Plant cells contain an enzyme which catalyses the breakdown of hydrogen peroxide, releasing oxygen, as shown in Fig. 1.1. enzyme hydrogen peroxide ⎯⎯⎯⎯→ water + oxygen [Figure 1.1] You will investigate the effect of pH on the activity of this enzyme. You are provided with the materials shown in Table 1.1. Table 1.1 | labelled | contents | hazard | volume /cm³ | |---|---|---|---| | H | hydrogen peroxide solution | harmful irritant | 130 | | E | enzyme solution | harmful irritant | 20 | | B5 | pH5 buffer | none | 20 | | B6 | pH6 buffer | none | 20 | | B7 | pH7 buffer | none | 20 | | B8 | pH8 buffer | none | 20 | | B9.5 | pH9.5 buffer | none | 20 | If any solution comes into contact with your skin, wash off immediately with cold water. It is recommended that you wear suitable eye protection. When discs of filter paper that have been soaked in E are put into hydrogen peroxide solution, H, the discs rise to the surface as H is broken down. The higher the activity of E, the faster the discs rise. A student investigated the effect of pH on the activity of enzyme E. The student suggested the hypothesis: As the pH of the buffer containing E increases, the activity of E decreases. Carry out step 1 to step 16 to test the student's hypothesis. step 1 Label 5 beakers with E5, E6, E7, E8 and E9.5. step 2 Put 10 cm³ of B5 buffer solution into the beaker labelled E5. step 3 Repeat step 2 by putting the appropriate buffer solution into the beakers labelled E6, E7, E8 and E9.5. step 4 Stir E and put 2 cm³ of E into each of the labelled beakers used in step 2 and step 3. Swirl each beaker to mix the contents. step 5 Leave the beakers for 2 minutes. step 6 Label a small beaker H and put 60cm³ of solution H into this beaker. step 7 After 2 minutes (step 5) stir E9.5. step 8 Pick up one disc of filter paper using forceps. step 9 Continue to hold the disc in the forceps and: • dip the disc in E9.5 for 5 seconds • put the disc at the bottom of the liquid in the beaker labelled H. step 10 Immediately release the disc from the forceps and start timing. step 11 Stop timing when the disc reaches the surface of the liquid. You may find it helps to observe the disc with a piece of black card behind the beaker. step 12 Record in (a)(ii) the time taken for the disc to reach the surface of H. If the time taken for the disc to rise to the surface is longer than 180 seconds, record as 'more than 180'. step 13 Remove the disc from the beaker using the forceps and put it on a paper towel. step 14 Dip the forceps in the water in the container labelled For washing. Dry the forceps with a paper towel. step 15 Repeat step 7 to step 14 two more times, using E9.5. step 16 Repeat step 7 to step 15 using E8, E7, E6 and E5. (b) Temperature is another factor that affects the activity of an enzyme. You will show the effect of preheating the enzyme at two different temperatures, before measuring the activity of this enzyme. You will select two temperatures and standardise the pH by choosing an appropriate buffer.

1. (a)(i)Explain why the discs rise.[1]
2. (a)(ii)Record your results in an appropriate table, including raw results and processed (mean) results.[5]
3. (a)(iii)The student suggested the hypothesis: As the pH of the buffer containing E increases, the activity of E decreases. Tick (✔) one box to show whether or not your processed results support this hypothesis. support do not support Explain how the trend in your processed results provides evidence for this decision.[1]
4. (a)(iv)State the independent variable in this investigation.[1]
5. (a)(v)Describe an appropriate control for this investigation.[1]
6. (a)(vi)One possible source of error in this investigation is reusing the same hydrogen peroxide solution for all the discs. Suggest a reason why reusing the same hydrogen peroxide solution is a possible source of error. State how you could reduce this source of error.[2]
7. (a)(vii)Describe how you could modify your procedure to determine the optimum pH for this enzyme.[1]
8. (b)(i)Measure the room temperature. room temperature[1]
9. (b)(ii)Select two temperatures between room temperature and 50°C. State the temperatures you have selected.[1]
10. (b)(iii)You will need to standardise the pH by choosing an appropriate mixture of buffer and enzyme, E5, E6, E7, E8 or E9.5. State the mixture of buffer and enzyme you will use.[1]
11. (b)(iv)Record your results for T1 and T2. T1 T2[1]
12. (b)(v)Explain the effect of increased temperature on the activity of enzyme E.[3]

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