Experiment: Protein and Pepsin Digestion

The purpose of this lab is to determine the optimum conditions for digestion in the stomach. This lab will focus on chemical digestion by gastric fluid, using pepsin and egg along with either an acid, a base, or a neutral solution to model conditions in the stomach.

Materials:

1. -1 tube of pH paper
2. -16 mL of 1% pepsin
3. -4 mL of 0.2% hydrochloric acid
4. -4 mL of 1% sodium bicarbonate
5. -4 mL of distilled water
6. -4 test tube stoppers
7. -4 test tubes
8. -8 grams of boiled egg white
9. -Balance (to measure the egg)
10. -Graduated cylinder
11. -Pencil (for marking test tubes)
12. -Scalpel (to cut pieces of egg)
13. -Test tube rack

Discussion:

As shown in Figure 1, each test tube produced different results. Some had a murky solution, some had condensation on the side of the test tube, and other had the egg float in the solution. Every test tube had at least minute dissolution of the egg, likely due to time spent in the solution. However, while the egg dissolved very slightly in Test Tubes A, C, and D, the egg dissolved by far the most in Test Tube B. Gastric fluids are made up of mucus, hydrochloric acid, and pepsinogen (Holt, Rinehart, Winston, 2009, page 988). Pepsinogen is inactive until hydrochloric acid converts it into the digestive enzyme pepsin (Holt, Rinehart, Winston, 2009, page 988). Also, pepsin needs to be at a low pH, approximately 2, for digestion to occur most effectively, and that is why Test Tube B had its egg dissolve the most ("Experiment 10-Enzymes", 2012). The egg would partially dissolve in the water on its own, as it would in the stomach, but the rate of dissolution is much slower without pepsin, and it is inactive without the hydrochloric acid to convert it from pepsinogen. That is why Test Tubes A, C, and D saw their egg dissolve slightly. Sodium bicarbonate is a base, and Test Tube A is therefore too far away from the optimum pH of 2 for the egg to be dissolved by the pepsin effectively. Distilled water and the control group are neutral, and Test Tube D and C, respectively, are therefore too far away from the optimum pH of 2 for the egg to be dissolved by the pepsin effectively. Hydrochloric acid is an acid, and Test Tube B is therefore close enough to the optimum pH of 2 for the egg to be dissolved by the pepsin effectively. The group hypothesis was: if distilled water is added to a solution of pepsin and egg, then it will digest the egg more effectively than the acid (hydrochloric acid) or the base (sodium bicarbonate) because food must be broken down by hydrolysis in order to be digested. The data rejected the hypothesis. The group did not realize that the enzyme can complete hydrolysis on its own, and it was not until the end of the lab that the optimum conditions for pepsin were realized. Then, the students saw why the hydrochloric acid dissolved the egg most efficiently, not the distilled water. Biuret's solution is used to test for the presence of protein. The reagent is at first blue, but turns violet if it detects the presence of peptide bonds, the chemical bonds that attach amino acids to each other (Bank, n.d.). While the amount of proteins being digested in the body is more than in the test tube, proportionally the surface area of the organs involved in digestion and absorption (for example the villi of the small intestine) is much larger. There are more digestive enzymes and the reaction is catalyzed much faster. In the test tube, the egg comprised a large part of the test tube. Inside the human body, even if an entire egg was eaten, the size of the egg would still be dwarfed by the amount of digestive enzymes and secretions. Also, the body has the optimum environment for pepsin (pH of 2), which would help the digestive process move much faster. After the body has broken down protein (long chains of amino acids) into peptides and amino acids, they are broken down into individual amino acids and absorbed into the bloodstream via the villi, the highly folded lining of the small intestine (Holt, Rinehart, Winston, 2009, page 990). The conditions for pepsin to break down protein are the same as any other enzyme: an optimum temperature (that of the body, 98.6 degrees Fahrenheit) and an optimum pH (in the case of pepsin, 2, the pH of the stomach). Any variations outside of these optimum values will result in decreased pepsin digestive performance. Therefore, in Test Tubes A, C, and D, it makes sense that the egg was not dissolved to the same extent of Test Tube B, because they were not near pepsin's optimum pH range of 2.

Conclusion:

This lab was conducted to teach the students about the digestion, the current unit in class, and to reinforce the material learned earlier in the year on enzymes and their optimum conditions. It also gave the students a better understanding of what happens to food in their body after the students ingest it, and how the students digest it and get nutrition from it. The participants in the lab observed how the hydrochloric acid converted the inactive pepsinogen into the digestive enzyme pepsin, and how the pepsin, when at its optimum pH of 2, dissolved the egg over the course of several days. The control group gave a basis for comparison, and showed how much a difference the presence of an acid was in dissolving the egg. There were some possible sources of error, however. The temperature in the test tubes should have been 98.6 degrees Fahrenheit, as it is in the body, but it was room temperature, which was likely different. This would matter because pepsin's optimum temperature is 98.6 degrees Fahrenheit, so with the proper temperature likely the egg would have been dissolved to a greater extent. The size of the egg, although weighed before being put in the test tubes, were not exact and could have affected the rate of dissolution by the pepsin. The test tubes were exposed to oxygen when the test tube stoppers were taken off, albeit briefly, for pH readings. Also, the amount of solution in Test Tube B was slightly less than Test Tubes A and D. Lastly, there was 4 mL less solution in the control group (Test Tube C) due to the lack of an independent variable. However, the egg size was the same, so most probably the rate of dissolution was severely hampered due to the large difference in the amount of digestive enzymes in the test tube.