As a result this will be a variable that I will keep constant in order to make it as much of a fair test as I can.
- Concentration of the substrate:
The concentration of the substrate that is used in the reaction will affect the rate at
which the two reactants react.
This is because the more concentrated the substrate is the more particles there are, this increases the frequency at which they collide the opposite is also true. If there less substrate particles they will not collide with the enzyme particles as often, this will increase the time it takes to react and you will get less product at the end of the experiment.
This is the variable that I have decided to use as the one I change. This is because it is the easiest one that I can control reliably, and be very precise as to the percentage of substrate in the solution into which the enzyme is put.
Using this variable I believe that I will be able to come a firm and reliable conclusion as to what affects the rate of reaction.
When an enzyme reacts it is believed to use the lock and key hypothesis. This is when the substrate particle fits into the active site of the enzyme, the substrate is held there until the reaction is completed, it is then released. When it is released the bits released from the active site are the products of the reaction. This works because each enzyme has a specific shape, very similar to how a lock has a special shape that only the right key can fit and open . Each enzyme has an active site; this is where the ‘key’ (substrate particle) goes in and reacts with the enzyme, the enzyme separates the substrate particle into the products. In the case of catalase and hydrogen peroxide the enzyme splits the hydrogen peroxide particles into water and oxygen.
To get reliable evidence to justify a valid conclusion at the end I need to carry out this experiment as precisely as I can. To do this I will be using 5 different concentrations of the substrate: 20%, 40%, 60%, 80% and 100%. I will use each concentration three times and average out the results in order to come up with as reliable a result as I can in the time allowed.
To begin with I am going to carry out a preliminary experiment, this I hope will help me to work out the best way to get results, and the best way to record them.
Preliminary experiment:
In this experiment I am going to measure the rate of the reaction by the amount of foam that is produced during the reaction. I will record this by measuring the height in centimetres.
For the first experiment I will use a concentration of 100%, this I hope will give me some idea of the speed at which the reaction will take place.
First of all I put 10cm³ of 100% hydrogen peroxide into a test tube; I then clamped this onto a retort stand. I cut up 8 pieces of potato each measuring 5 millimetres in width and 1 cm in diameter. I dropped these into the substrate and put a bung on the top of the test tube. After 10 minutes I measured to see how far the foam had gone up the test tube, it appeared that it hadn’t reacted very much considering that the substrate was at 100%, I took it as an anomalous result so I repeated the experiment.
The second time round the same thing happened, not much foam was produced. I decided to measure the temperature of the substrate to see if the problem was the temperature wasn’t at the optimum temperature to get the enzyme to work well. It turned out that the substrate was at 22ºC, I decided that a better temperature would be 30ºC because this is not quite at the point of denaturation but is hot enough to get the enzyme particles to move faster and react quicker.
To get the substrate to 30º C I put a beaker (water bath) filled with water at 30ºC below the test tube and dipped the test tube in it. I waited 2 minutes to allow the substrate to be heated up and put the potato chips in it, this time the reaction was much faster and more foam was produced.
After I did this once more I saw that the results I was getting were inconsistent, this was because the foam was always higher on one side than it was on the other. I decided to find another way to measure the rate of reaction, I looked in a science book and saw that the experiment could be carried out using the oxygen given off to measure the rate of reaction. I decided that this would be more reliable and so I set up some new apparatus. I used the same water bath and test tube system but this time I used a delivery tube and collecting tube to collect the oxygen that was being given off. I also decided that the chips of potato were too big and didn’t have a large enough surface area to allow the hydrogen peroxide particles to react with, so I cut them into quarters so I ended up with 32 potato chips. This allowed the substrate to react with more enzyme particles and cause a faster reaction.
The oxygen collecting method worked much better than the measuring foam method, I did it twice, one with 100% concentration and the other with 60% concentration to make sure it worked I got consistent results to what I expected to happen, this helped
me to make my prediction of what I believe is going to happen.
Preliminary results
As a result I decided to use this method in my real experiment.
Below is a diagram of the oxygen collecting apparatus that I will use in my actual experiment:
From doing the preliminary work I believe that I can make a realistic prediction as to what will happen in the real experiment.
I think that as the concentration of the substrate is increased the more oxygen will be produced in the time.
I believe that the amount of oxygen produced for each concentration will increase proportionally until the concentration is at 100%, at this point the reaction rate will not be able to get any faster, this is because at this point all the substrate and enzyme particles are working at their fastest possible rate, known as the Vmax.
I predict that the graph at the end of the experiment will look like the one below. It shows that as one side increases the other increases proportionally, until such time as all the particles are working as efficiently as they can, at this point line would, if I carried on the experiment with 100% concentration, straighten out. This is because it cannot go up any further, this point is called the Vmax.
The hypothesis that I am now investigating is:
The rate of reaction will be faster with a higher concentration than with a low concentration of the substrate hydrogen peroxide.
To get the results that I want I will be doing 15 experiments 3 for each concentration. Each experiment will last 10 minutes at the end of this time I will measure how much oxygen has been collected.
In order that I carry out the experiment safely I am going to have to take some precautions. It states on the information sheet for hydrogen peroxide that it is corrosive and an irritant, in order to stop it harming if an accident occurs I am going to wear safety goggles and gloves when handling the substance.
To make my results are as reliable as they can be I have to make sure that no water escapes when I am putting the collecting tube into the water tub, I will do this by having a thumbs over the end and not letting go until it is under the water. I have to make sure that the end of the delivery tube is securely in the bottom of the collecting tube so it won’t fall out and lose of oxygen, if this happened it could create an anomalous result and cause me to write a false conclusion.
To get the correct concentrations of substrate I am going to measure it out in a measuring cylinder. When I want the 40% concentration I will measure out 60 cm³ of water and mix in 40 cm³ of hydrogen peroxide, this will give me an accurate concentration and help to get the reliable results that I need to come to a valid conclusion.
Below are the results for the experiment, I carried out the experiment exactly as I did in the preliminary one, I kept the water bath at 30ºC by adding hot water when it was needed and I kept the potato chips the same size.
Below are the results for the experiment:
This graph shows the amount of oxygen that was collected for each concentration in each experiment. There are three coloured dots because I carried the experiment out three times for each concentration. You can see that the results are quite reliable because all the points are very close to each other, and because we did it three times I don’t believe it could have been a fluke. The graph shows the line of best fit, it shows that there is a fairly strong positive correlation between the concentration of the substrate and the rate of reaction i.e. as one goes up the other goes up as well.
These results are more or less exactly what I expected to get. They show that as the concentration of the substrate is increased the amount of oxygen being produced also increases, therefore the higher the concentration is the faster the reaction is. This proves my prediction to be correct, in order to prove it further I am going to average the results and put the results into a graph, this should also prove my theory for the graph as well.
I have averaged out the results so that they are as accurate as they can be.
These results that I have produced support the original prediction I made was correct.
The graphs show that as the concentration of the substrate increases the rate of reaction increases proportionally. The explanation that I give for this happening is that because the higher concentration had more particles for the enzyme to react with and produce more of the products, water and oxygen. This certainly seems to be the case as lower concentrations have fewer particles for the enzyme to react with, and the rate of reaction was slower, producing less oxygen.
In conclusion to the experiment I believe that the hypothesis that I made is correct. This can be seen in the graphs and in the tables. The reason that I believe all the evidence is correct is because it fits in with the idea that when there are more substrate particles for the enzyme to react with the reaction is faster, therefore the lock and key theory.
The evidence that I produced in the experiment, I think, is very reliable. This is because I did everything to make sure that we didn’t get any anomalous results such as, starting the clock the second the enzyme went into the substrate, I kept the temperature of the substrate at a constant 30ºC.
The only anomalous result that we got was on the 100% concentration, we got a reading of 19 when the other two experiments came up with 17 and 17.5, a larger difference than any of the other concentrations. I can only say that this was probably caused by not all the enzyme being wiped off the surface I used to put them into the test tube or some oxygen being left in the delivery tube from the prior experiment. It didn’t, however, seem to affect the overall results as the graphs turned out exactly how I had expected them to.
I think that the results that I got from the experiment are enough to justify a valid conclusion, this is because they reflect the lock and key theory, and they also reflect the research I did into the experiment from an AS level biology book.
I think I could have done some things to make the evidence I got a bit more reliable. I could have extended the enquiry to get more results from the concentration experiment, this would have given me more reliable results and helped me come up with a more accurate conclusion. I could also have tried a different experiment such as the size of the enzyme and how it affects the rate of reaction. Although I said that this would have been a difficult variable to control I feel that if I had been careful to get the right sizes of enzyme each time it would have given me results that I could use to come a more extensive conclusion about what affects the rate of reaction.
In this experiment I could have made the results more reliable by getting the enzyme into the substrate by using a syringe to inject it into the test tube. This would have deleted the problem of losing oxygen whilst I put the bung onto the test tube. This would have meant I would have to cut the potato to a very small size and would have made controlling that variable quite difficult because I couldn’t be sure to cut it to the same size each time.
Overall I think that this experiment went very well, I got the results I expected and the experiment went without major incident other than the one small anomalous result, this was quite good although it would have been better if I’d got none. The anomalous result didn’t affect the outcome very much and so it didn’t seem too significant. The evidence that I got allowed me to come to a good conclusion about how the concentration of a substrate affects an enzyme.
