The degradation of albumen using pepsin
Pepsin, and Trypsin are two enzymes that degrade proteins found in our food stuff. Each of the these enzymes cleave the proteins at a different location and together they degrade the proteins to short peptides and to the building blocks: amino acid, which are readily absorbed by the intestine lining.
Pepsin is produced in the mucosal lining of the stomach. It is secreted in an inactive form, trypsinogen, and are then converted to an active form, pepsin, under very low pH conditions: 1.0-3.0. Pepsin optimal activity is obtained at this range of pH. Pepsin is used in the preparation of cheese and other protein-containing foods.
In this experiment, degradation of egg white proteins by pepsin is followed using a colorimeter. Egg white proteins are first heated to create a turbid solution. Upon degradation the solution becomes clear.
- One Egg White
- 100ml 0.2N HCl solution
- 20ml of 0.5% Pepsin solution
- Bunsen burner
- Thermometer (or a Temperature sensor)
- 400-600ml flask
- 5ml and 1ml pipettes
- Stand with 10 tubes
- colorimeter. Digital or analogue
Before commencing this practical you should be familiar with the set up and operation of a colorimeter.
Prepare the Egg White Solution then add 40ml distilled water to 10ml Egg white. Mix it rapidly with a fork and filter it through a few layers of muslin cloth. Heat the solution up to 55-60°C (not above this temperature!!) with constant stirring till a turbid solution is obtained. At this stage the solution should resemble diluted milk. This solution is the substrate used in the experiment. Keep it in a small flask.
Continue as follows;
- Calibrate the colorimeter using. Use the Red Filter (~650nm)
- Prepare a blank solution (ie., no albumen present): to 3ml distilled water, add 1ml enzyme solution.
- Pour the blank into a cuvette and insert it into the colorimeter.
- Close the cover well.
- Adjust your colorimeter to give 100% transmission.
- Measure the pH of the reaction solution
- Add to a tube - 2.4ml Egg white solution, 0.6ml 0.2N HCl, 1.0ml of water
- Measure the pH of the solution using the pH probe. It should be in the range of 2.0-3.0 If necessary, adjust the pH by changing the volume of 0.2N HCl you add.
- Measure Rate of Protein Degradation:
- Add to the cuvette - 2.4ml Egg white solution 0.6ml and 0.2N HCl
- Add to the cuvette 1ml Enzyme solution.
- Mix well with a small wooden stick and insert the cuvette immediately into the colorimeter. Close the cover well.
Follow changes in light transmittance registered on the colorimeter display during the experiment.
Repeat above with at least 2-4 different enzyme concentrations.
Data Analysis. The rate of Protein degradation is calculated from the rate of change in
- Finding the optimum pH for two protease enzymes (trypsin and pepsin) A slightly different version to the one described above.
- Test for Proteins
--Ssmith 10:11, 28 March 2007 (BST)