Enzyme Specificity
Activity #2

Digestion of Lipids by Lipase

In this portion of the investigation, you will experiment with the enzyme lipase. This enzyme assists in the breakdown of lipids (fats are a subclass of lipids) into smaller molecules called carboxylic acids or "fatty" acids. With the production of fatty acids in the reacting solution, the pH of the solution should change to a more acidic value, that is, the pH value should decrease.

Because you will be working with a water solution, it will be hard for a fat to mix with the solvent, water. In the digestive system, the same problem exists but is solved with the body's production of a fat “mixer” or emulsifier called bile. This is produced in the liver and added to the upper portion of the small intestine in the digestive tract. It is much like soap (technically speaking, detergent) in its molecular structure and activity.

What is the pH of the small intestine where fat digestion takes place? In reading the procedural instructions, you can find out whether or not your test tube's “intestinal” solution is acidic or basic (alkaline). The formation of the breakdown products of fat digestion changes the pH of the solution and is used to determine whether or not the fat has been broken down into its subunits of fatty acid.

Materials and Apparatus

  • Vegetable oil, whole milk, or fresh cream
  • Pancreatin solution (see formula), a source of lipase
  • Bile salts
  • Phenol red solution or phenolphthalein (pH indicator solution)
  • Beral pipettes
  • Thermometer
  • Distilled water
  • 3 test tubes (18 x 150) with white patch for marking
  • Test tube rack

Procedure

  1. Set up the three test tubes in a rack and label them 1, 2, and 3.
  2. To each of the test tubes, add 5 ml of your fat source (vegetable oil, whole milk, or cream). To each test tube, add 8-10 drops of phenol red or phenolphthalein indicator. Shake well. (The color should be pink. If it is not pink, add 0.01 M NaOH solution drop-wise until a pink color is obtained)
  3. To test tube #1, add 5 ml of distilled water and a “pinch” of bile salt (an amount that would fit on the broad end of a flat tooth pick). What is the function of bile salt?
  4. To test tube #2, add 5 ml of pancreatin solution and a pinch of bile salts.
  5. To test tube #3, add 5 ml of pancreatin solution.
  6. Shake each tube to mix well. All tubes are placed in a beaker containing warm water in the range of 35ºC–40ºC. Watch for any color changes (some may be rapid). RECORD THE TIME REQUIRED FOR CHANGE IN COLOR (alkaline to acidic; for phenol red, pink to yellow; and for phenolphthalein, pink to colorless). What chemical product is responsible for changing the pH from alkaline to acidic?

Data Analysis

  1. What is the purpose of setting up the particular combination of ingredients in test tube #1? Compare with test tubes #2 and #3? What is being tested in tube #1?
  1. What is the reason for either adding bile salts or omitting them as in test tubes #2 and #3?
  1. What do your test results show in terms of bile salts—are they needed for fat digestion?
  1. Find the molecular structure of animal fat. What other molecule besides a fatty acid is produced when a fat molecule is “digested” (chemically speaking, hydrolyzed) in the presence of a lipase?