Carbohydrates, Lipids, Proteins

Carbohydrates

Introduction

You may wish to read about Carbohydrates before you begin this part of the lab. Read up to the "Lipids" section, then push the "back" button on your browser to return here.

Cleaning Up

It is important to keep the lab in the same clean condition that is in when you arrive. After the experiments, rinse all of the equipment and glassware and wipe down the lab bench.

The contents of test tubes can be disposed down the drain. The tubes should be rinsed with tap water and put upside down in a test tube rack. Leave used glassware near the sink area.

Monosaccharides

Some sugars such as glucose are capable of reducing other compounds and are called reducing sugars. When reducing sugars are mixed with Benedicts reagent and heated, a reduction reaction causes the Benedicts reagent to change color. The color varies from yellow to green to dark red, depending on the amount of and type of sugar.

Mark six test tubes one centimeter from the bottom using a wax pencil. Put a second mark on each tube approximately three cm from the bottom. Number the test tubes 1 through 6 near the top of the tubes. It is important that these numbers be located somewhere near the top of the tube.
Fill four of the test tubes to the 1 cm mark with the following solutions:
Test tube #1: water
Test tube #2: glucose solution
Test tube #3: sucrose solution
Test tube #4: starch solution
Use a mortar and pestle to crush a piece of onion. Add several drops of water while crushing. Put several drops of the onion juice in test tube #5 and then fill it to the 1 cm mark with water.
Be sure to clean the mortar and pestle when you have finished.
Use a mortar and pestle to crush a piece of potato. Add several drops of water while crushing. Put several drops of the potato juice in test tube #6 and then fill it to the 1 cm mark with water.
Fill each of the test tubes (1 through 6) to the 3 cm mark with Benedicts reagent and put the tubes in a boiling water bath for 5 minutes.
Record your results in Table 1. A change in color indicates the presence of reducing sugars.

Below: The test solutions and Benedict's reagent are boiled in a water bath for five minutes.

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Below: Results of several solutions tested with the Benedict's test

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Starch

Iodine solution (IKI) reacts with starch to produce a dark purple or black color.

Use a wax marker to mark two test tubes 1 cm from the bottom.
Fill one of the tubes to the 1 cm mark with water and fill the other to the 1 cm mark with a 1% starch solution. Be sure to stir the starch before filling your tube.
Add two drops of IKI solution to each tube and note the color change.

Record your results in Table 2.

Below left: starch solution and IKI - Iodine turns dark in the presence of starch.

Below right: distilled water and IKI

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Put a drop of IKI solution on a small slice of potato. Note the color after 30 minutes and record your observation in Table 2.
Put a drop of IKI solution on a small slice of onion and note the color. Record your observation in Table 2.
Put a thin slice of potato on a slide and stain it with IKI. The potato should be sliced as thin as possible; thinner than paper is best. If you cannot get it thin enough, press down on the cover glass to crush the specimen.

Draw a potato cell from the slide that you prepared in the previous step. Label the cell wall and starch granules.

1a) Which macromolecule are the dark granules within the potato cells composed of? [Hint – What caused the iodine to turn dark?]

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Potato cells stained
with IKI X 100 Potato cells stained
with IKI X 200

Prepare a wet mount of onion stained with IKI. Try to get a piece that is thinner than paper; the thinner it is, the better the image will be. A thin piece can usually be obtained by

Draw an onion cell in the space provided.

1b) Does onion store food as starch?

Left: Onion stained with IKI X 100 - The nuclei of these cells are light brown in this photograph. The numerous starch granules seen in potatoes are absent.
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Lipids

Read about lipids in the class notes before you begin this part of the lab. Read up to the "Proteins" section, then push the "back" button to return here.

Emulsification

Lipids are nonpolar and therefore do not dissolve in water. Emulsifiers are molecules have both polar and nonpolar parts and thus are capable of dissolving in or interacting with both lipids and water. When emulsifiers are mixed with lipids and water, they may act to suspend small droplets of the lipid in water. The lipid is not dissolved in water, but is broken into smaller fragments that may remain suspended for long periods of time.

Bile salts are emulsifiers that are produced by the liver and assist in the digestion of lipids by enabling lipids to be broken up into small particles so that enzymes can break them down quicker.

Tween and liquid soap used in the experiment below are emulsifiers.

Add 2 cm of vegetable oil to two test tubes and add another 2 cm of water to each tube.

Sudan IV is a stain used to stain lipids. Add six drops of Sudan IV to each tube and mix the contents by swirling the test tubes.

1) Describe what happens to the oil and water mixture.
Add 5 drops of tween to one of the test tubes. If tween is not available, use two drops of a liquid soap solution.
Cap each test tube with your thumb and shake them vigorously. Observe each of the tubes immediately after shaking. Put the tubes in a rack and observe them after 1 minute, 5 minutes, and 30 minutes. Record your observations in table 3.

Below: The tube on the right contains oil and water. The one on the left contains oil, water, and a detergent. Both tubes were shaken to mix the oil and water. The oil can be seen floating on the water in the tube on the right. The tube on the left shows that the oil droplets remain mixed with the water longer before separating.

Below left: oil and water X 40 - Note the large fat droplet on the upper, right half of the photograph. The smaller bubbles scattered throughout the photograph are air bubbles due to vigorous shaking.

Below right: oil, water and detergent (emulsifier) X 40 - The large oil droplets have been broken up into smaller droplets after shaking.

Proteins

Read about Proteins before you begin this part of the lab. Read up to the "Nucleic Acids" section, then push the "back" button to return here.

Biuret Test

The copper atoms of Biuret solution (CuSO₄ and KOH) will react with peptide bonds, producing a color change. A deep violet color indicates the presence of proteins and a light pink color indicates the presence of peptides.

Color Indication

Light blue No protein or peptides

Violet Protein

Pink Peptides

We will perform the biuret test on egg albumin, a protein found in chicken eggs. In a second experiment, we will also study how pepsin, an enzyme found in the stomach, is capable of breaking protein down into smaller fragments called peptides.

Pepsin is normally found in the warm (37º C) acidic environment of the stomach. To simulate these conditions, HCl will be added and the test tube will be incubated at 37º C.

Mark three test tubes at 2 cm.
Fill one of the tubes to the 2 cm mark with water, the second one to the 2 cm mark with albumin solution (a protein), and the third one to the 2 cm mark with starch solution.
Add 5 drops of 3% copper sulfate solution (CuSO₄) to each tube.
Add 10 drops of 10% potassium hydroxide solution (KOH) to each tube.

Record the final color of each test tube in Table 4.

Below:

Tube 1: Water (control)

Tube 2: Albumin (protein)

Tube 3: Starch

Mark three test tubes at 2 cm, 4 cm, and 6 cm.
Add water to the 6 cm. mark to test tube 1. Add albumin solution to the 2 cm mark to test tubes 2 and 3.
Add pepsin to the 4 cm mark to tubes 2 and 3.
Add water to the 6 cm mark of test tube 2.
Add 0.2% HCl to the 6 cm mark of tube 3.
Obtain 3 strips of pH paper and measure the pH of each tube. This can be done by inserting the paper into the liquid and then comparing the color of the paper to the chart on the side of the pH paper container. Record your results in table 5.

Incubate all of the tubes at 37 degrees C for 45 minutes. This step will allow the pepsin to digest the protein.
Add 5 drops of 3% copper sulfate solution (CuSO₄) to each tube.
Add 10 drops of 10% potassium hydroxide solution (KOH) to each tube.

A violet color indicates the presence of protein. A lighter, pinkish color results in the presence of peptides.
Record the final color of each test tube in Table 5.

Below:

Tube 1 (left): water

Tube 2 (center): albumin, pepsin, water

Tube 3 (right): albumin, pepsin, HCL

Explain why tube 3 was incubated at 37 degrees C (this is body temperature).

What is the function of pepsin in the stomach?

Explain why HCl was added to tube 3? (Hint: What is the pH of the stomach?)

What is the name of the enzyme involved in this experiment?

What is the optimal pH range of this enzyme (acid, neutral, or base)? What happens to enzymes when the pH is not appropriate for the enzyme?

Trypsin is an enzyme found in the small intestine. It cleaves larger peptide fragments into smaller peptides. The pH of the small intestine is slightly alkaline. Knowing this, approximately what pH range (acid, neutral, or base) do you predict trypsin to function best?

Based on your answer to the two previous questions, what can you conclude about the optimal pH of enzymes. Does it depend on the enzyme?
Explain why you expect tube 2 to contain protein and tube 3 to contain peptides. [Hints: 1. HCl does not break down protein. 2. See your answers to the previous four questions above.]