3.2: Lab 3 Procedures

PROCEDURE 1: PRACTICE WITH BIOMOLECULES

1. Before moving on to the experiments for today, complete Table 1 on the Answer Sheet to review the differences between the four classes of biomolecules.

PROCEDURE 2: POSITIVE AND NEGATIVE CONTROLS

Today we will be designing an experiment to test for the presence of proteins (in a solution of albumin, a.k.a. egg whites), carbohydrates (glucose & starch), and lipids in a mystery food item. To test for the presence of these biomolecules, we will be using reagents and solutions called indicators. Indicators produce a physical change, often a change in color, in a solution when mixed with a particular molecule. We will be using iodine potassium iodide (IKI; iodine for short) solution, Biuret's reagent, and Benedict's solution. But in order to determine which molecule they test for (protein, glucose, or starch), we have to set up an experiment. Then, we can use the indicators to test for the presence of these molecules in the mystery food item.

For certain experiments, there are two types of control groups - positive and negative. The positive control is a solution where we expect to see a positive result (in this case color changing). The negative control is a solution where we expect to see a negative result (in this case no color change or no difference at all). Follow the procedures below to create positive and negative controls for each of the biomolecules and determine the biomolecules that each indicator tests for.

Equipment we’ll need:

• 12 test tubes and a test tube clamp
• Two test tube racks
• Grease pencil
• Iodine potassium iodide (IKI) solution, Benedict’s reagent, and Biuret’s reagent
• Flasks of water, glucose, starch, egg whites, dropper bottles with lipids, and the mystery food
• Two small paper towel squares
• Two 400 mL or 600 mL beakers
• One hot plate with beaker of water

Before we start this experiment: Fill a 600 mL beaker with about 400 mL of tap water. Put the beaker on the hotplate and set the heat to about level 5. We will need this water on the hotplate for the duration of the lab today, so if the beaker gets low on water, fill it back up.

• Do not let the water in the beaker completely evaporate! An empty beaker on a hotplate will break and may send shards of glass shooting across our desk!

Lipids

The following experiment will set up a positive and negative control for lipids.

1. Obtain two small paper towel squares. With a pen, label each square “L” or “W”.
2. On the “L” square, put a single drop from the lipid dropper bottle.
3. On the “W” square, put a single drop of water.
   • We may need to gently dab the drop of lipid or water into the paper using a finger.
4. Set them aside. In 30 minutes, hold them up to the light and see if there is a stain.

Iodine solution

The following experiment will determine which biomolecule the iodine solution tests for.

1. Obtain four test tubes. Use a grease pencil and ruler to mark lines 1 cm and 2 cm from the bottom of each test tube. Label the test tubes 1-4, where the numbers refer to:
   • 1 = Albumin
   • 2 = Glucose
   • 3 = Starch
   • 4 = Water
2. Fill each tube with the iodine solution to the 1 cm line.
3. Fill each with its corresponding solution to the 2 cm line.
   • For example, fill tube 1 with albumin to the 2 cm line, tube 2 with glucose to the 2 cm line, etc.
4. Swirl each tube to mix the contents.
5. Record the color of each tube in Table 2 on the Answer Sheet.

Biuret’s reagent

The following experiment will determine which biomolecule the Biuret’s reagent tests for. We will repeat the same steps as above.

1. Obtain four test tubes. Use a grease pencil and ruler to mark lines 1cm and 2cm from the bottom of each test tube. Label the test tubes 1-4, where the numbers refer to:
   • 1 = Albumin
   • 2 = Glucose
   • 3 = Starch
   • 4 = Water
2. Fill each tube with the Biuret’s reagent to the 1cm line.
3. Fill each with its corresponding solution to the 2cm line.
4. Swirl each tube to mix the contents.
5. Record the color of each tube in Table 2 on the Answer Sheet.

Benedict’s reagent

The following experiment will determine which biomolecule the Benedict’s reagent tests for. Note the difference in step 4!

1. Obtain four test tubes. Use a grease pencil and ruler to mark lines 1cm and 2cm from the bottom of each test tube. Label the test tubes 1-4, where the numbers refer to:
   • 1 = Albumin
   • 2 = Glucose
   • 3 = Starch
   • 4 = Water
2. Fill each tube with the Benedict’s reagent to the 1 cm line.
3. Fill each with its corresponding solution to the 2 cm line.
4. Put each test tube into the beaker with hot water on our hot plate for 5 minutes.
   • Make sure there is enough water in our beaker.
5. After 5 minutes in the hot water bath, record the color of each tube in Table 2 on our Answer Sheet.
   • The test tubes will be very hot! Use the test tube clamp to move the test tubes from the hot water bath to the test tube racks.

Clean up/Disposal

1. Pour the solutions from the test tubes into an empty 400 mL beaker.
2. Pour the beaker into the hazardous waste container next to the sink.
3. Rinse the test tubes and beaker thoroughly in the sink.
4. Scrub off the grease pencil lines and any other markings. Remove any tape labels.
5. Put the clean test tubes in the metal box next to the sink and take the beaker back to the desk.

PROCEDURE 3: MYSTERY FOOD ITEM

Based on the results from Procedure 3, we should now know which biomolecules each indicator tests for and what the positive and negative controls look like. Now, we are going to design an experiment to find out which biomolecules are in a mystery food item.

Equipment we’ll need:

• 3 test tubes and a test tube clamp
• Test tube rack
• Iodine solution (IKI), Benedict’s and Biuret’s reagents, and a paper towel square
• One hot plate with beaker of water

Before we start: Make sure there is enough water in the beaker on the hotplate.

1. Answer questions in the Answer Sheet to build this experiment.
2. Obtain a paper towel square. Place a single drop of the mystery food solution on the square. Set aside and review in about 30 minutes.
3. Obtain three new test tubes. Use a grease pencil and ruler to mark lines 1 cm and 2 cm from the bottom of each test tube. Label the test tubes 1-3, where the numbers refer to:
   • 1 = Iodine solution
   • 2 = Biuret’s reagent
   • 3 = Benedict’s reagent
4. Fill each tube with the appropriate chemical indicator to the 1 cm line.
5. Fill each test tube with the mystery food solution to the 2 cm line.
6. Swirl each tube to mix the contents. Place test tube #3 with Benedict’s solution into the hot water bath.
   • Do not put test tubes #1 or #2 in the hot water bath.
7. Record color changes and answer questions in the Answer Sheet.

Clean up/Disposal

1. Pour the solutions from the test tubes into an empty 400 mL beaker.
2. Pour the beaker into the hazardous waste container next to the sink.
3. Rinse the test tubes and beaker thoroughly in the sink. Using a wire brush remove any mystery food remnants from the bottom of the test tubes.
4. Scrub off the grease pencil lines and any other markings. Remove any tape labels.
5. Put the clean test tubes in the metal box next to the sink and take the beaker back to the desk.

Introduction: Enzymes

Many chemical reactions can occur in a beaker or test tube. Generally, these reactions either need a large amount of time to occur or need to be heated to high temperatures. When thinking about biology, life does not have large amounts of time to digest its food for energy and life cannot be heated to high temperatures. So for cells to be able to perform chemical reactions at a reasonable rate, life needed to evolve something to help. The result was enzymes. Enzymes are a type of protein that speed up chemical reactions. Their three dimensional shape is essential to their ability to speed up reactions.

Enzymes have a special pocket called the active site where the reactant(s), called substrate(s), connect or bind. Due to the specifically shaped active site, only certain substrates can fit. Once the substrate binds to the active site, the enzyme will either help build or break a chemical bond, but the enzyme itself is unchanged. Then, when the new molecule(s) are released, another substrate enters the enzyme and the process continues. When we write chemical equations, we put the name of the enzyme on top of the arrow to show that it is involved but it is not changing like the substrates are changing into products.

Today we will be performing an experiment on the enzyme catalase. Catalase breaks down the molecule hydrogen peroxide (H2O2) into water (H2O) and oxygen gas (O2). When oxygen gas is present in liquid, it forms bubbles. Catalase is found in nearly every single organism, such as in the human liver or in potatoes. We will use blended potatoes as a source of catalase. During the experiment, the oxygen gas produced by this chemical reaction will be visible as bubbling (and large amounts of it). We will be designing an experiment to answer the question “how does temperature affect the function of an enzyme?” Hint: recall that shape is essential to the enzyme functioning properly and that various environments could cause the folding of the enzyme protein to fall apart thus making the enzyme non-functional. We will be measuring and comparing the amount of bubbling to determine whether the enzyme catalase is still working.

PROCEDURE 4: ENZYME EXPERIMENT

Part 1: Positive and negative controls

Just as we did with the biomolecules experiments, we will set up positive and negative controls for the enzyme catalase. Positive and negative controls are important to not only compare future, unknown results to, but also to verify that the materials are working correctly. Here, we will test that the enzyme, substrate, and indicator do what they should and we will have positive and negative results to compare our experiment to.

Equipment we’ll need:

• 2 test tubes
• Test tube rack
• Potato juice (as a supply of catalase), hydrogen peroxide, and water

1. Answer the questions in the Answer Sheet prior to setting up this experiment.
2. Label 2 glass tubes with a grease pencil. Label one with “+” and the other with “-” at the top.
3. Measure 2cm from the bottom of each tube and draw a line. Mark another line on each tube at 5cm.
4. Fill both test tubes up to the 2cm line with the Catalase enzyme solution (potato juice).
5. Fill the “+” tube to the 5cm line with hydrogen peroxide.
6. Fill the “-” tube to the 5cm line with pure water.
7. Swirl both tubes carefully to mix for 10 seconds.
8. After 3 minutes, measure the height of the “bubble column” in centimeters (cm)- from the top of the liquid to the top of the bubbles.
9. Record the height of the bubble columns in Table 4 on the Answer Sheet.

Part 2: Perform the experiment

Equipment we’ll need:

• 3 test tubes
• Test tube rack
• Potato juice and hydrogen peroxide
• One hot plate with beaker of water

1. Answer the design questions in the Answer Sheet to build an experiment looking at temperature’s effect on enzymes.
2. Obtain three test tubes. Number these tubes 1-3 using a grease pencil. Measure and mark lines for 2cm and 5cm from the bottom of each tube.
3. Fill all three tubes with potato juice up to the 2 cm line. Apply the independent variable at this time. Allow the independent variable to affect the enzymes in the potato juice for at least 10 minutes.
4. Retrieve the test tubes and put them into the test tube rack. IMMEDIATELY add hydrogen peroxide up to the 5 cm line and swirl for 10 seconds.
   • It is important to add the hydrogen peroxide right away before the different temperature treatments do not return to room temperature.
5. After 3 minutes, measure the amount of bubbles in the tube.
6. Record the height of the bubble columns in Table 5 and answer the questions in the Answer Sheet.

Clean up/Disposal

1. Pour the solutions from the test tubes down the sink and rinse the test tubes thoroughly. Use a wire brush to remove any potato remnants from the bottom of the test tubes.
2. Scrub off the grease pencil lines and any other markings. Remove any tape labels.
3. Put the clean test tubes in the metal box next to the sink and place the beaker on the towel to dry.