How does the reaction between the enzyme and substrate molecule occur?
Basically the enzyme and substrate molecule are moving around in their corresponding area (wherever that might be) and when they hit each other the enzyme locks the substrate molecule in and splits it or does whatever it needs to do (remember the reactions vary). The enzyme then wanders around until it hits another substrate molecule and then splits that. This goes on and on.
The only comment I can make about this is the kinetic theory. This is when heat is applied to molecules and they move faster as a result (the heat provides energy and this energy is put into kinetic energy which make the molecules move or go faster). So the kinetic theory within this experiment is the higher the heat temperature the faster the enzymes and substrate molecules will move.
Information about the enzyme and substrate molecule that I’m going to use
Catalase is present in the peroxisomes (microbody organelles that house various oxidation reactions in which toxic peroxides are generated as side products) of nearly all aerobic cells. It serves to protect the cell from the toxic effects of hydrogen peroxide by catalysing its decomposition into molecular oxygen and water without the production of free radicals (An atom or a group of atoms with an unpaired electron. Radicals are unusually reactive and are capable of causing a wide range of biological damage).
Catalase is a very fast reacting enzyme; it is found in many living cells, it breaks down hydrogen peroxide to water and oxygen. In fact one molecule of it can deal with six million molecules of hydrogen peroxide in 1 minute. Hydrogen peroxide is toxic so needs to be changed into harmless substances so as to protect the body from it.
Catalase + Hydrogen peroxide = water + oxygen
From looking at the equation I found out that catalase reacts with hydrogen peroxide to give out water and oxygen. Oxygen bubbles produce froth on the surface of the solution. In my forthcoming experiment I will expect to see froth being produced.
The only other point to mention about this enzyme is that its optimum temperature is 40oC. This means that I will have to base my recordings around this value.
Method-
Apparatus
I have decided to use the following equipment in order to carry out my experiment, their uses will be explained later:
Water Baths
Thermometer
Test Tube
50 ml Measuring cylinder
Potato (catalase)
Hydrogen peroxide
Bung with rubber tubing coming out of it
Cylinder that measures up to 100 ml
Water
Tub to hold the water in
Borer
Knife
Ruler
Stopwatch
How’s it set up?
The first step to take will be setting up the experiment, and then I will explain how to do the experiment later. First of all I will get all the above equipment. The potato will have to be as large as possible and I will need a large amount of hydrogen peroxide. The next thing to do is to fill up the tub and baths with water (around ¾ full). The potato will be bored and cut into specific lengths of 10mm by 40mm. A test tube will then be put into the bath and a temperature will be chosen (this will depend on where I am in my experiment) which will be measured by a thermometer- the intervals and range of the temperature will also be found out in the preliminary test. On this test tube the bung with the rubber tube will be put on it. Once that is done I will fill up the 100ml cylinder with water from the tub (making sure that the water stops at a measurable point) and place it upside into the water in the tub (making sure that I don’t tip any water out while putting it upside down). Once the 100ml cylinder is upside down in the water I will record, on the meniscus, where the water stops. The rubber tubing will then be lead from the test tube to the 100 ml cylinder and up into it. The next thing that I will do is fill up the test tube with 15cm3 of hydrogen peroxide (this will be measured using the 50ml measuring cylinder) and put in the potato (catalase) with it- the amount of hydrogen peroxide will also be found out within the preliminary test. Once the enzyme and substrate is placed I will give it a specific time. At the end of this time I will stop the experiment and record where the meniscus is again on the cylinder. This time will be the last thing to find out in my preliminary test.
Diagram
How’s it done?
The diagram above should have explained my method in a much simpler form. To actually make something out of this I will take recordings on the reaction rate of the enzyme and substrate molecule. The reason I put the apparatus like this is because it manages to work upon the reaction between the hydrogen peroxide and the catalase.
When I was thinking this up I thought, how could I make an experiment from the catalase reaction? So I worked upon what happens during and after the experiment and the most prominent things were that after the reaction different products appeared- water and oxygen. I decided to base the reaction around these products. To do this I could have either set the experiment like above, except no 100ml measuring cylinder, weigh the water used before the experiment and then again after the experiment and the difference between the two recordings would be my reaction rate. Or I could have seen how much oxygen appeared during the reaction.
I chose the oxygen to measure because it is easier (there is less of it to measure than the water). Not only that but the water one would also be impractical- how could I get the water out from the test-tube so that I could measure it?
Now I will explain how my diagram works in measuring how much oxygen is released. Once the apparatus is set up like in the diagram the reaction occurs, which releases oxygen (and water). This oxygen travels through the pipe and into the 100ml cylinder. As the oxygen goes into the cylinder water is pushed out. The difference between the measurement of the water in the cylinder at the beginning of the test and the end of the test is the measurement of the reaction rate at whatever temperature the water bath was between catalase and hydrogen peroxide.
The factor of this experiment that I have chosen to vary is the temperature. This is why I have a chosen to include the apparatus, the water bath. This bath has a heating mechanism that varies the temperature of the water in it, which is done by turning a knob on the side. So to get the different results I will start at my lowest temperature and do this test. Then I will increase the temperature by my chosen interval and do the test again. I will repeat this several times and get the temperature up to my maximum value. All these temperature values will be found out within my preliminary experiment. I will repeat this entire experiment 2 more times getting 2 more tables of results. I will average the corresponding results to make one average table. This will be the table that I use to create my further graphs.
Once I have my results of the reaction rates at different temperatures I will compare them and plot a graph, which has the rate of reaction against the temperature. Using this graph I will analyse my results and find out any patterns. Afterwards I will prove any predictions that I have made with my results and then I will consider how to improve my experiment.
There are a couple of things that I must note when I do my experiment. I must make sure that the last thing I do is put in the potato, because if the reaction happens before I start recording I may lose some of the oxygen that is given of and so my recordings will be inaccurate. The second thing to note is that the bath has a heating mechanism, not a cooling mechanism, so the lowest it can go is room temperature. To get it below room temperature I will use ice to cool the water. These tests will have to be done quickly so that the ice doesn’t melt and the temperature change before the test is over. The last thing to note is that when I place the test-tube in the water the water line will have to be over, at a minimum, the liquid line for the hydrogen peroxide inside the test-tube. This is so that it will then give the temperature to the all the liquid not just a part of it, which would distort the results.
Fair test
A fair test is when all the results from varying tests can be compared as equals or fairly. If the results have been changed or distorted from test to test in any way then they cannot be fairly compared. The points of a fair test are used to prevent this.
In order to keep my method fair I will have to keep some things in order. The main things are the variable. Since all the variables can change my results I will have to keep them constant so that the tests stay the same and can be fairly compared.
- Heat- because of the energy it gives and so speeds up the reactions. This will affect my results. pH- because enzymes work best at optimum temperatures. By changing the pH I will also change my results.
- Time given for the enzyme to react with the substrate molecule- the longer I give the enzyme the more reactions that occur. So time will also affect my results.
- Concentration of enzyme- the higher the concentration of the enzyme (basically the more enzymes there are) the quicker the substrate molecules will be reacted with (because there’s more of them there). So the concentration will also affect my results. The measurements of the hydrogen peroxide and the amount of potato fall under this category.
- Potato- different types of potatoes have different amounts of hydrogen peroxide. The more hydrogen peroxide there is the longer the experiment will take and so the potato can also affect my results (this is why I want a large potato so that the potatoes properties don’t change from test to test).
- The surface area of the potato- the larger the surface area the more hydrogen peroxide there is. This means that the test will take longer to finish because more reactions will occur. So the surface area can also distort my results.
Since my variable is temperature this is the only factor of my experiment that can change. This is not to say though that the temperature can vary any way it wants, it still needs to be controlled and within a test it will also have to be constant. All the other factors, which are named above, will stay constant throughout the experiment. The less they change the better and more reliable my results will be.
As a result of the potato going mouldy and changing over time (which varies the concentration of the hydrogen peroxide) the experiment will have to be done as quickly as possible so that the potato remains as constant as possible. With this in mind I will have to get a very large potato so that every bit of potato put into the test tube is from the same source, this will keep the concentration of hydrogen peroxide as constant as possible (as it might vary if I change the potato for every test, distorting the results). If I get a small potato then there will not be enough of it to cover my whole experiment and so it will have to be changed, along with its concentration of hydrogen peroxide.
I will keep my variables the same by making sure that I have implied the same strategy for each test taken and that I use equipment that’s as accurate as possible and also use the equipment as accurately as possible. So this means constant and careful measurements and clean equipment.
Safety-
To keep this a safe test a will have gloves when handling the chemicals and be careful not spill any onto myself. The knife will always be cut away from me and no one else and when I am testing on a high temperature I will not touch the water to prevent me from burning myself.
Prediction:
Now that I have made a method that’s reliable because it’s based on proven scientific knowledge I can guess what is going to happen. From the above scientific knowledge I can now make a fair and reliable prediction.
I predict that the catalase will work best at around 40oC. This is because this temperature is the enzyme’s optimum temperature. If the temperature goes above 40oC then the efficiency of the enzyme will drop dramatically because it denatures.
I will also predict that as the temperature goes from 0oC to 40oC there will be a constant rise in the reaction rate. This is because as the temperature rises the particles vibrate more from the heat energy being transformed into kinetic energy. This means that the hydrogen peroxide molecules move faster as the temperature increases, which means that more collisions will occur between the hydrogen peroxide molecules and the catalase. This means that the reaction rate will also increase. I see no reason why there shouldn’t be a constant rise in the reaction rate up to 40oC so therefore my predictions stays. After 40oC the reaction rate will go steadily down as the enzymes gradually denature one after the other:
Predicted graph:
40oC
Temperature
Reaction rate
Preliminary Experiment:
Within this experiment I will find out what range of temperatures I will use on my actual experiment and how long I will leave the experiment before I take down the second result from the measuring tube.
I need a rough estimate of my temperature range and time so I will just guess. The temperature will have to be based around 40oC because the enzyme denatures at that temperature. So I think that I will do a preliminary experiment using my method as above that includes the temperatures 20oC, 40oC and 60oC. With these results I can decide what my actual temperature range will be. When doing this test I also need a time. This measurement can’t be based on anything and so I will merely guess. I will repeat the tests that include the temperatures 20oC, 40oC and 60oC up to three times at the times of 2 minutes, 4 minutes and 6 minutes.
Preliminary Results-
This preliminary experiment has helped me a lot. I have decided that my time will be 5 minutes (when deciding this I took into consideration the amount of time I have and what time measurement showed the most varying results). I decided my temperature range to be 0oC – 60oC. This is because I knew that if I went up above 60oC the denaturing process would just stop any reaction happening at all. Still, the results weren’t varied enough so I decided to extend the other end of the temperature scale, extending 20oC to 0oC. This will give me enough results to make a reliable graph on.
Experiment:
While I am actually doing the experiment I will apply all that I’ve said in my method and all that I found out in my preliminary experiment and so will hopefully come up with some very accurate results (please note that the averages in the average table of results will be rounded to 2 decimal places).
Results: tables-
First set of results
Second set of results
Third set of results
Average set of results
Results: Graphs-
Graphs Analysis-
The line of best fit here shows me that there is a slight curve in line going up to 40oC. This represents acceleration in the reaction rate up to 40oC. Then the line curves down and then rapidly decelerates to 0oC. Within this graph are no straight lines. This shows that there are no constant speeds as the reaction rate always either speeds up or slows down if it’s going to change speed. You can see that 40oC is the point where the reaction rate stops increasing and suddenly starts to decrease.
Conclusion:
First of all let’s remind ourselves of my aim; ‘to find out how temperature affects the breakdown of hydrogen peroxide by the enzyme catalyse.’
As an answer to that aim I will say that temperature affects the breakdown of hydrogen peroxide by catalase by first increasing the breakdown rate and then rapidly decreasing the breakdown rate after 40oC. As the temperature rises from 0oC to 40oC then breakdown rate also rises. This rate constantly accelerates very slightly until around 33oC and then the rate slows down until 40oC, where it stops increasing at all. From 40oC onwards the enzymes decrease in breaking down the hydrogen peroxide. It starts slowly at first, but then accelerates faster and faster until it reaches 64oC where the breakdown rate stops all together. This is how temperature affects the breakdown of this certain combination of enzymes and substrate molecule.
The reason for this is because as the temperature raises the kinetic energy in all the particles increase. This means that more collisions will occur between the two types of particles, which means that more breakdowns will occur. This explains the accelerating increase in the line of best fit before 40oC. However at temperatures after 40oC the enzymes start to denature and lose their shape. This denaturing process increases as more enzymes lose their shape and ability to fit their substrate molecule with the increasing temperature. This explains the line that decreases rapidly after 40oC.
My predictions were that the enzyme would reach its highest breakdown rate at 40oC and that there would be a constant rise in the breakdown rate until 40oC. One of these predictions was right while the other was wrong. The correct prediction was that the highest breakdown temperature was 40oC. This is proven by the graph showing that the highest breakdown point in my experiment is 40oC. The reason for this is because this temperature the enzyme’s, catalase, optimum temperature. It is at this temperature that catalase works best and can break down as much hydrogen peroxide molecules as possible.
My second prediction was that there would be a constant rise in the breakdown rate until 40oC. This prediction is proved wrong in my graph because the line leading up to 40oC is curved slightly and then curves of when it approaches 40oC. This shows acceleration and deceleration in the breakdown rate leading up to 40oC. These factors mean that the rise isn’t constant. The reason why this prediction is wrong is because when the temperature rises I thought that each molecule would have had the same amount of kinetic energy each. If this were the case then there would have been a constant rise. Upon consideration though I realise that no test could be this accurate and that the molecules further inside the test-tube would have different kinetic energy compared to the molecules on the outside. This variance gives me the acceleration and the deceleration in the rise.
Evaluation:
Analysis of method and results-
I am pleased with my method because it turned out accurate reliable results that answered my aim.
Observations-
The main thing that I noticed was that the water didn’t heat up or cool down straight away. To compensate for this I had to wait for a couple of minutes for the water to reach a constant temperature. However when I waited on the water baths that required ice I noticed that the ice started to melt. This meant I had to use the time I had, when the water that needed ice was on the constant temperature, as efficiently as possible so that the temperature wouldn’t vary to much, which would have rendered the test unfair.
Changes that would be made if I were to repeat the experiment-
If I were to do the experiment again I would change the apparatus slightly so that I had a smaller water bath. This would mean that there less water could be held and yet the test tube could still fit. The less water though would mean that the temperature would be gained faster and could be controlled so that it would vary less. My second change would be a stand that would hold up the measuring cylinder and yet could fit a pipe through. This would mean that I wouldn’t have to hold the measuring cylinder for the entire test.
The reliability of my results-
I think that my results were very reliable. This is especially evident in my graphs where the results fit very well with the line of best fit. The fact that everything was based on proven science shows how accurate and reliable my results must be.
Do my results support a firm conclusion-
I think that I had enough results to make a firm enough conclusion on this experiments aim. This is shown by the way that my aim was answered very specifically. However one of my predictions was one wrong not by poor results but by poor knowledge on my part.
Further work to prove my conclusion-
For further work I could do different enzymes or substrate molecules using the same method. I could then compare the results to my catalase and hydrogen peroxide experiment and my extra scientific knowledge and see what makes the most efficient enzyme. I could also vary the time length and see how that affects the breakdown rate. This further work would give me a broader view of enzymes.
Overall though I think that this was a very successful experiment and that I have managed to gain and analyse some useful results that told, explained and answered me a lot. I have also set myself some more questions for me to answer in any other extra experiments that I may do in the future.