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Investigating the Effect of pH on the Activity of an Enzyme

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Investigating the Effect of pH on the Activity of an Enzyme

For this investigation, I will be finding out the effect of pH on the activity of the enzyme potato catalase. Catalase is found in all living organisms. It has one of the highest turnover rates of all enzymes; one molecule of catalase can convert millions of molecules of hydrogen peroxide to water and oxygen per second. Catalase occurs in many plant and animal tissues. It breaks down toxic hydrogen peroxide, a by-product of many bio-chemical reactions, into water and oxygen.

Variables

The variables that I will ensure remain constant through the experiment will be:

  • Substrate concentration and enzyme concentration
  • Temperature
  • Volume of substrate and volume of enzyme.
  • Volume of buffer solution used.

The independent variable (what I am changing) for my experiment, is the pH level.

I will use all 14 values of pH: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14. To make sure the solution is at the right pH I will use pH buffer solutions (solutions that maintain a constant pH). This is an easy and effective way of changing the independent variable.  

The dependent variable (what is measured as a result of change) will be the time taken for manometer fluid to rise by 5 cm due to the production of oxygen.

In experiments to confirm that one has isolated the right variable(s) they would use a control group.  

The control group will receive the same attention as the test groups; however, it will not be influenced by the variable(s) which the rest of the groups are testing.

This is advantageous in many ways.

  • Provides a baseline to which experimental results can be compared.
  • Enhances the reliability of results.
  • Prevents the effect of one variable from being drowned out by the known greater effects of other variables- this would apply if more than one independent variable was being used e.g. temperature and pH level.

However, I will not be using a control group for my experiments as it is impossible to carry an experiment out with having a “non pH” solution.

Apparatus and Chemicals

To carry out the experiment, I will need the following apparatus:

  • Stand, bosses and clamps
  • Beaker, tub, spring clip, stop clock, marker pen, forceps, ruler, ceramic tile and razor blade.
  • U-tube Manometer (easy and accurate technique of measuring the rate of production of oxygen from hydrogen peroxide in the presence of living tissue (in this case, potato)) tube- 3mm in diameter.
  • Manometer fluid-water. Oil and mercury are other options.
  • Syringes ( 5 cm3) x 2
  • Hydrogen Peroxide (3%)
  • 14 x pH buffer solutions: 1 one buffer solution has to have volume greater than 20 cm3  as each pH experiment will use 5cm3  buffer solution and will be carried out four times.
  • 14 x Test tubes/boiling tubes with rubber bung. Boiling tubes are preferred as they allow larger volumes to boil freely.
  • 56 x 1cm3 cubes of potato.

Safety Precautions

  • Wear goggles at all times; if hydrogen peroxide makes contact with the eyes, it can cause long-term damage. However, the solution I will be using will only be 3% concentration yet it can still pose a risk if used irresponsibly.
  • Wear a laboratory coat, to prevent spillages of hydrogen peroxide onto clothes.
  • Wear gloves to protect skin.
  • Take care to use the razor blade with caution.
  • When using the pH1 and pH14 buffer solutions, care must be taken as they are very hazardous solutions that can cause serious damage to eyes, skin and can sometimes lead to death.
  • Common sense i.e. no running in the laboratory, not eating or drinking e.t.c
  • Wash hands after experiment has been completed.

Method/Procedure (refer to Figure 1 for set up of apparatus)

  1. With the razor blade cut 56 potato cubes with a volume of 1 cm3 (using the ruler) and place them under water in a small tub.
  2. Assemble the apparatus as it is shown in Figure 1.
  3. With the marker pen, mark out on the right hand manometer tube, a line where the position of the meniscus is at and another line 5 cm above it.
  4. Remove the bung from the boiling tube. With one of the 5cm3 syringes, place into the boiling tube 5cm3 of pH1 buffer solution.
  5. Using the forceps add one of the potato cubes from the water tub.
  6. With the other syringe, add 5cm3 of hydrogen peroxide.
  7. Replace the bung immediately. Make sure it gives an airtight seal. Start the stop clock. Agitate the boiling tube to start the reaction. As the reaction commences, oxygen will be produced as the hydrogen peroxide is broken down. The manometer fluid should be pushed down on the left hand side and rise on the right. Time how long it takes for the fluid to rise through the 5cm mark on the right hand side.
  8. Open the clip at the top of the boiling tube. This should result in the manometer fluid returning back to its original position. Close the clip.
  9. Wash out the boiling tube and syringes and repeat this procedure 3 times. Then carry the same thing out with pH levels 2-14, making sure each one is replicated 3 times (total of 4 times).
  10. After the results have been recorded in a table (Figure 2), each pH level’s average time should be calculated.
  11. Finally, plot a graph showing the how the pH level affected the time taken for the manometer fluid to rise 5cm.

Possible factors that would affect reliability of my results:

  • When the Hydrogen peroxide is poured in Oxygen may be lost before bung is placed in. However as long as I do not take too long between the two, this should be minimal.
  • Timing may not be accurate as possible.    

Figure 1: Diagram of Apparatus- This is how the apparatus used for the experiment will be set up.  

Figure 2: Results table- This is how the results will be recorded.

pH level

Time it takes for manometer fluid to rise 5cm/seconds

Average time/seconds

Replicates

1

2

3

4

5

6

7

8

9

10

11

12

13

14

Figure3: Graph of results.

image00.png

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