Science

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GES 102 – ENZYME LAB In this lab you will investigate an enzyme called lactase and test whether its activity depends on temperature. The enzyme lactase breaks down lactose, a double sugar found in dairy products, into single sugars. People who are lactose-intolerant lack this enzyme, or its activity is greatly diminished. You will use the production of the single sugar glucose as a measure of lactase activity. This will be done by using a test strip that changes color in the presence of glucose. In addition you will investigate the effect of pH on the activity of the enzyme pepsin, which helps break down protein in your stomach. A demonstration has been set up for you in the lab where the ability of pepsin to digest egg white protein is tested in solutions with different pH. Background: All chemical reactions start with molecules called reactants, which are turned into products. Today, for example, you will use the double sugar lactose as the reactant, and it will be broken into two singles sugars, the products. Regardless of whether the overall reaction releases energy or requires it, every reaction needs a threshold energy amount to get started, called the activation energy. In organic chemistry one often adds heat energy to make the reactions occur more rapidly. This would be impossible inside living cells, however. The cells have to be very frugal about energy use and the heat might even kill the cell. So how can a large numbers of reactions take place in a cell with a minimum of energy expenditure and often at room temperature? Enzymes make it possible. Enzymes are protein molecules, made up of long chains of amino acids that fold together to form a very specific shape. One part of the enzyme molecule, the active site, perfectly matches the molecule it is intended to work on, called the substrate. The substrate binds

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temporarily to the active site, which provides a perfect environment for the substrate to undergo the chemical reaction. Note that the enzyme doesn’t add any energy, it only lowers the activation energy, and the reaction can proceed immediately. Enzymes are usually very fast in their action. A substrate will be worked on, released and another substrate molecule can then bind to the active site. The enzyme is not changed at all by this process (it serves like a catalyst), and can be used over and over continuously. Enzymes are often named in a specific way, ending with –ase, and often the name is related to their particular substrate. For example, you will be working with lactase today, which is the enzyme that has the double sugar lactose as its substrate. As mentioned above, enzymes can only work on substrates that fit perfectly into the active site. If the shape of the enzyme changes, it would be less able to interact with its substrate. The three-dimensional shape of enzymes (as for any proteins) depend on weak bonds, such as hydrogen bonds, and this make their shape sensitive to temperature, salt and pH. There are enzymes that work fine in boiling water, and others that have the perfect shape in strong acids. However, when you move enzymes into an environment that is different from what they were engineered for, they will become less efficient. If the environment is changed drastically enough, enzymes may irreversibly lose their shape, and they are then said to be denatured. This is true for all proteins. You would expect enzymes to have an optimum temperature and pH, and you would also expect this to coincide with the environment in which they are naturally found. In this week’s lab, you will investigate the effect of temperature on the activity of lactase, and the effect of pH on the activity of pepsin. Materials: Lactase experiments:

• Test tube rack • 7 test tubes • 5 mL Pipettes • Disposable pipettes • Dairy milk • Lactase solution, fresh • Lactase solution, boiled • Sucrose solution • Glucose solution • Glucose strips (with color chart) • For whole section: water bath at 37 degree Celsius; ice bucket

Pepsin demonstration:

• Test tube with egg white in distilled water • Test tube with egg white and pepsin in distilled water • Test tube with egg white in pH 2 solution

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• Test tube with egg white and pepsin in pH 2 solution Experiment: ACTIVITY OF LACTASE: Please start with the lactase experiments, You can then evaluate the pepsin demonstration, while you are waiting. Lactase breaks down lactose (a double sugar) to glucose and galactose (single sugars). You will check for the presence of glucose in a test tube by using glucose strips, they will react to glucose only. Since enzymes are very specific, you would not expect lactase to be able to break down other double sugars, such as sucrose (table sugar). Obtain 7 test tubes, number them from 1-7, label them with your initials and put them in your test tube rack.

1. Put 5 mL of glucose solution into the first test tube. Be very careful to use only the pipette marked Glucose for this purpose. Glucose is the product we will be looking for and it must not get into any of your other test tubes by mistake.

2. Put 5 mL of sucrose solution into test tube 2.

3. Put 5 mL of dairy milk into test tube 3.

Test Tube 1: Glucose solution only Test Tube 2: Sucrose solution only Test Tube 3: Dairy milk only

Dip glucose strips into each of these test tubes and compare their color to the color chart on the container. The first test tube should obviously test positive. What about test tubes 2 and 3? Why is this? Note that sucrose is also a double sugar, and if it were to be broken down, one of the products is glucose. You now have a baseline and are ready to start your experiments.

4. Use a pipette to add 5 mL of dairy milk to test tubes 4-7. You are now going to add some lactase solution to these test tubes. Make sure to mix the contents of each of test tubes 4-7 thoroughly by wearing disposable gloves and holding your finger over the opening while inverting the test tube a couple of times. Record the starting time.

Test Tube 4: Dairy milk + 2 drops of lactase solution and place in the test tube rack Test Tube 5: Dairy milk + 2 drops of lactase solution and place in the ice bucket Test Tube 6: Dairy milk + 2 drops of lactase solution and place in the warm water bath Test Tube 7: Dairy milk + 2 drops of boiled lactase solution and place in test tube rack

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(make sure to use the boiled lactase already made for you) If you have not already done so, record your expectations from each of the Test Tubes. You will use glucose strips to read each of the test Tubes after 20 minutes has passed, and again at 60 minutes. Record your results in a table. If you need advice about how to make the table, please ask your lab instructor. Please note. In Test Tube 2 you showed that the sucrose solution did not test positive for glucose, which was expected. But sucrose is a double sugar, and maybe lactase is able to break it down. To find out, add 2 drops of lactase solution to Test Tube 2 and place it back in the test tube rack. You need to try Test Tube 2 only once with a glucose strip after 60 minutes. ACTIVITY OF PEPSIN: A pepsin demonstration has been set up for us, but don’t look at it until you have considered this experiment and decided what your expectations are. You need to consider what pH environment pepsin is engineered to work in. For this experiment, pieces of boiled egg white were suspended in test tubes with distilled water and a solution at pH 2. Egg white (albumin) is pure protein and if pepsin is able to break down protein, it should make the piece of egg white smaller.

• Test tube with egg white in distilled water • Test tube with egg white and pepsin in distilled water • Test tube with egg white in pH 2 solution • Test tube with egg white and pepsin in pH 2 solution

Write your expectations in your laboratory notebook. You are now ready to look at the demonstration. Make sure to write down your observations in your lab notebook. For both the lactase experiments and the pepsin demonstration you need to present and analyze your results. You need to be clear about your expectations before each experiment, this corresponds to your hypothesis in the Scientific Method. Did the experiments turn out as you expected? Your lab syllabus calls for a Results and Analysis section. In addition, you need to answer the following questions in the lab notebook. As before, don’t copy the question, instead write a complete sentence that includes the information asked for.

1) In which test tube was lactase expected to be the most effective? Why?

2) Why did you add boiled lactase to one of the Test Tubes? What results do you expect from this?

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3) Was lactase able to break down sucrose? Why is this?

4) Using the naming rule mentioned in the lab, what name would you expect is given

to the enzyme that is able to break down sucrose?

5) People who are lactose-intolerant often take a particular digestive supplement before eating ice cream, for example. Why is this? What do you expect is the active ingredient in the digestive supplement?

6) In which test tube was pepsin expected to be the most effective? Why?

7) Give examples of household products that contain enzymes.

References

This lab was developed and adapted from classic General Biology labs.

© John Hakanson (2014)

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