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Investigation Into the Effect of Substrate Concentration On The Activity Of An Enzyme

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GCSE Biology Coursework: Investigation Into the Effect of Substrate Concentration On The Activity Of An Enzyme

Background Information

Enzymes are made of proteins and are catalysts to break up different chemicals, for example Amylase helps to break down Glucose this is done through the Lock and Key theory. This theory states that the substrate (the key in this case the Hydrogen Peroxide) fits exactly into the active site (the lock in this case the Catalase enzyme) of the enzyme. As there is usually only one key that fits a lock, there is usually only one substrate or group of substrates that fit the active site of a certain enzyme. This theory supposes that enzymes have active sites that do not change shape easily. So the way in which the substrate fits is purely a result of the shape of the substrate and the active site. The process can be seen in the figure 1 Catalase acts as a catalyst to break down Hydrogen Peroxide, which is a poison to living organisms. Catalase is a common enzyme found in living organisms. Hydrogen peroxide is formed as a waste product of metabolism in many living organisms. It is toxic and must be quickly converted into other, less dangerous, chemicals. To manage this problem, the enzyme catalase is frequently used to rapidly catalyse the decomposition of hydrogen peroxide into harmless oxygen and water. Catalase is also used in the textile industry, removing hydrogen peroxide from fabrics to make sure the material is peroxide free. A minor use is in contact lens hygiene - some lens cleaning systems sterilise the lenses by soaking them in a hydrogen peroxide solution, and catalase is used to decompose the peroxide before reinserting the lenses in the eye. (http://en.wikipedia.org/wiki/Catalase). Hydrogen peroxide is a substrate though. Hydrogen peroxide usually decomposes exothermically into water and oxygen gas this means that it produces energy and so may get hotter during the reaction. The decomposition happens more rapidly in alkali. Hydrogen Peroxide is a flamable liquid and Hydrogen peroxide vapour is explosive and will explode if it goes above 70ºC. hydrogen peroxide is a strong bleach and is used to sterilise products however the hydrogen peroxide must be removed before it is sold for manufacture as it is also an irritant. (http://en.wikipedia.org/wiki/Hydrogen_peroxide - Physical_properties) . Enzymes work best in certain conditions her is the list:

Here are a list of things that may help enzymes work faster:

  • Temperature: The higher the temperature the faster the enzyme works until temperature rises above 40-50°C because this denatures (means that they begin to die) many enzymes.
  • PH: The PH scale is used to measure how much the acid/alkali the solution contains using litmus paper if the number is below 7 then it is acidic and a number above 7 is alkali and 7 is neutral. Enzymes work best at a 7 neutral.
  • Concentration of Substrate: If the Substrate is in excess then this means that every Enzyme will join to one of the substrate’s this also means that there is more of a chance of an enzyme and substrate colliding so the reaction will occur faster.
  • Concentration of Enzyme: Much the same as the concentration of the substrate but this time if there is an excess of enzymes then the substrates will all join with an enzyme there will also be a higher chance of a collision between substrate and enzyme.

(http://www.seps.org/cvoracle/faq/catalase.html)

Figure 1

image00.png

Hypothesis

I think that more oxygen (in the form of the foam) will be produced with the higher the concentration of Hydrogen peroxide. I think this will happen because the more reactant (in this case the Hydrogen Peroxide) the more product that is produced (oxygen and water).

Equipment list

  • Hydrogen Peroxide: H2O2 we will use this as the substrate and this is what the Catalase will react will to produce the Oxygen and water
  • Water: I will use distilled water, as it contains no impurities that may alter the experiment and is used to dilute the Hydrogen peroxide. The hydrogen peroxide shouldn’t react with the water because water is one of the products formed by the reaction between hydrogen peroxide and catalase.
  • Potato Extract (Catalase): This will react with the Hydrogen Peroxide to produce Oxygen and water.
  • Test tubes: we will put the different concentrated solutions in different test tubes to make it a fairer test.
  • Test tube racks: we will use the test tube racks to stop the test tubes from falling over this will help us get a fairer result by not controlling the mixing of the solution.
  • Stop Watch: we will need a stopwatch to record how fast reaction takes.
  • Syringes: We will use two syringes one syringe to transport the Hydrogen Peroxide and the other to transport the water so that the Hydrogen Peroxide is not diluted in water before it is added to the Catalase this will make the test fairer.
  • Thermometers: We will measure the temperature of the different test tubes. This is because Catalase will work faster in a hotter environment (but not over 40°C as this will cause the enzyme to denature).
  • Beakers: This is where we will mix the Hydrogen peroxide to get different concentrations of Hydrogen Peroxide.
  • Marker pens: We will use the marker pens to write numbers on the test tubes to make sure we remember which test tube contains which concentration of Hydrogen Peroxide we need to do this as Hydrogen peroxide and water are both colourless so this will prevent us from guessing which test tube contains what.
  • Goggles: We will need goggles because Hydrogen Peroxide is not only poisonous but is an irritant if we get it on our skin and will damage our eyes if we splash some in our eyes.
  • Ruler: We will need a ruler to measure a line where we can stop the stop watch when it reaches this point this will act as a control and will make gathering results easier than measuring the amount of foam produced.
  • Measuring cylinder: We will need measuring cylinders to measure the amount of Hydrogen peroxide and water solutions. We will use a separate measuring beaker for both water and Hydrogen peroxide this will stop the hydrogen peroxide from being diluted before its is added into the test tube.

Safety

To make sure that my experiment is safe I will wear an apron to protect my clothes and skin from being burned by the Hydrogen peroxide and I will wear eye protection to protect my eyes from Hydrogen Peroxide. We will be careful when moving glass so that it does not fall and break.

Plan

I will be making three experiments to make sure that my results will be accurate. I will take the average of these three results to get the final accurate result I will measure the amount of water and Hydrogen peroxide in cm³. I will have 3cm³ of catalase (potato extract) in each test tube. I will make all of the solutions 20cm³ of Hydrogen peroxide. I will have 3cm³ of catalase (potato extract) in each test tube. I will put the concentrations in the beakers so for example instead of adding 1.6cm³ (80%) of Hydrogen peroxide and 0.4cm³ (20%) of water (this would be incredibly difficult if not impossible to measure without accurate measuring equipment) we will simply add 8cm³ (80%) of Hydrogen peroxide and 2cm³ (20%) of water into one of the beakers, where we will be able to mix it easier then instead of measuring out the quantities out again after every test we could simply use the solution for all three tests as this would save error from adding a wrong concentration and receiving an anomaly. All of the test must be carried out at the same temperature (room temperature) and at the same pH level (7).

In the table below are the amounts of Hydrogen peroxide and water I would add to the beakers:

Test Tube

Amount of Hydrogen peroxide (%)

Amount of water (%)

Amount of Hydrogen peroxide to put in beaker (cm³)

Amount of water to put in beaker  (cm³)

1

100

0

10

0

2

80

20

8

2

3

60

40

6

4

4

40

60

4

6

5

20

80

2

8

6

0

100

0

10

Method

  1. First of all we must get goggles and aprons before we handle the hydrogen

            peroxide.

  1. We then must set up our apparatus so the 6 test tubes in the test tube holder and must use the marker pen to label them sensible things so that we know which test tube will contain which concentration of hydrogen peroxide. We must get the Hydrogen peroxide, potato extract (catalase), beakers, syringes and stopwatch. We must then mark a line at 8cm away from the bottom of the test tube this will be measured with a ruler so that it is accurate. This will act as a control so that all 6 test tubes in all three tests have this line marked on them.
  2. Next we must put 3cm³ of potato extract (catalase) into each of the test tubes.
  3. We then must mix the concentrations as appropriate (see the table above) in the beakers (one beaker per concentration). We will use a syringe to measure and transfer the hydrogen peroxide and water from beaker to beaker we will remember to use a different syringe
  4. Next whilst one other person is recording the time on the stopwatch the other person will prepare to add the 100% of Hydrogen peroxide concentration to the 3cm³ of catalase in the test tube. The person timing the reaction must start the stopwatch as soon as the Hydrogen peroxide comes into contact with the catalase.
  5. The two people then crouch down level with the opening of the test tube the stopwatch is still going when it reaches the line marked on earlier 8cm from the bottom of the test tube. When the oxygen (in the form of foam) reaches this line then the stopwatch is stopped this time is then recorded in a table of results.
  6. We then must repeat steps 5-6 except this time instead of adding 100% of Hydrogen peroxide we must add the 80% of Hydrogen peroxide and 20% of water solution.
  7. We must repeat steps 5-6 for the last 4 concentrations.
  8. When we have got all these results we must start on the second test and copy steps 3-8.
  9. For the last test copy steps 3-8

Obtaining Evidence

The reason I am collecting this data is to show that the concentration of Hydrogen peroxide changes the amount of oxygen (foam) produced. I think that if the concentration of Hydrogen peroxide is increased then the amount of oxygen (foam) should also increase with it.

In the tables below are the results that I collected:

Test 1

Test Tube

Concentration of Hydrogen Peroxide (%)

Concentration of water (%)

Time taken for the oxygen (foam) to rise 8cm (seconds)

1

100

0

9.59

2

80

20

13.75

3

60

40

23.91

4

40

60

33.57

5

20

80

1.43.36

6

0

100

- (the water did not react with the catalase)

We are not going to count this test as it used a different potato extract (catalase) to the other three tests and as you can see must have contained less catalase than the other tests so we are not going to count this, as it will make the test unfair.

Test 2

Test Tube

Concentration of Hydrogen Peroxide (%)

Concentration of water (%)

Time taken for the oxygen (foam) to rise 8cm (seconds)

1

100

0

7.85

2

80

20

10.76

3

60

40

12.78

4

40

60

26.85

5

20

80

1.45.96

6

0

100

- (the water did not react with the catalase)

Test 3

Test Tube

Concentration of Hydrogen Peroxide (%)

Concentration of water (%)

Time taken for the oxygen (foam) to rise 8cm (seconds)

1

100

0

6.06

2

80

20

8.22

3

60

40

12.16

4

40

60

28.40

5

20

80

1.51.23

6

0

100

- (the water did not react with the catalase)

Test 4

Test Tube

Concentration of Hydrogen Peroxide (%)

Concentration of water (%)

Time taken for the oxygen (foam) to rise 8cm (seconds)

1

100

0

6.79

2

80

20

6.60

3

60

40

12.65

4

40

60

28.34

5

20

80

1.52.01

6

0

100

- (the water did not react with the catalase)

Averages for all of the tests (excluding test 1)

Test Tube

Concentration of Hydrogen Peroxide (%)

Concentration of water (%)

Time taken for the oxygen (foam) to rise 8cm (seconds)

1

100

0

6.90

2

80

20

9.10

3

60

40

12.53

4

40

60

27.86

5

20

80

1.49.73

6

0

100

- (the water did not react with the catalase)

My results support my hypothesis showing that a higher concentration of Hydrogen Peroxide the more oxygen (in the form of foam) is produced.

Analyzing and Considering Evidence

In my 3 results I can see that the results are all vaguely similar. For example in test 2 it took 26.85 second for a 40% concentration of Hydrogen Peroxide to rise 8cm and in test 3 it took 28.40 seconds for the same solution to rise the same distance and finally in test 4 it took 27.86 seconds for the same solution to rise the same distance as the other tests. These results are all relatively identical taking into account human error (we might have started the stopwatch a couple of tenths of a second early/late which would affect the results or we might have stopped the stopwatch a couple of tenths of a second late/early).

In my graph I have found that the results seem to be very fast compared to a 20% concentration and it takes longer and longer and the gap between the last results gets bigger the less concentrated the solution becomes the longer it takes for the oxygen (foam) to rise 8cm.

In the reaction between Hydrogen Peroxide and Catalase I think the higher the concentration the more chance there is of a catalase enzyme and a Hydrogen Peroxide molecule hitting and therefore reacting. This can be seen in my drawing below:

However if the concentration is increased (more Hydrogen Peroxide molecules) then the more chance there is of the Catalase and Hydrogen Peroxide hitting. The more Hydrogen Peroxide and Catalase hitting then the faster the reaction will be and the faster it will take the foam to move 8cm up the test tube.

But if there is a lower concentration then there will be fewer connections between the Hydrogen Peroxide and Catalase this means that it takes longer for the oxygen (foam) to move 8cm up the test tube.

Evaluating

I think that our tests have been very successful as we have achieved reliable results and we have corrected any flaws that we think were not meant to be there (the anomaly). My experiments showed that my prediction was right. In test 4 we found an anomaly where the time was faster than the reaction before and this did not seem normal compared to the other tests that we had done so I went back to change it. I think that the experiment could have been made better if we had done the experiment in larger groups because then we could have had numerous stop watches and then we could have taken the average time of all the stop watches this would mean that we would receive few anomalies or none at all. Having more people in the group would also help prepare the experiment quicker. We could have weighed the amount of hydrogen peroxide and catalase and then at the end of the experiment we could weigh the solution and see if any of the oxygen had gone in the form of the gas. Another way of making the experiment better would be if we timed the reaction until it had stopped and then we measured how far it had moved (we would need a very long test tube for this) this would allow us to see that if a low concentration of Hydrogen peroxide was added then if it would be an experiment where the foam would be slow but would move further up the tube compared to a higher concentration of Hydrogen Peroxide where it would be a violent reaction but would not go very far up the test tube I doubt this but it would be good to see if my prediction is true. I think that my method was as suitable as I could have made it with the equipment that we were given. I think this was a fair test because we put the same amount of catalase in the test tubes and the same amount of solution in each test tube. We also used distilled water to dilute the concentration of Hydrogen peroxide so that there weren’t any unknown chemicals that might have changed the reaction in some way. We did not find any difficulties during the investigation. I think that my results may have only been 90% correct because we did not use multiple stopwatches and there is always a slight margin for human error when we were recording the time.    

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