Question 121 marks
Question
Sucrase is an enzyme which hydrolyses the disaccharide sucrose into reducing sugars, as shown in Fig. 1.1. sucrase sucrose reducing sugars Fig. 1.1 The progress of this reaction can be followed by measuring the concentration of reducing sugar produced. To do this, samples can be taken at time intervals and the action of sucrase stopped in the sample. The concentration of reducing sugar in the sample can then be tested using Benedict’s solution and compared to known concentrations of reducing sugar. You are provided with the materials shown in Table 1.1. Table 1.1 volume labelled contents hazard / cm3 R 2.0% reducing sugar solution none 50 harmful B Benedict’s solution 25 irritant W distilled water none 150 unknown concentration of reducing harmful U 2 sugar, sampled at 2 minutes irritant If any solution comes into contact with your skin, wash off immediately under cold water. It is recommended that you wear suitable eye protection. (a) You need to carry out a serial dilution of the 2.0% reducing sugar solution, R, to reduce the concentration by half between each successive dilution. Fig. 1.2 shows the first two beakers you will use to make your serial dilution. (i) Complete Fig. 1.2 by drawing as many extra beakers as you need for your serial dilution. For each beaker: • state, under the beaker, the volume and concentration of reducing sugar solution available for use in the investigation • use one arrow with a label, above the beaker, to show the volume and concentration of reducing sugar solution added to prepare the concentration • use another arrow with a label, above the beaker, to show the volume of W added to prepare the concentration. 0.0 cm3 of W 20.0 cm3 of …… 2.0% reducing …… sugar solution, R 10.0 cm3 of 2.0% reducing sugar solution, R, available for use Fig. 1.2 [3] Carry out step 1 to step 8. 1. Set up a water-bath and heat to boiling ready for use in step 6. 2. Prepare the concentrations of reducing sugar solution decided in (a)(i) and shown in Fig. 1.2. Use a glass rod to mix the reducing sugar solutions and water. 3. Label test-tubes with the concentrations of reducing sugar solution prepared in step 2. 4. Put 2 cm3 of each concentration of reducing sugar solution into an appropriately labelled test-tube. 5. Put 2 cm3 of Benedict’s solution, B, into each of these test-tubes. Shake gently to mix. 6. Put the test-tube labelled 2.0% into the boiling water-bath. Start timing. 7. Measure the time taken to the first colour change. Record the result in (a)(ii). If there is no colour change after 90 seconds, record as ‘more than 90’. 8. Repeat step 6 and step 7 using each of the concentrations of reducing sugar solution you prepared in step 2, instead of 2.0%. Record your results in (a)(ii). (ii) Record your results in an appropriate table. [5] You are provided with an unknown concentration of reducing sugar solution in the test-tube labelled U. 9. Put 2 cm3 of B into the test-tube labelled U. 10. Repeat step 6 to step 7, using the test-tube labelled U instead of 2.0%. Record your result for U in (a)(iii). (iii) Record your result for U. result for U = …… [1] (iv) Using your results in (a)(ii) and (a)(iii), estimate the concentration of reducing sugars in U. U = …… [1] (v) Suggest how you would make improvements to this investigation to obtain a more accurate estimate of the concentration of reducing sugars in U. …… [2] (b) A student wanted to determine the Michaelis-Menten constant (Km) for sucrase during the hydrolysis of sucrose, as shown in Fig. 1.1. The student measured the initial rate of reaction at different concentrations of sucrose. The results are shown in Fig. 1.3. 5.0 4.0 3.0 initial rate of reaction / arbitrary units 2.0 1.0 0 5 10 15 20 25 30 40 concentration of sucrose / mmol dm–3 Fig. 1.3 (i) Use the graph in Fig. 1.3 to estimate the Michaelis-Menten constant (Km). Show your working on the graph and in the space below. Km = …… mmol dm–3 [3] (ii) The Km value for another enzyme, Z, is 0.95 mmol dm–3. State which enzyme, Z or sucrase, has a lower affinity for its substrate. Give a reason for your answer. enzyme …… reason …… …… [1] (iii) Explain why the initial rate of reaction does not increase between 30 mmol dm−3 and 40 mmol dm−3 of sucrose. …… [1] (c) A scientist carried out some research into the sugar content of five different fruit juices. The results are shown in Table 1.2. Table 1.2 concentration of sugar type of fruit juice / arbitrary units red grape (RG) 21.25 white grape (WG) 18.50 orange (OR) 11.75 pineapple (PA) 10.25 grapefruit (GF) 14.00 Plot a bar chart of the data in Table 1.2 on the grid in Fig. 1.4. Use a sharp pencil for drawing bar charts. Fig. 1.4 [4] [Total: 21]
Mark scheme
1(a)(i) 1 labels under correct sequence of beakers: 1.0, 0.5, 0.25, 0,125 and % at least once ; 3 2 shows transfer of 10 (cm3) from previous beaker transferred to the next beaker in sequence and cm3 once ; 3 shows 10 (cm3) of, W / water, added to each beaker and cm3 once ; 1(a)(ii) 1 heading for independent variable: percentage concentration sugar and before heading for dependent variable) and no 5 units in body of table ; 2 heading for dependent variable: time and s(econds) and no units in body of table ; 3 times for all concentrations ; 4 correct trend in results ; 5 results recorded to nearest whole second ; 1(a)(iii) records the time for U and s(econds) ; 1 1(a)(iv) estimates the correct concentration of reducing sugar in U from the results given in 1(a)(ii) ; 1 1(a)(v) any two from: 2 1 using more concentrations of reducing sugar between two concentrations that lie either side of the estimate ; 2 (dilutions made) using proportional dilution ; 3 plot a graph of known concentrations and read off the value of sample ; 1(b)(i) 1 reads off Vmax correctly from the graph at 4.3 au ; 3 2 calculates ½ Vmax correctly ; 3 reads off the value for Km mmol dm–3 correctly from the graph ; 1(b)(ii) identifies enzyme sucrase has a lower affinity for its substrate and Km higher for sucrase ; 1 1(b)(iii) all active sites are occupied by substrate / AW ; 1 1(c) 1 x-axis: type of fruit juice 4 and y-axis: concentration of sugar / a.u ; 2 scale on x-axis: even width of bars and scale on y-axis: 5 a.u to 2 cm., labelled at least every 2 cm and origin at zero ; 3 correct plotting of all bars ; 4 separate bars drawn with horizontal and vertical lines joined precisely ;