
250cm3Conical flask- this is where the reaction between the hydrogen peroxide solution and enzyme catalase (in potato discs) will take place.
Bung with two holes – the bung will be inserted onto the conical flask and sealed tightly so that no oxygen will escape. One hole is for the delivery tube and the other for the 10cm3 syringe.
10cm3 syringe – the sole purpose of this piece of apparatus is to make sure no oxygen gas is lost, the hydrogen peroxide in inserted directly into flask via the syringe instead of taking off the bung and inserting it, which would result in some gas escaping.
Waterbath – essential in order to regulate the temperature required.
3 thermometers – to measure the temperature in the waterbath (one on the right side and one on left side for precision) and for the test tube containing the hydrogen peroxide solution.
50cm3 beaker – the hydrogen peroxide is kept in here until ready to be measured.
10cm3 measuring cylinder – the 10cm3 of hydrogen peroxide is measured here.
Test tube – this is where the hydrogen peroxide will be kept when heating it to the required temperature in the waterbath.
Test tube stand – to hold the test tube.
Stop clock- this is needed to time the reaction for 300 seconds for each reading.
Potato – this contains the enzyme catalase (contains the active site), needed to decompose hydrogen peroxide.
20cc of Hydrogen peroxide – this is the substrate needed in order for a reaction to occur.
Cork borer/knife – for cutting the potato and making the 1mm by 7mm shaped discs.
Petri dish – this is where the potato discs will be kept in until ready to be out in the conical flask.
Tile – this is the surface where the potato is cut on.
Clamp stand - to hold the gas syringe in place.
Ice – use as an aid for lowering the temperature of the water.
By using the bung with the two holes and the 10cm3 syringe, more reliable results can be obtained. This is a more accurate technique rather than removing the bung, adding the hydrogen peroxide and then putting the bung back on. By inserting the hydrogen peroxide through a syringe no oxygen gas would escape.
During preliminary work, the enzyme to substrate ratio was investigated and it was found that a ratio of 1: 2 worked best which is 20 potato discs (containing the enzyme) to 10cm³ of hydrogen peroxide. This is because the active sites of the enzyme are occupied more quickly.
Method
- Set up apparatus needed as shown below. Set the waterbath to the temperature required.
- Cut the potato and make several cylinders with the cork borer and slice 20 approximately 1mm by 7mm (measure with ruler if needed) shaped discs with a knife. Put them into a petri dish. 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.
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Measure 10cm3 of hydrogen peroxide (20cc) in a measuring cylinder and pour this into a test tube. Place the test tube in the water bath standing in a test tube rack. Place thermometer in the test tube and leave to equilibrate for 5 minutes.
- Transfer the 20 potato discs into the conical flask, place the bung back on and leave in the water bath. Leave water to equilibrate for 10 minutes.
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Put the hydrogen peroxide in a beaker. Remove 10cm3 of the solution using a 10cm3 syringe.
- Place the syringe into the hole in the bung and add the hydrogen peroxide to the potato discs in the conical flask.
- Immediately start the stop clock. Record the volume of oxygen gas produced in the gas syringe every 30 seconds for a total of 300 seconds.
- Repeat this method and increase temperature by 10°C each time until readings for 60°C is collected.
Equilibration of the water in waterbath is very important because the temperature is known to fluctuate. For example there may be a temperature difference within the waterbath, therefore to ensure that the temperature of the water is the same in the waterbath, the water must be left for a period of time to come into balance. Using a couple of thermometers on either side can help in determining whether temperature is the equalised and stabilised.
It is important to find out whether the hydrogen peroxide will react even when no enzyme is present and to see if any oxygen is given off - this will be the control.
Diagram
Variables
1. Independent Variable:
- Temperature of solution,
- Temperature of the water in the waterbath measured in °C using thermometers.
2. Dependant variable:
-
The volume of oxygen gas produced by the breakdown of hydrogen peroxide, which will be measured in cm3 in a gas syringe for 300 seconds.
3. Control Variables:
- These variables, which can alter the rate of reaction, need to be kept constant all
the time:
Concentration of enzyme catalase: the higher the concentration, the higher the rate of decomposition of hydrogen peroxide. With a larger number of catalase molecules, the more chance of frequent collisions between enzyme and substrate molecules. Therefore the amount of potato discs (20 discs) needs to be controlled. This also applies to the surface area. In order to keep this constant, making sure the same size of potato discs, 1mm by 7mm is essential using a ruler each time I conduct the experiment.
Concentration of the substrate hydrogen peroxide: volume of hydrogen peroxide (10cm3) using a measuring cylinder and the concentration (20cc) must be kept constant.
pH – this needs to remain constant because at a high pH, the loss of hydrogen ion (H+) causes the enzyme to denature. At a very low pH, many H+ ions attaches to the negative regions of the enzyme. This results in a change in shape and denaturisation.
Room temperature – this may be a factor that will affect our results. Therefore we will have to try our bests to insure the test is carried out under the same conditions. This includes the room temperature.
It is not necessary to take into account regard for living organisms and the environment, as the potato to be used is already available.
ANALYSIS
Calculation of the average volume: Based on the example for 10°C
Average volume (cm3) = 26 + 25 + 26
= 25.7 cm3
3
Calculation of the rate of reaction: Based on the example for 10°C
Average volume (cm3)
Rate of reaction (cm3 s-1) = x 102
Time (seconds)
25.7
= x 102 = 8.57 cm3 s-1
300
The reason why the rate of reaction should be multiplied by 102 is so that it is easier to use them in a graphical form.
According to the data, the results supported the hypothesis and so were be accurate and reliable. The rate of the decomposition of the substrate hydrogen peroxide increases until its optimum temperature, which is 40ºC, is reached. Beyond this, the rate of enzyme activity slows down. This shows that the temperature factor is a great influence on the rate of activity.
2H2O2 (aq) 2H2O (l) + O2 (g)
From studying the graph, it can be seen that the enzyme catalase’s optimum temperature is 40ºC (at the peak), it is after 50ºC that the rate of reaction drops rapidly. This is shown by a steep declination in the graph. At this point and beyond the three-dimensional shape of the active site becomes distorted and due to this the enzymes become inactive.
It was predicted that an increase in the temperature would result in an increase in kinetic energy. Since their speed increases, they have more successful collisions and therefore the rate of reaction increases. This is proved correct until the optimum temperature.
At lower temperatures (10ºC), the enzymes are not destroyed, thought their activity is slowed down. The particles of reacting substances do not have much energy. However when heated the particles take in energy.
Reactions controlled by enzymes work in the same way, but because they are proteins, the enzyme will denature if temperature exceeds 40ºC and will no longer activate.
As shown on the graph, the rate of reaction is not directly proportional to the temperature. If the temperature is doubled, it might be expected that that rate of reaction will double, but this is not true.
At 10°C, there is a great increase in the rate of reaction (8.6cm3 s-1) shown by a steep line progressing upwards. It is at this point where the substrate and enzyme molecules gain kinetic energy. Due to the fast moving molecules, many collisions occur and so the rate of reaction rapidly increases. The substrate molecules are active and rush to occupy the active sites of the enzyme. The rate of decomposition of hydrogen peroxide is quite slow; hence the production of oxygen gas in the gas syringe increases steadily over time.
At 20°C and 30°C, a same process takes place, but due to an increase in temperature, the rate of reaction is greater (at 20°C it is 11.4cm3 s-1). The active sites of the enzymes catalase are occupied more quickly here, as the molecules are provided with more kinetic energy so move at a faster rate. The decomposition of hydrogen peroxide occurs more rapidly and therefore more oxygen gas is produced.
At 40°C, the rate of enzyme activity is at its greatest (14.2cm3 s-1), this is the catalase’s optimum temperature shown by the peak on the graph. The rate of decomposition works best and the volume of oxygen gas is produced more rapidly over 300 seconds. This is 42.7cm3.
At 50°C and 60°C, there is a rapid downfall shown by the graph. The rate of reaction falls from 14.2 cm3 s-1 to 3.4 cm3 s-1. This is because when the temperature rises above the enzyme’s optimum temperature, the enzymes denatures or becomes inactive. The high temperature breaks the hydrogen bonds and other bonds holding the structure together and due to this it’s three-dimensional structure is destroyed. The active site of the enzyme can no longer hold the substrate molecule, so the enzyme-substrate complex (lock and key mechanism) cannot be formed for a reaction to occur. When the enzyme (protein) is denatured it becomes less effective as a catalyst and soon the enzyme activity gets slower and then finally it stops.
EVALUATION
Limitation – It is quite different to make sure each and every potato disc is of the same size even though a ruler is used.
Improvement - Vernier callipers, or a micrometer screw gauge can be used to measure the diameter of the potato discs- this would be more accurate than a ruler.
Limitation – there were a few anomalies (see results table on page 7 highlighted in orange). Possible reasons might have been due to human error or contamination in the substrate. It could also be the temperature of the room. The experiment carried out on different days, some times the temperature in the room varied, from 23°C to 30°C.
Improvement - Repeating the whole experiment – to eliminate anomalies and ensure the corrections of the optimum temperature.
Limitation - Even though there were only a few anomalies, there was no way of determining the pH. Knowing the pH is essential as hydrogen ions are involved.
Improvement ph meter could be used to determine the pH of the substrate hydrogen peroxide. A buffer solution could also be used,
I can improve my method to give me better results by using a wider range of concentration of hydrogen peroxide. This would then give me more results in which I can use to draw up a better conclusion
Temperature close to the optimum temperature, which is 40°C, could be investigated such as 39°C or 41°C to ensure the correct optimum temperature. A thermometer with a high resolution could be used for accuracy.
