Pepsin is an enzyme that is present in gastric juice. Gastric juice is secreted into the stomach of humans and many other animals. Pepsin catalyses the hydrolysis of proteins into peptides, as shown in Fig. 1.1. [Figure 1.1] Egg albumen (egg white) contains a high proportion of protein. 10% albumen solution is a cloudy-white colour. This becomes colourless when pepsin is added to the 10% albumen solution. A student used a colorimeter to follow the progress of the hydrolysis of protein by pepsin.

1. (a)(i)The student used a blue filter in the colorimeter. Outline one other step the student should carry out to prepare the colorimeter so that correct measurements of absorbance can be obtained.[1]
2. (a)(ii)After preparing the colorimeter, the student: • made a pH2.0 albumen solution by mixing 10cm³ of 10% albumen solution with 10 cm³ of pH2.0 buffer solution • added 3.0 cm³ of the pH2.0 albumen solution to a colorimeter tube • added a small volume of pepsin solution to the colorimeter tube • immediately placed the colorimeter tube in the colorimeter • recorded the absorbance of the mixture every 20 seconds for 5 minutes • calculated the rate of change in absorbance as a measure of the rate of protein hydrolysis. The results are shown in Fig. 1.2. [Figure 1.2] Use Fig. 1.2 to calculate the rate of change in absorbance at pH2.0 between 40s and 120s. Show your working and include appropriate units. rate of change in absorbance at pH2.0 = .....................................................[3]
3. (b)(i)The student decided to investigate the effect of pH on the rate of protein hydrolysis by pepsin. From the internet the student found out that pepsin is inactive at pH 6.5 and above. The student was provided with a colorimeter and standard laboratory apparatus. Describe a method that the student could use to investigate the effect of pH on the rate of protein hydrolysis by pepsin and to determine the optimum pH of pepsin. Your method should be set out in a logical order and be detailed enough to allow another person to follow it. Details of how to prepare and use the colorimeter should not be included.[7]
4. (b)(ii)Complete the sketch graph in Fig. 1.3 to predict the effect of pH on the rate of protein hydrolysis by pepsin. Include a label for the y-axis in your answer. [Figure 1.3][2]
5. (c)(i)Laryngopharyngeal reflux (LPR) is a disease that occurs when gastric juice from the stomach moves up the oesophagus and into the larynx, as shown in Fig. 1.4. [Figure 1.4] Gastric juice can damage the laryngeal epithelium. Gastric juice contains hydrochloric acid and pepsin. Some scientists identified a reduction in the quantity of two proteins, CA3 and Sep70, present in the laryngeal epithelium of people with LPR. The scientists obtained 6 sections of mammalian laryngeal epithelium to investigate how different test conditions affect the quantity of CA3 and Sep70 in the laryngeal epithelium. Each section of laryngeal epithelium was exposed to different test conditions for 20 minutes, as shown in Table 1.1. Pepsin is inactive in the presence of the inhibitor, pepstatin. [Table 1.1] Proteins were then extracted from the sections of laryngeal epithelium. The proteins were separated using electrophoresis. CA3 and Sep70 were identified using fluorescent antibodies. Fig. 1.5 shows the results of the protein electrophoresis. [Figure 1.5] Use Table 1.1 and Fig. 1.5 to state the conclusions that can be made from the results of the protein electrophoresis.[2]
6. (c)(ii)The results of this investigation were published in a scientific paper. A student who read the scientific paper concluded that pepsin causes damage to the laryngeal epithelium in people with LPR. Suggest why the results of this investigation might not support this conclusion.[2]

This question uses a diagram from the original paper, which isn't shown here. Open the full paper from the past papers archive to see it.