IB

Biology Grade 11 HL Internal assessment Victor von Hundt, Konstan;n Frank

Lab 0: Concentra.on of subtrate (Hydrogen-Peroxide) aec.ng enzyme catalase rate of reac.on. Lab. date: 25th November 2011

Introduc.on
We inves.gated our research Ques.on How does the concentra.on of the substrate (Hydrogen-peroxide) aect the rate of reac.on of the enzyme catalase?. Catalase is an enzyme that converts hydrogen peroxide to water and oxygen in cells that are confronted with oxygen. The formula is

2 H2O2 2 H2O + O2

The hydrogen peroxide needs to be converted as it is highly toxic to cells and kills them as they reach a certain concentra.on. We expect the reac.vity of the catalase (produc.on of gas) to increase as the concentra.on of the substrate (hydrogen-peroxide) increases, as there is more substrate the catalase can break down. Therefore our hypothesis is :

The rate of reac.on of catalase increases as the concentra.on of hydrogen peroxide increased.

Our independent variable is the concentra.on of hydrogen peroxide. Our dependent variable is the produc.on of H2 and O2. We tried to keep the room temperature the same, as well as the size, weight and surface area of the liver samples. Furthermore the pressure did not aect the experiment as we conducted it on the same day and not at dierent places. We also used the same dis.lled water when we had to dilute the hydrogen peroxide.

Material
We used
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 50g fresh liver, 100 ml Hydrogen peroxide 300 ml 1 stop watch, 5 glass cylinders with a volume of 250ml, 6 small plas.c trays, delivery tubes, rubber stopper, one big plas.c container, rubber connec.on tubes, sharp knife, scale, one test tube 50ml, one test tube 100ml, one test tube 250ml tripod in
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order to conduct our experiment.

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3 2

1 2 3 4 5 6 7 8

Liver Substrate Glass cylinder Rubber stopper Delivery tubes Water container lled with water Beehive Test tube

Methods
To begin with we cut the liver each into 5cm3 pieces, taking care that the surface area was about the same, so that it was only one piece, formed like a cube, and not several pieces. We used a scale and the pieces weighted as follows

Sample 1 2 3 4
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Weight 5.06 5.06 5.07 4.93

Sample 5

Weight 4.94

Then we placed them on small plas.c trays, labeling the trays from 1 to 5. The average weight was 5.012 g ((5.06g+5.06g+5.07g+4.93g+4.94g)/5). Aeerwards we labelled the glass cylinders from 1 to 5. Every glass cylinder was lled with 25ml of substrate. Glass cylinder 1 with 25ml of hydrogen peroxide, glass cylinder 2 with 12.5ml of hydrogen peroxide and 12.5ml of dis.lled water, glass cylinder 3 with 6.25ml of hydrogen peroxide and 18.75ml of dis.lled water, glass cylinder 4 with 3ml of hydrogen peroxide and 22ml of dis.lled water and glass cylinder 5 with 1.5ml of hydrogen peroxide and 23.5ml of dis.lled water. We measured the volume by using a measuring cylinder with 1ml labels so a maximal inaccuracy of 0.5ml is possible. Aeer this we set up the delivery tubes and connected them with a rubber stopper, which we put on each glass cylinder to deliver later-on the gas, which will be produced in the glass cylinder to the big water container. In the water container we put a beehive, that collected the gas from the deliver tubes and made the gas rise into the completely water lled test tube, which was labelled, to show how much water was displaced. The volume of water displayed equals the amount of gas formed. Aeer everything was set up we prepared the stop watch and put the rst piece of liver into the glass cylinder. Immediately aeer throwing the piece into the substrate we put the rubber stopper on it, started the stop watch and xated it with a tripod. We let the experiment run for 2 minutes and then pulled the rubber stopper out to stop the gas delivery. We recorded the result (volume of water displaced of the test tube) by reading the test tube scale and wrote it into our recording sheet.We started with glass cylinder 5 and did the same procedure as described to all the other glass cylinders. We changed the water lled test tubes aeer every experiment run (aeer every glass cylinder) and had to adjust the
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size of the test tubes for glass cylinder 1 and 2, as we expected the amount to increase tremendously and wanted to collect all the gas.

Data collec.on

Concentration in ml 0.05 25 12.5 6.25 3.125 1.5 Table 1. - Raw data

Volume Displayed in cm3 0.05 239 112.8 18.3 8.2 5.2

Time in seconds 0.5 120 120 120 120 120

Table 1 displays all the results we recorded during the experiment. What can be seen straight ahead is a strong posi.ve correla.on, meaning that as the concentra.on increases the volume displayed and therefore the amount of gas produced increases as well.

Data processing and presenta.on

Amount of Hydrogen peroxide in ml 0.05 25 12,5 6,25 3,125 1,5

Volume Displayed in cm3 0.05 239 112,8 18,3 8,2 5,2

Time in seconds 0.5

Rate of reactivity

120 120 120 120 120

1,9916666666667 0,94 0,1525 0,0683333333333 0,0433333333333

Amount of hydrogen peroxide in 25ml of substrate against formation of gas 300,0 Volume of gas formed in cm3
239,0

225,0

150,0
112,8

75,0
5,2 8,2 18,3

0 1.5 3 6.25 12.5 25 Amount of hydrogen peroxide in 25ml of substrate in ml

Conclusion
To begin with, let us state the hypothesis we created before the experiment once more ;

The rate of reac.on of catalase increases as the concentra.on of hydrogen peroxide increased. Looking at the results the predicted hypothesis was true. We can see that at a concentra.on of 1.5ml in 25ml substrate the volume of gas formed was 5.2 cm3, at a rate of 12.5ml of hydrogen peroxide in 25ml substrate the volume of gas formed was 112.8cm3 and at 25ml pure hydrogen peroxide we collected 239cm3 of gas. This shows that our hypothesis is true!

Evalua.on
I was able to prove my hypothesis experimentally but there are some situa.ons which may lead to my results not being very accurate. Some of them can include the following. Because of .me limita.on (80 minutes) I was not able to do duplicate or more sets of the same inves.ga.on. This would have made my results more reliable as the average of several trials will be used in the conclusion. I only prepared a range of 5 hydrogen peroxide samples. If I would have used more samples, the range and accuracy of our samples would have increased.

The sample could have been impure, because the liver was transported to the school in an unsterile container. This could have lead to a reac.on taking place before the liver was used in the experiment, or the liver decomposing, because it was not cooled and the transport took too much .me.

Again due to .me reasons it was dicult to completely remove air from the measuring cylinder where the gas was formed. The volume of .ny air bubbles was dicult to determine and hence added up with the gas formed during the reac.on and may have aect the rate.
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We were not able to let the reac.on (pumng the liver into the substrate) happen when the glass cylinder was connected to delivery tubes. So same gas might already have escaped by the .me we connected the glass cylinder to the delivery tubes. This might have aected the volume formed and therefore the rate of reac.on.

Human error such as reading measurements on the measuring cylinder while preparing the dilu.ons may have altered the expected acid concentra.on. Also, the volume of gas collected in the measuring cylinder may have been slightly incorrectly read. There was no special tool for cumng the liver samples accurate; I used a scalpel to cut the pieces. This could have caused the rst reac.on (using low concentra.on of the acid) to be inaccurate since one of the reactants did not have the accurate volume.

Sugges.ons for improvements

To reduce error and make my results more reliable, mul.ple of the experiment need to be done. This will help to iden.fy any anomalies. For example duplica.on of the inves.ga.on would have reduced errors.

To make my results more reliable more dilu.ons with an equal amount of liver samples would be an advantage. This will help to construct detailed graphs and also reduce the chance of big errors.

Care must be taken to make sure that no air is lee in the gas collec.on measuring cylinder. This can be done by completely lling the measuring cylinder with water and covering with a small piece of paper and then the covered end is quickly immersed in water in the trough and the paper removed.

To make sure that the reac.on starts only once the stopper is in place is to .e liver samples so that it is not in contact with the acid. Once the stopper is in place then the liver sample can be lee to fall and reac.on to start. Alterna.vely, if a long necked ask is used, the ribbon can be placed at the neck aeer the ask has been slightly .lted. Once the stopper is in place the ask is lee to stand normally which will cause the liver to fall into the acid and the reac.on will start.

To reduce reading errors, only one person should do the reading and the measuring cylinder which was used to measure volumes of acids should always be on the at bench and ver.cal. To avoid parallax, one should take readings below the meniscus at right angle to the measuring cylinder. The measuring cylinder which collects gas should be xed on a stand and made sure that it is accurately ver.cal.

To reduce the risk of an impurity, the liver sample should be carried in a sterile container which has a cooling func.on in order to avoid dirt and decomposing inuencing the pureness of the sample.
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Safety
During the whole experiment we wore goggles and gloves, as we worked with chemicals. Furthermore we wore a lab coat and conducted the experiment only while standing. We poured the hydrogen peroxide in special containers aeer we nished the experiment.

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