Some fruits contain protease enzymes. These enzymes can denature the proteins in milk, causing the milk to clot. You will investigate the effect of protease concentration on the time taken for milk to clot. You will use your results to estimate the concentration of protease in a fruit extract. You are provided with the materials shown in Table 1.1. Table 1.1 labelled | contents | hazard | volume/cm³ --- | --- | --- | --- P | 100% protease solution | none | 50 M | milk | none | 30 W | distilled water | none | 100 U | fruit extract containing unknown protease concentration | none | 20 If any solution comes into contact with your skin, wash off immediately under cold water. It is recommended that you wear suitable eye protection. You will need to make different concentrations of protease solution, using proportional dilution of the 100% protease solution, P. You will need to prepare 10cm³ of each concentration, using P and W. Table 1.2 shows how to prepare one of the concentrations of protease you will use. Decide which other concentrations of protease you will use. Carry out step 1 to step 8. step 1 Stir the 100% protease solution, P. In the beakers provided, prepare the concentrations of protease as shown in Table 1.2. step 2 Label test-tubes with the concentrations of protease stated in Table 1.2. step 3 Put 2 cm³ of milk, M, into each labelled test-tube. step 4 Put 1 cm³ of the 100% protease solution, P, into the appropriately labelled test-tube. Start timing. step 5 Hold the test-tube at an angle and slowly rotate the test-tube as shown in [Figure 1.1]. Hold a piece of black card behind the test-tube and observe the thin layer of milk on the side of the test-tube. step 6 As soon as a number of small clots appear, stop timing and record the value in (a)(ii). If there are no clots after 180 seconds, stop timing and record as 'more than 180'. milk drains back smoothly from the sides of the test-tube small clots stick to the sides of the test-tube no clotting clotting [Figure 1.1] step 7 Repeat step 4 to step 6 with each of the other concentrations of protease you prepared in step 1. Record your results in (a)(ii). step 8 Repeat step 2 to step 7 using clean test-tubes.

1. (a)(i)Complete Table 1.2 to show how you will prepare the concentrations of protease you will use. Table 1.2 percentage concentration of protease | volume of P /cm³ | volume of W /cm³ --- | --- | --- 100 | 10.0 | 0.0[2]
2. (a)(ii)Record the two sets of results in an appropriate table.[5]
3. (a)(iii)Suggest one source of error in the procedure described in step 6 of this investigation.[1]
4. (a)(iv)Suggest why the procedure was repeated.[1]
5. (a)(v)To estimate the concentration of protease in fruit extract U, you will need to test a sample of the extract. State the volume of fruit extract U that you will use. volume = ..................................................... cm³[1]
6. (a)(vi)Record the time taken for clots to appear using fruit extract U. time taken = ....................................................[1]
7. (a)(vii)Use your results from (a)(ii) and (a)(vi) to estimate the concentration of protease in fruit extract U. concentration of protease in fruit extract U = ...................................................... %[1]
8. (a)(viii)With reference to your estimate in (a)(vii), suggest how you would modify this procedure to obtain a more accurate value for the concentration of protease in fruit extract U.[2]
9. (b)(i)Plot a graph of the data shown in Table 1.3 on the grid in [Figure 1.2].[4]
10. (b)(ii)Use the data in Table 1.3 and your graph in [Figure 1.2] to explain the effect of pH on the activity of protease.[3]

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