MI 2023 H2 Biology Paper 4 ANS

Class Adm No Candidate Name: This question paper consists of 19 printed pages, including 1 blank page. [Turn over] 2023 End-of-Year Examination Pre-university 3 BIOLOGY HIGHER 2 9744/04 Paper 4 Practical 29 August 2023 Candidates answer on the Question Paper 2 hour 30 minutes READ THESE INSTRUCTIONS FIRST Do not open this booklet until you are told to do so. Give details of the practical shift and laboratory, where appropriate, in the boxes provided. Write in dark blue or black pen. You may use an HB pencil for any diagrams and graphs. Do not use staples, paper clips, highlighters, glue or correction fluid/tape. Answer all questions in the spaces provided on the Question Paper. The use of scientific calculator is expected, where appropriate. You may lose marks if you do not show your working or if you do not use appropriate units. At the end of the examination, fasten all your work securely together. The number of marks is given in brackets [ ] at the end of each question or part question. Shift Laboratory For Examiner's Use 1 /20 2 /21 3 /14 Total /55

2 9744/PU3 BIO EOY/2023 Candidates with access to microscope at the start of the paper are given the first 1 hour 15 mins to use it. Please answer question 3, within this time frame. Candidates with no access to microscope at the start of t he paper will be given access 1 hour 15 minutes after the start of the paper. You may proceed with Question 1 first. Answer all questions. Question 1 You are provided with a solution labelled E containing an enzyme which coagulates (clots) milk. Enzyme E hydrolyse peptide bonds between certain amino acids in a protein found in milk and this results in the coagulation of the milk. Calcium ions are needed for this coagulation. When a mixture of milk, calcium chloride solution and E is gently turned in a test -tube, the coagulation goes through the stages shown in Fig. 1.1. Stage 3 is the end-point of the enzyme-catalysed coagulation. Fig. 1.1 You will investigate the effect of temperature on the time taken to reach the end-point. You will test the activity of enzyme E at 30oC and other temperatures up to a maximum of 50oC. You are provided with: • 70 cm3 of 100% milk, labelled M • 20 cm3 of enzyme solution, labelled E • 20 cm3 of calcium chloride solution, labelled C If C or E comes into contact with your skin, wash off immediately under water. It is recommended that you wear suitable eye protection. (a) (i) List the temperatures that you will be using to investigate the effect of temperature on the enzyme of enzyme E. You must include 30 oC and 50oC in the range of temperatures. ……………………………………………………………………………………………………… [1] 30oC, 35oC, 40 oC, 45 oC, 50oC;

3 9744/PU3 BIO EOY/2023 [Turn over] Read steps 1 to 12. Proceeds as follows. 1 Set up a water bath at 30oC to be used later in step 5. 2 Put 10 cm3 of M into a test-tube. 3 Put 1 cm3 of C into the test-tube. 4 Gently shake the test-tubes to mix M and C. 5 Put the test-tubes into the water bath and leave for 3 minutes. (ii) Explain why the test-tubes are left in the water bath for 3 minutes in step 5. …………………………………………………………………………………………………………. ……………………………………………………………………………………………………… [1] 6 Remove the test-tubes from the water bath. The process of clotting will start when E is added to the test-tube. 7 Put 1 cm 3 of E into the test-tube, so that it runs down the side of the test -tube and forms a layer on the surface of the mixture, as shown in Fig. 1.1. Fig. 1.2 To ensure temperature of milk and calcium are the same/equilibrated to prevent significant change in temperature upon adding E;

4 9744/PU3 BIO EOY/2023 8 Gently shake the test-tube to mix the solutions and start timing with a stop-watch. 9 Rotate the test-tube as shown in Fig. 1.3. Continue to rotate it while observing the mixture until the end-point shown in stage 3 of Fig. 1.1. Stop timing when small clots are observed. If stage 3 is not observed by 180 seconds, stop timing and record this as ‘more than 180’. Fig. 1.3 10 Record in (a)(iii) the time to reach the end-point. 11 Set up the water-bath at the next temperature after 30oC stated in (a)(i). 12 Repeat step 2 to step 11 for all the temperatures stated in (a)(i). (iii) Record your results in an appropriate table. Calculate the rate of reaction for each temperature and show your answer clearly in the table. [5] Temperature (oC) Time taken for clotting (s) Rate of reaction (s-1) 30 68 0.0147 35 40 0.0250 40 27 0.0370 45 20 0.0500 50 25 0.0400 1. Headings with appropriate units; 2. Record time for all temperature; 3. Highest temperature the fastest time; 4. Time taken recorded to nearest second; 5. Rate calculated and shown in same precision (3 s.f)

5 9744/PU3 BIO EOY/2023 [Turn over] In order to deduce the optimal temperature of the enzyme involved in this enzyme-catalysed reaction, the errors in the experimental procedure need to be reduced. (iv) Describe 2 significant sources of error in the procedure that reduce confidence in your result. …………………………………………………………………………………………………………. …………………………………………………………………………………………………………. …………………………………………………………………………………………………………. …………………………………………………………………………………………………………. …………………………………………………………………………………………………………. ……………………………………………………………………………………………………… [2] (v) Describe two improvements to this procedure that would enable the deduction of the optimal temperature of this enzyme involved. …………………………………………………………………………………………………………. …………………………………………………………………………………………………………. …………………………………………………………………………………………………………. ……………………………………………………………………………………………………… [2] Reduce error 1. Did not equilibrate temperature of enzyme, so temperature of enzyme may not be same as milk; 2. Different speed of rotating test tubes and this affect rate of blood clotting; 3. Lack of replicates to ensure results is reliable; 4. Subjective/difficult to assess if end point is reached 5. Temperature not maintained throughout the incubation 1. Difficult to assess if end point is reached, so either take video of all experiment and decide end-point clearly, or compare results with an image of end-point; 2. Measure mass of clotting after a fixed duration instead of measuring end-point; 3. Greater range of temperature , such as 0 to 100oC instead of 30 to 50oC; 4. Greater number of temperature, such as 10 temperatures instead of 5 temperatures) 5. Draw results on graph to derive highest rate of reaction;

6 9744/PU3 BIO EOY/2023 (b) A student carried out an investigation into the effect of enzyme concentration on the clotting of milk. The student calculated the activity of the enzyme for each concentration of enzyme. The results are shown in Table 1.1. Table 1.1 enzyme concentration (%) activity of enzyme (A.U.) 0.05 19 0.10 34 0.15 50 0.20 65 0.30 96 (i) Plot a graph of the data in Table 1.1 on the grid provided. [4] 1. x-axis: % enzyme concentration and y-axis: activity of enzyme (/) au ; 2. scale on x-axis: 0.05 to 2 cm, labelled at least every 2 cm and scale on y-axis: 20 to 2 cm, labelled at least every 2 cm ; 3. correct plotting of all five points using small crosses or dots in circles ; 4. five plots joined with thin line passing through all points (line is either smooth curve or joined plot to plot) ;

7 9744/PU3 BIO EOY/2023 [Turn over] (ii) Use your graph to determine the activity of the enzyme when the enzyme concentration is 0.25%. Show on your graph how you obtain the answer. activity of enzyme: …………………. a.u.[2] (iii) Describe and explain the trend shown in your graph. …………………………………………………………………………