Seeds of many plant species contain an enzyme that is used to hydrolyse sucrose into reducing sugars. This enzyme is essential to provide the reducing sugars needed for the seeds to grow. When seeds are soaked in sucrose solution, some of this enzyme diffuses from the seeds into the surrounding solution and hydrolyses the sucrose, as shown in Fig. 1.1. [Figure 1.1] You will investigate the release of this enzyme from the seeds of two different species of plant, G and H. Seeds from the two different species of plant, G and H, were put into sucrose solutions at 20 °C for 24 hours, as shown in Fig. 1.2. All conditions, including the mass of seeds used, were the same. [Figure 1.2] After 24 hours, a sample of the sucrose solution was removed from each beaker. You are provided with the materials shown in Table 1.1. Table 1.1 labelled contents hazard volume/cm³ R 1.00% reducing sugar solution none 50 W distilled water none 150 G1 sample of the sucrose solution taken after 24 hours from plant G none 10 H1 sample of the sucrose solution taken after 24 hours from plant H none 10 Benedict's Benedict's solution harmful irritant 25 If any solution comes into contact with your skin, wash it off immediately with cold water. It is recommended that you wear suitable eye protection. You will determine the concentration of reducing sugars in G1 and H1 by: • preparing different concentrations of reducing sugar solution • carrying out a semi-quantitative Benedict's test on each of the concentrations of reducing sugar • carrying out a semi-quantitative Benedict's test on G1 and H1 • using your results to estimate the concentration of reducing sugars in G1 and H1.

1. (a)(i)You will need to carry out a serial dilution of the 1.00% reducing sugar solution, R, to reduce the concentration by half between each successive dilution. You will need to prepare four concentrations of reducing sugar solution in addition to the 1.00% reducing sugar solution, R. After the serial dilution is completed, you will need to have 10cm³ of each concentration available to use. Complete Fig. 1.3 to show how you will prepare your serial dilution. Each beaker should have: • a labelled arrow to show the volume of reducing sugar solution transferred • a labelled arrow to show the volume of distilled water, W, added • a label under the beaker to show the concentration of the reducing sugar solution. [Figure 1.3][3]
2. (a)(ii)Carry out step 1 to step 9. step 1 Set up a water-bath using the beaker of water labelled water-bath and heat it to boiling, ready for step 6. step 2 In the beakers provided, prepare the concentrations of reducing sugar solution as shown in Fig. 1.3. step 3 Label five test-tubes with the concentrations you prepared in step 2. step 4 Put 2 cm³ of each reducing sugar concentration into the appropriately labelled test-tube. step 5 Put 2 cm³ of Benedict's solution into each of the test-tubes. Shake gently to mix. step 6 Put the test-tube containing 1.00% reducing sugar solution into the boiling water-bath. Start timing. step 7 Record, in (a)(ii), the time taken to the first appearance of a colour change. If there is no colour change after 120 seconds, stop timing and record the result as 'more than 120'. step 8 Remove the test-tube from the boiling water-bath. step 9 Repeat step 6 to step 8 with the remaining concentrations of reducing sugar. You will need the boiling water-bath again in step 13. Record your results in an appropriate table.[5]
3. (a)(iii)State the dependent variable in this investigation.[1]
4. (a)(iv)You will now collect results to estimate the concentration of reducing sugars in samples G1 and H1. Carry out step 10 to step 16. step 10 Label two test-tubes G1 and H1. step 11 Put 2 cm³ of G1 into the appropriately labelled test-tube. step 12 Put 2 cm³ of Benedict's solution into the test-tube. Shake gently to mix. step 13 Put the test-tube into the boiling water-bath. Start timing. step 14 Record, in (a)(iv), the time taken to the first appearance of a colour change. If there is no colour change after 120 seconds, stop timing and record the result as 'more than 120'. step 15 Remove the test-tube from the boiling water-bath. step 16 Repeat step 11 to step 15 with H1. Record your results for G1 and H1. result for G1 .............................................................. s result for H1 .............................................................. s[1]
5. (a)(v)Fig. 1.4 shows a scale of reducing sugar concentrations from 1.00% to 0.00%. [Figure 1.4] Complete the scale in Fig. 1.4 so that it shows, in the correct positions, all the reducing sugar concentrations you prepared in step 2.[1]
6. (a)(vi)Use your results in (a)(ii) and (a)(iv) to estimate the concentrations of reducing sugars in G1 and H1. Show your estimates for G1 and H1 on Fig. 1.4 by drawing arrows (↓) at the correct positions on the scale. Label one arrow G1 and the other arrow H1.[1]
7. (a)(vii)Suggest modifications to this procedure that would allow you to obtain a more accurate estimate of the concentrations of reducing sugars in G1 and H1.[3]
8. (a)(viii)A student repeated the investigation by soaking seeds from several other species of plant in sucrose solutions for 24 hours, either at a temperature of 20°C or at a higher temperature. All other conditions were kept the same. After soaking at 20 °C for 24 hours, the concentrations of reducing sugars were different for the seeds from different species of plants. However, after soaking at the higher temperature for 24 hours, the concentrations of reducing sugars were the same for all the seeds tested. Suggest and explain why, at a higher temperature, the concentrations of reducing sugars were the same for all the seeds tested.[2]
9. (b)(i)Amylase is another enzyme released by germinating seeds. A scientist investigated the release of amylase from germinating seeds by measuring amylase activity over four days. Amylase activity was measured in arbitrary units (au). The results are shown in Table 1.2. Table 1.2 time after germination /hours activity of amylase /au 0 1.6 24 7.5 48 5.4 72 2.6 96 1.7 Plot a graph of the data in Table 1.2 on the grid in Fig. 1.5. Use a sharp pencil. [Figure 1.5][4]
10. (b)(ii)Use your graph to estimate the activity of amylase at 60 hours after germination. activity of amylase = ...................................... au[1]

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