Research on the Theory
At low temperatures in enzyme/substrate solutions, the molecules have little energy due to the low temperatures. This results in few collisions between the enzyme and substrate and thus few reactions. Therefore at low temperatures, the enzyme is still active, it just is not colliding enough to cause lots of reactions.
As temperature increases the number of collision increases due to the energy from heat. More collisions result in more reactions. Initially, as you increase temperature for every 10 degrees Celsius increases the reaction rate doubles.
Above the optimum temperature the collisions become increasingly violent- this results in damage to the 3D shape of the enzyme due to the collision and temperature breaking the bonds. The enzymes are being denatured and therefore there is a decrease in the number of reactions.
Finally at high temperatures the enzyme will be completely denatured due to a complete collapse in its 3D structure.
Method- measuring the volume of oxygen
1. I will set up the apparatus shown above.
2. Using the electronic weighing scale, I shall weigh 0.5g of liver.
3. Using the tile and knife, I will place the liver on the tile,
4. I will measure the hydrogen peroxide using a measuring cylinder.
5. I will place 0.5g of liver into the conical flask.
6. I will place hydrogen peroxide and place it inside the tube with a beaker full of ice and with a thermometer.
7. As concurrently as possible, I shall add the liver to the hydrogen peroxide, place the bung in the neck of the conical flask.
8. The selected temperature that I am going to study is 5ºC, 10ºC 15ºC, 25ºC and
45ºC. I decided my temperature by:
5ºC- iced water
10ºC-thermometre reading
15ºC-water bath required
25ºC-water bath required
45ºC-beaker of hot water
9. Start the stopwatch and record volume of gas collected every 30-60 seconds.
10. When the volume of evolved gas stops and no longer moves, I shall stop the whole experiment.
11. I shall repeat the above 7 steps three times to produce an average, if there is enough time.
Apparatus:
Retort stand, Boss, Clamp, conical flask, small 100cm3 beaker, 50ml measuring cylinders, stopwatch, delivery tube with bung, electronic weighing scales, 10 vol. hydrogen peroxide, liver [contains enzyme catalase].
Fair test
In this investigation I will keep constant the following
· The mass of the liver will be measured and weighed accurately. Reading the scale number would do weighing accurately. I will add more liver if it weighs less than 0.5 and add less liver if it is higher than 0.5.
· 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 liver 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 liver 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
· Wear work shirt
· Handle the hydrogen peroxide with care as it is corrosive and an irritant
I shall take care when handling the hydrogen peroxide, as the substance is poisonous. In order not to confuse beakers of water and hydrogen peroxide, I shall clearly label each of them.
I shall follow general lab procedures, i.e. no running; bags under worktops etc.
Predictions and Reasons
The chosen temperatures that I am going to study are 5ºC, 10ºC, 15ºC, 25ºC, and 45ºC. I predict that the enzyme catalase will denature at 45ºC.
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. 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.
Average Table
This s table show the average measurement that we recorded for each temperature.
Analysing results and Conclusion
From my results it appears that catalase works best at 15ºC, and it is virtually denatured at 45ºC.
Looking at the first part of the reaction it is clear that the gradient at the beginning gets steeper when looking at the temperatures between 15ºC-25ºC. At each temperature the line levels off towards the end of 330 seconds. 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 liver 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,
This stops the substrate from ‘fitting´ and so no product is formed.
My results do not totally support or to weaken my original prediction. The reason being that on line of best-fit graph, my results suit my prediction. It shows that the temperature, 15ºC was the fastest and 45 was the slowest as well it is when the enzyme denatures. My results undermine my original prediction as at 45ºC the reaction still takes place where as in my prediction I stated that enzymes would denature at 45ºC approximately, I didn’t expect this to happen.
Evaluation
The problem that I observed is 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 strange results. Ideally I should have brought the temperature of the hydrogen peroxide up to the needed temperature before adding to the liver.
When I did the experiment-using beaker, ice and measuring syringe, I tried to keep the temperature steady, for example when I was experimenting 25ºC, the temperature either goes lower or higher during the experiment, this makes the test unfair, therefore I ended up getting strange results, however I did the experiment all over again.
Looking back at my results I found some strange 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 15ºC this is as we expected. But at 45ºC 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 25ºC-45ºC. 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.
