Digestion of Protein Lab

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Teacher:	________________________________	Period:	___________

Aim
To illustrate the effects of pH on the activity of an enzyme.

Materials
test tubes, test tube holders, a test tube rack, a ruler, a boiling water bath, 1x1 cm squares of exposed and developed film (equally dark all over), distilled water, .5% pepsin solution, .1% NaOH (sodium hydroxide) solution, .8% HCl (hydrochloric acid) solution, pH sticks, and markers.

Overview
Pepsin is an enzyme found in the highly acidic environment of the stomach. It is used to break down proteins into smaller molecules called polypeptides. In this experiment, you will measure the activity of pepsin based upon the change of transparency in the film (which contains proteins that the pepsin will break down).

Procedure

1. Number five test tubes near the top with a marker. Make a mark on each tube 4 cm and 8 cm from the bottom.
2. To tube #1, add distilled water to the first mark and .8% HCl to the second mark.
3. To tube #2, add .5% pepsin to the first mark. Place the tube in the boiling water bath, making sure that the water in the tube is below that water level of the boiling water. After 10 minutes, remove the tube, allow it to cool, and then add .8% HCl to the second mark.
4. To tube #3, add .5% pepsin to the first mark and .1% NaOH to the second mark.
5. To tube #4, add .5% pepsin to the first mark and distilled water to the second mark.
6. To tube #5, add .5% pepsin to the first mark and .8% HCl to the second mark.
7. Using pH sticks, approximate and record the pH of the solution in each of the test tubes by carefully tilting each tube and dipping the pH stick into the solution. Make sure you use a different pH stick for each test tube. Place the used sticks in a special beaker on the side for disposal.
8. To each of the five tubes, add a 1x1 cm square of film. You may have to shake the tubes gently to ensure that the film sinks to the bottom.
9. Place all of the tubes in a 37 degree Celsius water bath. Remove the tubes after 15 minutes.
10. Place the tubes in a test tube rack. Check them every 10 to 15 minutes until the dark color on one of the squares of film has almost disappeared.
11. Carefully remove the film from each tube. Assign each film a number, 1 through 5, where 1 is the least amount of coating and 5 is the most coating. Fill in this information in the chart below. Then, assign a similar rating for the relative pepsin activity, where 1 is the least activity and 5 is the greatest activity.

Observations
Record the data you gathered in the table below.

Pepsin activity
Tube #	Contents of tube	pH	Relative amount of coating	Relative pepsin activity
1
2
3
4
5

Questions

1. In this experiment, what was tube #1's purpose?
   
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2. Did the pepsin in tube #2 have a low activity? If it did, why?
   
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Conclusions
In the space below, write down any conclusions you can draw from this experiment. Include your initial hypothesis and reasoning as to which tubes would have the greatest and least activity of pepsin, and whether or not the data you gathered supported this hypothesis.

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Sources of Error
Identify any possible sources of error which may have affected the results of this experiment.

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