You are provided with a solution labelled E containing an enzyme which coagulates (clots) milk. Enzyme E hydrolyses (breaks) 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 milk will coagulate, producing lumps of a white solid in a liquid. The end-point of the enzyme-catalysed coagulation is when all the milk coagulates, as shown in [Figure 1.1]. You will investigate the effect of temperature on the time taken to reach the end-point. You are provided with the materials shown in Table 1.1. Table 1.1 labelled | contents | hazard | volume/cm³ M | 100% milk | none | 150 E | enzyme solution | harmful irritant | 20 C | calcium chloride solution | harmful irritant | 20 If C or E comes into contact with your skin, wash off immediately under cold water. It is recommended that you wear suitable eye protection. You will test the activity of enzyme E at 30°C and other temperatures up to a maximum of 50°C. Carry out step 1 to step 14. step 1 Set up a water-bath ready for step 6. The starting temperature of the water-bath should be 30°C, as shown in Table 1.2. step 2 Put 10 cm³ of M into a test-tube. step 3 Repeat step 2 so that you have two test-tubes containing M. step 4 Put 1 cm³ of C into each test-tube. step 5 Gently shake the test-tubes to mix M and C. step 6 Put the test-tubes into the water-bath and leave for 3 minutes. step 7 Remove one of the test-tubes from the water-bath. The process of coagulation will start when E is added to the test-tube. step 8 Put 1 cm³ 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 [Figure 1.2]. step 9 Gently shake the test-tube to mix the solutions and start timing. step 10 Rotate the test-tube and continue to rotate it while observing the mixture until the end-point is reached. Stop timing when the end-point is reached. If the end-point has not been reached by 180 seconds, stop timing and record this as 'more than 180'. step 11 Record in (a)(iii) the time to reach the end-point. step 12 Repeat step 7 to step 11 with the other test-tube in the water-bath. step 13 Set up the water-bath at the next temperature after 30°C stated in Table 1.2. step 14 Repeat step 2 to step 13 for each temperature stated in Table 1.2.