2024 RI H2 Bio P4 MS

©RI 2024 Preliminary Examination 9744/04 [Turn over RAFFLES INSTITUTION 2024 Year 6 Preliminary Examination Higher 2 BIOLOGY 9744/04 Paper 4 Practical 15th August 2024 2 hours 30 minutes Candidates answer on the Question Paper. Additional Materials: As listed in the Confidential Instructions . READ THESE INSTRUCTIONS FIRST Write your index number, CT group and name on all the work you hand in. Give details of the practical shift and laboratory, where appropriate, in the boxes provided. Write in dark blue or black pen. You may use a 2B pencil for any diagrams or graphs. Do not use staples, paper clips, highlighters, glue or correction fluid. Answer all questions in the spaces provided on the Question Paper. The use of an approved 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, hand in the Question Paper conta ining your answers. The number of marks is given in brackets [ ] at the end of eac h question or part of the question. This document consists of 19 printed pages and 1 blank page. CIVICS GROUP CANDIDATE NAME INDEX NUMBER 4 S 0 3 2 For Examiner’s Use 1 2 3 Total Shift Laboratory Raffles Institution Internal Examination

2 ©RI 2024 Preliminary Examination 9744/04 For Examiner’s Use Answer all questions. 1 You are advised to read the whol e of the question before starting the practical work, as you will need to make decisions about how to obtain high quality results using the apparatus and materials provided. You are going to investigate the clotting of milk. Young mammals are fed on milk. To enable efficient digestion of milk it is clotted in the stomach using the protease enzyme, rennin. Rennin converts the soluble protein caseinogen in the milk to an insoluble form called casein. The manufacture of cheese also requires the clotting of milk. This can be done in two ways by:  acidifying the milk  using the enzyme, rennin You are provided with the materials shown in Table 1.1. Table 1.1 labelled contents hazard M 100% milk none E 1% rennin harmful irritant A 1 mol dm-3 hydrochloric acid corrosive If E or A comes into contact with your skin, wash off immediately under cold water. It is recommended that you wear suitable eye protection. In Part 1 you will observe the clotting of milk using two methods: Method 1 involves the use of A and Method 2 involves the use of E. In Part 2 you will investigate the effect of various temperatures on the clotting activity of E.

3 ©RI 2024 Preliminary Examination 9744/04 [Turn over For Examiner’s Use Part 1 – observing clotting Method 1 – using acid 1. Add 5 cm3 of M to a test-tube. 2. Add 1 cm3 of A into the same test-tube. 3. Put a bung into the test-tube and mix the contents by inverting the test-tube three times. 4. Tilt the tube and rotate it slowly such that the contents touch all of the internal glass surfaces as shown in Fig. 1.1. Do not shake the tube. Fig. 1.1 (a) Record your observations. [2] 1. larger white / creamy solid precipitate in supernatant/ liquid; A: white clotted milk A: white clumps A: white suspension 2. small white / creamy specks stick to wall of test-tube ; 3. supernatant / liquid, becomes less opaque/ becomes translucent/ turns clearer; R: white liquid or solution / clear solution 5. Keep the test-tube for later reference.

4 ©RI 2024 Preliminary Examination 9744/04 For Examiner’s Use Method 2 – using rennin 1. Prepare and maintain a water-bath at 55 °C. 2. Put 5 cm3 of M into a test-tube. 3. Put 1 cm3 of E solution into a separate test-tube. 4. Place both test-tubes in the 55 °C water bath for 3 minutes. It is important to maintain the temperature of the water bath during the entire 3 minutes. 5. Add the 5 cm3 of M into the test-tube containing E. 6. Put a bung into the test-tube and mix the contents by inverting the test-tube three times and immediately start the stopwatch. 7. Tilt the tube and rotate it slowly such that the contents touch all of the internal glass surfaces as shown in Fig. 1.1. Do not shake the tube. 8. Continue to rotate the tube and record the time taken for clott ed milk particles to first start to form on the walls of the tube as shown in Fig. 1.2. (b) Time taken for the milk to clot. ………7………s [1] A:7-78s Fig. 1.2 9. Keep the test-tube for later reference. (c) Suggest the advantage of adding the milk to the rennin solution rather than adding the rennin solution to the milk. [1] 1. the volume remaining in the milk test-tube is a lower propor tion of the total volume / ORA ; 2. smaller percentage error;

5 ©RI 2024 Preliminary Examination 9744/04 [Turn over For Examiner’s Use (d) Use the universal indicator paper to test the pH of:  m i l k  milk with acid (use the test-tube from Method 1 step 5)  milk with rennin solution (use the test-tube from Method 2 ste p 9)  rennin solution Record your results in the space below and explain the advantag e of carrying out this test before investigating the effect of concentration of rennin on the clotting of milk.[2] pH of milk:…………7…………………….…….. pH of milk with acid:……2……….…….……… pH of milk with rennin:……7………………….. pH of rennin: …………6………………………. A: 8, 2, 8, 7/6 Explanation: Since the pH of the milk and renin is the same as the pH of milk, clotting is not due to the acidity* of rennin but due to its enzymatic activity; (e) Caseinogen molecules are hydrophilic and therefore soluble in w ater. When formed, they are incorporated, along with certain other molecules and i ons, into spherical structures called micelles. Each micelle has a hydrophobic core and a stabilising outer layer of hydrophilic caseinogen molecules. During clotting the micelles are broken open and their contents clump together to form clots. Suggest why the contents tend to clump together.[1] 1. the hydrophobic contents come together due to hydrophobic in teractions and are shielded from water by clumping ; (f) Suggest why the time taken for clotting was different in the two methods.[3] 1. Clotting was faster with the acid/ in Method 1; 2. [Method 1] H+ ions / protons which interact with charged or ionisable amino acid side chains / R groups* on protein, disrupting the (hydrogen and ionic) bonds in the protein, leading to denaturation / clotting ; 3. [Method 2] Rennin activity r elies on (time needed for) successful effective collisions between substrate and active site / forming enzyme-substrate complex with caseinogen 4. Larger number of protons compared to enzymes; 1mark;

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