In many reactions in our bodies a chemical called hydrogen peroxide is produced as a bi-product. It is a metabolic poison and if not broken down results in cells death.
H20 2 ⇔ H2O + O2
Substrate + Catalase (Enzyme) ⇔ H2O + O2
The enzyme catalase can bind with hydrogen peroxide molecules, split them into water and oxygen and release these products at a rate of 107 molecules per second.
Does temperature affect the rate of reaction when hydrogen peroxide is added to liver chunks? I am going to investigate this in an experiment using 15cm3 H202 (10vol) and 1.5 grams of liver chunks.
I believe that the enzyme will work best at the temperature 37°C because this is optimum human body temperature. I think the reaction will be slower before 37°C and also after it as it should start to denature from this point onwards.
H202 (10vol)
1.5 grams of liver chunks
Water Bath
Basin
Conical Flask
Rubber Bung
Delivery Tube
Measuring Cylinder (x2)
Test Tube (x8)
Burette
Stand
Mercury Thermometer (x2)
Stopwatch
Electric Scales
Before starting the main experiment I will carry out two preliminary experiments, one to check that I am using adequate amounts of liver and that the vol is sufficient for high-quality results. The second preliminary experiment will be to see how much the heated H202 breaks down by itself so we can subtract any necessary seconds off the times on the main experiment. In the preliminary tests I will be using 15cm3 H202 (10vol) and 1.5 grams of liver chunks preheated to 37°C. I am using liver chunks rather than a solution because I would need to dilute the solution and I feel that I will get better results using a pure form of the substrate, also the solution would have a larger surface area and I would not want the reaction to go too fast.
The temperatures I will use are 22°C, 27°C, 32°C, 37°C, 42°C, 47°C and 52°C, three temperatures on either side of the optimum. To give myself a wide range of results in order to prove my prediction.
The results of the preliminary tests showed that the vol used was inadequate and caused the liver chunks to be broken down too fast. There was a larger amount of substrate than enzyme. As I believed that the amount of liver was sufficient for the experiment I decided to change the 10vol H202 to only 5vol H202.
In the second preliminary experiment to test how much the heated H202 breaks down by itself I followed the same process as the first preliminary experiment but with out adding any liver. The results showed that at the predicted optimum temperature the 15cm3 H202 (5vol) did not beak down at all.
Before starting the main experiment you should make sure you are wearing goggles and that long hair is tied back, you should also make sure that any bags are out of the way and all stools are underneath tables to ensure you have a safe working environment. As the hydrogen peroxide is an irritant, then wearing gloves would be an option and the goggles will protect your eyes. You must wash your hands before and after you come into contact with the liver as not to contaminate any of the chemicals used or the liver itself. You must be careful that your hands do not have any contact with your mouth or eyes before washing in case of any irritation or disease from the raw liver.
The first thing you need to do is to collect all of the equipment and set it up. It will be easier to start by going from the lowest temperature to the highest, as it is easier to heat up the water bath than to cool it down. The reason you should use a water bath and not just a Bunsen burner is because with a water bath you can have the water at a set temperature where as using a Bunsen burner the temperature can fluctuate. The first temperature needed is 22°C so you can preset the water bath and leave it to heat up whilst you set up the other equipment. After measuring out 15cm3 H202 (5vol) and placing in a test tube you then need to cut up to four or five chunks of liver with a total weight of 1.5 grams, each piece of liver needs to be of approximately the same size to make each test fair. Place the liver chunks in a sealed food bag and weigh down in the water bath, if the liver gets wet then it may effect the results. Place the test tube into the water bath and wait for the solution to reach 22°C (using one of the mercury thermometers). After the H202 has reached the correct temperature, start the stopwatch and leave the H202 and the liver in the water bath for a further five minutes, to give the liver more chance to reach the correct temperature. It will take the liver longer to heat up as it has a greater thickness and surface area than the H202. After five minutes has passed place the liver chunks into the conical flask, then pour in the H202 quickly replacing the rubber stopper as not to let any oxygen escape. It takes a minute to extract the liver chunks from the bag so if you empty them into the conical flask first there is less chance of you losing O2 before the rubber stopper is in place. As soon as the stopper is in position you should then start the stopwatch and time how long it takes for the burette to empty. A burette should be used rather than a measuring cylinder as a burette is much more accurate. Repeat this process for each temperature then repeat the whole experiment two more times.
To ensure there are no variables, I will repeat the experiment twice more after the first one. I will place both the liver chunks and the 15cm3 H202 (5vol) into the same water bath, with the same type of thermometer. I will be using an electric scale in order to ensure that the liver chunks have a total weight of almost exactly 1,5 grams. Also each piece of liver will be cut to approximately the same size. The temperatures I will use are 22°C, 27°C, 32°C, 37°C, 42°C, 47°C and 52°C with three temperatures on either side of the optimum. Also there will be preliminary tests to make certain that the 15cm3 H202 (10vol) does not break down by itself when heated.
H202 (10vol), H202 (5vol)
Liver Chunks
Water bath, Conical Flask
Basin, Mercury thermometer (x2)
Test tube, Measuring Cylinder
Stopwatch, Clamp
1 Food bag, A Weight
Burette, Rubber Bung
Delivery Tube
In the preliminary tests I will be using 15cm3 H202 (10vol) and 5cm3 liver solution (50% pure) preheated to 37°C. The burette will be filed with water and placed upside down in the stand. The top of the burette will be submerged in the tub of water and from this the pipe attached to the bung will lead. The 15cm3 H202 (10vol) and 5cm3 liver solution will be poured into the conical flask. As the bung is place into the mouth of the flask the stopwatch is started and is only stopped once the burette is empty or the reaction stopped.
The burette emptied too quickly which would give inadequate results in the main experiment also the measuring method was inaccurate so instead of measure how much the burette emptied in 5 minutes I will measure how long it takes for the burette to empty, with a maximum time of 5 minutes. I repeated the preliminary test this time using 15cm3 H202 at 5 vol instead of ten and also instead of liver solution I will be using 1.5 grams of liver chunks.
This produced sufficient results and so this is the process that will be used in the final experiment.
Rate of Reaction = 50 cm3
Time taken to empty
the burette
My results followed my prediction and show that temperature is one of the factors that affect the rate of an enzyme catalysed reaction. The results also show that 37°C is the optimum temperature for this particular enzyme/substrate reaction. At 37°C the temperature is high enough for both the enzyme and substrate to have more kinetic energy. Kinetic energy is the energy of motion. An object that has motion - whether it is vertical or horizontal motion - has kinetic energy. The more you heat the object, the more energy it will have, up to a point. This kinetic energy causes the enzyme and substrate to collide and it is during these collisions that the enzyme is splitting the substrate into its products. When you heat the enzyme/substrate you give them more kinetic energy. The enzymes and substrate collide more often causing the reaction to occur more often thus increasing the reaction rate. If you heat the enzyme/substrate too much the bonds holding the enzyme molecule in its shape begin to break, causing it to denature and the lock and key mechanism to no longer work.
My first graph shows that the more the temperature was increased the faster the reaction got, until 37°C where it started to slow down again, the reaction increased slowly but decreased rather rapidly due to denaturisation. In my second graph it also shows how the reaction rate steadily increases up to 37°C then dramatically slows down again after wards. Although Graph 1 and 2 seems different they actually support each other, as in graph 1 it shows that at 37°C it takes less time for the burette to empty and in graph 2 is shows the reaction rate is at its fastest.
In the first repeat of the experiment there was an anomalous result at 42°C, this could have been due to an incorrect reading on the thermometer or the liver/solution could have cooled whilst being transferred from the water bath to the conical flask. When calculating the averages I did not include the anomaly, as it was obviously an anomalous result and if included would have affected the accuracy of my results.
I think the experiment was successful with good results but it could still have been more accurate. To double-check my results I could have repeated the experiment a few more times and if not the whole experiment then I should have at least repeated the part of the experiment where an anomaly occurred (42°C). This could have been due to the fact the temperature of the liver was not necessarily exact due to inadequate temperature measurement or the liver/solution could have cooled whilst being transferred from the water bath to the conical flask. The concentration of the substrate and the enzyme were controlled by using the same amount, H202 (5vol) and 1.5 grams of liver chunks, for each experiment and for each repeat. One factor that was not controlled was pH; this may have affected the results because if the acidity was not suitable for the enzyme it may have cause the enzyme to denature. The pH factor could have caused the anomaly but also so could a contaminated test tube or piece of liver.
An extraneous variable was that each person working on the experiment has different reactions for example one person may have slightly faster reactions when starting the stopwatch. One way to control this is by getting each person to do just one or two of the jobs the same person to start the stopwatch, the same person to weigh the liver, the same person to check the temperature and also the same person to measure out the hydrogen peroxide. Another factor that needs improvement is that the temperature of the liver could not be guaranteed as you cannot test the temperature of a chunk of liver with a thermometer and it may have been better to have used a temperature probe in order to receive more accurate results.
On hindsight it may have been better to used a liver solution than chunks, it would have been easier to test the temperature of a solution than a solid, also a liver solution would have had a larger surface area mean there would have been more area for the enzyme active site to come into contact with. Over all though my results did back up my hypothesis even despite the fact that my experimental procedure could have used some more accurate equipment.
Cambridge Advanced Sciences: Biology 1
Mary Jones
Richard Fosbery
Dennis Taylor