To Investigate the Effect of Temperature On the Enzyme Catalase.
Aim: To investigate the effect of temperature on the enzyme catalase.
Prediction: I think that at 40ºC, the reaction of hydrogen peroxide to oxygen and water will be the fastest. And I think that at 20ºC and at 60ºC, the reaction will be the slowest!
Diagram:
Hypothesis: Enzymes are "biological catalysts". They speed up chemical reactions in all living things, and allow them to occur more easily. They occur in plant cells and animal cells. Without them we would not be alive. Although they work powerfully, enzymes are just chemical molecules, made up of proteins. They are too small to be seen either when they are inside cells or after they have been released from them, for example in the digestive system.
Each particular enzyme has a unique, 3-dimensional shape shared by all its molecules. Within this shape there is an area called the "active site" where the chemical reactions occur.
There are many different types of enzymes in the body and each are suited to their surroundings and to the job they need to perform. You see, enzymes can
a.) Break down large molecules into smaller ones which are much easily absorbed by the body and these enzymes are usually called "breaker" enzymes.
b.) Join small molecules together to form large molecules that are used inside the body's cells. These enzymes are called "builder" enzymes.
Although enzymes can do fantastic thins they are sensitive and work best under specific conditions. Each type of enzyme has its own specific optimum conditions under which it works best.
Enzymes work best when they have a high enough substrate concentration for the reaction they catalyse. Of too little substrate is available the rate of the reaction is slowed and cannot increase any further. Sometimes, it too much product accumulates, the reaction can also be slowed down. So it is important that the product is removed.
The pH must be correct for each enzyme. If the conditions are too alkaline or to acidic then the activity of the enzyme is affected. This happened because the enzyme's shape, especially the active site, is changed. It is "denatured", and cannot hold the substrate molecule.
Temperature is a key factor, too. If it is too cold the enzymes will move around too slowly to meet the substrate molecules, so the reaction rate is slowed. They need a lot of kinetic energy, which is supplied by the heat, to get them to move around and perform their tasks at the optimum rate. Likewise, if it is too warm they do not work properly either. This is because extra heat energy shakes them around so much (kinetic energy) that the bonds holding the molecular structure of the enzyme together will break and the active site loses its shape and will no longer work.
All reactions that take place in the body proceed because the products have less energy than the substrates. However, most substrates require an input of energy to get the reaction going, the reaction is not spontaneous. The energy required to start the reaction is called the "activation energy". When the substrate(s) react, they need to form a complex called the transition state before the reaction actually occurs. This transition state has a higher energy level than either the substrate or the products (it's the peak of the curve on the graph!). Outside the body, high temperatures often supply ...
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All reactions that take place in the body proceed because the products have less energy than the substrates. However, most substrates require an input of energy to get the reaction going, the reaction is not spontaneous. The energy required to start the reaction is called the "activation energy". When the substrate(s) react, they need to form a complex called the transition state before the reaction actually occurs. This transition state has a higher energy level than either the substrate or the products (it's the peak of the curve on the graph!). Outside the body, high temperatures often supply the energy required for a reaction. This clearly would be dangerous inside the body, though! Fortunately we have enzymes that provide an alternative way with a different transition state and lower activation energy. The rate of the reaction without any external means of providing the activation energy continues at a much faster rate with an appropriate enzyme than without it. The maximum rate that any reaction can proceed at will depend, among other things, upon the number of enzyme molecules and therefore the number of active sites available.
Enzymes control all kinds of reactions in all cells. For example, they help control respiration, photosynthesis, and our digestion, amongst many others. We also make use of enzymes elsewhere. Protease and lipase enzymes are used in biological washing powders to remove those stubborn stains. Enzymes are also used in making foods and drinks. The enzyme pectinase helps to break down the cells in fruit to release more of their juice. But probably the fastest enzyme known is called CATALASE.
The catalase enzyme breaks the chemical hydrogen peroxide down to water and oxygen. Hydrogen peroxide is a very toxic chemical, which is a by-product of respiration in your cells. If the enzyme catalase did not exist, these toxins would poison the cells in your body and eventually you would die. The catalase enzyme is found in all cells and protects the cells by speeding up the chemical reaction of turning the toxic chemical from hydrogen peroxide to oxygen and water. This process occurs very naturally!
Hydrogen Peroxide Water + Oxygen
The substrate (hydrogen peroxide) and the catalase molecules are continuously on the move. Every so often they will bump into each other so that the substrate molecule firs into the enzymes' active site (the lock and key mechanism). Then the substrate is broken down into the two products, oxygen and water. However, the enzyme is not used up in the reaction. It is ready to work again! No matter what job an enzyme does, it will work in a similar way!
I know that temperature affects the rate of reaction with any enzyme because of a previous experiment where I tested to see if a change in temperature would affect the rate of reaction concerning a starch enzyme (amylase). The results that I had recorded were pretty substantial and from these I could tell that 40ºC was the optimum temperature as it is really close to the body's temperature (37ºC) and the reaction went here the quickest. At 25ºC (room temperature) and at 80ºC the reactions took a very long time and in the end I realised that at 80ºC, the enzyme had denatured! From these, I am using the same outlook to come to my prediction in this experiment.
Apparatus for making Liver Juice:
- 200ml-distilled water
- Pinch of sand
- Liquidised liver
- Pestle and Mortas
- Big conical flask
Method for making Liver Juice:
- First you pour the liquidised liver into the mortas with the sand and grind it all together.
- Gradually you add the water into the mortas, stirring it with the liver until the juice you end up with is smooth and runny.
- You pour the water that you have left over (the water that would've spilt on the table if you had poured it into the mortas.) into a big conical flask.
- Next you pour the diluted liver juice into the big conical flask and pop a sturdy bun into the opening.
- And there you have your liver juice.
Apparatus for experiment:
- One Test Tube
- Delivery Tube
- Measuring Cylinder
- Bath filled with water
- Three Water Baths heated at different temperatures
- Stopwatch
Before the Experiment:
- Fill three test tubes with 10cm³ of Liver juice and place into first heated water bath.
- Fill next three test tubes with 2cm³ of the Hydrogen Peroxide and also place into first heated water bath.
- You wait until the liver juice and the hydrogen peroxide in each test tube has reached the same temperature as the heated water bath, ie. If the water bath is at 40ºC, the test tubes should also all be at 40ºC before the experiment takes place.
- You prepare each experiment at different temperatures exactly like above.
Now you are ready to start the experiment. Set up your apparatus in the order as shown in the diagram on page 1.
Method:
- Take one test tube filled with the liver juice and one test tube filled with the hydrogen peroxide.
- Make sure someone is ready with the stopwatch and someone is ready to whack the delivery tube bum onto the test tub.
- Then, tip the hydrogen peroxide into the test tube of liver juice very quickly.
- The person with the delivery tube bum must press it into the test tube and the person with the stopwatch has to time how long it takes for the bubbles in the measuring cylinder to stop.
- This HAS to be done very carefully and very quickly!!!!
- After the bubbles have stopped, the person with the stopwatch stops timing and everyone records the time in a results table.
- You do this with every temperature and be extremely careful!
Fair Test:
To make this experiment fair and also to make it a success, you must do the following:
- First of all, make plenty of liver juice. There should be enough to carry out 16 experiments in total, and there should be enough in case you make a mess or spill anything. It's also that all your catalase is at the same concentration.
- Make sure you know what you are testing for. In this experiment, you are timing how long it takes for the oxygen bubbles to settle/stop. The reactions can be very fast (so pay a lot of attention to it) or very slowly (so be patient and stick to your experiment).
- For each temperature you must carry out three experiments. This is so you can see if you've gone wrong somewhere by identifying anomalous results.
- Make sure you have put the correct amount of liver juice and hydrogen peroxide into the test tubes.
- Check to see that all the test tubes are at the right temperature and also that the heated water baths are at the temperatures listed above.
- You must have the same pH level for the enzymes
- You must have the same concentration of enzyme and substrate throughout the experiment.
- Try and have at least three to four people in your group. It makes the whole experiment a lot easier and clearer because it speeds up the process of whacking bums on test tubes and timing. It also leaves less space for mistakes to occur because one person doesn't have to do everything.
- With that last one, make sure everyone in your group is ready to execute the experiment.
Results Table:
Temperature
Exp. 1
(in seconds)
Exp. 2
(in seconds)
Exp. 3
(in seconds)
Average Time
25°C
1.75
9.77
1.94
1.15
40°C
5.62
3.84
6.07
5.18
60°C
4.00
6.60
6.69
5.76
70°C
3.75
4.00
2.00
3.25
Conclusion: As you can see from the results table, the results that we recorded were pretty accurate. At 25°C the average time for the hydrogen peroxide to react with the catalase enzyme was the slowest and that at 70°C the reaction was the fastest. At 40°C, I actually expected the reaction to be the fastest because that temperature is closer to the actual body temperature, but from the results recorded, it seems like the reaction was slower by 1.93 seconds. I think my prediction was right, but my results are wrong. I clearly know that at 70°C, the reaction should be one of the slowest as the enzymes are starting to denature at this point. But I was right about the reaction being slow at 25°C because the average time is much longer than the rest of the averages. It also means that at 25°C enzymes are still moving around too slow to meet the substrate molecules.
In this experiment we investigated the effect of temperature on the enzyme catalase. Previously we knew that any reaction involving enzymes depend on ideal conditions in their environment to work. If these are not to the enzymes satisfaction, they do not work properly. For this we increased the temperature and timed how long it takes for a reaction to be complete. We did this by timing how long it takes for the oxygen bubbles to stop. Previously we knew that enzymes work really slowly when the surrounding temperature is below the body's temperature (37°C) because there is not enough kinetic energy for the enzymes to move and meet the substrate molecules. At 37°C this is ideal temperature and enzymes work most efficiently here. The reaction is the fastest. Above 45°C, the reactions start to slow down because the enzymes can not perform their jobs anymore. They are slowly deteriorating, ie. Denaturing, and this means that the substrate molecule can no longer fit into the enzymes active site as it has been deformed. It becomes deformed when there is too much kinetic energy (from the heat) supplied and the enzymes are shaken and rattled around which breaks the molecular bond structure of the enzyme and so they loose the shape of their active site.
Evaluation: Our results were pretty accurate except for the last temperature. I think that the average time for the experiments at 70°C is far too low because at this stage the enzymes should start to be denaturing, so I think this is the anomalous result. I think this also occurred because we had performed the experiments over a period of four lessons, always using the same concentration of liver juice, but because we stored it in the fridge the cold might have affected the catalase enzymes in the liver juice. At first, we were very clumsy with our experiments. It was really hard, as we had never performed an experiment like this. At first we had trouble putting the bun on, timing accurately or missing oxygen bubbles. We re-tested one experiment for the 70°C temperature because we had missed the oxygen bubbles and so had forgotten to time.
If I were to redo this experiment, I would:
- Practise putting the bun onto the test tube,
- Pay careful attention to the experiment,
- Check temperatures accurately,
- And carry out some re-tests to check what went wrong
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Stefanie Frager
Biology Coursework
