4: Testing for Plant Macromolecules

Plant cells are composed of a broad array of different, chemical compounds. Perhaps the most common of these compounds are macromolecules, that is, “large” molecules. Macromolecules account for the majority of the structures within plant cells. There are four types of macromolecules found in plants: carbohydrates, lipids, proteins, and nucleic acids.

Macromolecules consist of smaller subunits. Carbohydrates are composed of monosaccharides. Many lipids, including fats, are composed of a glycerol molecule and long fatty acid chains. Proteins are composed of amino acids. The two nucleic acid types, DNA and RNA, are composed of nucleotides. Each of these subunits has some combination of carbon (C), hydrogen (H), and oxygen (O) atoms. The bonds between the atmos represent chemical energy.

Macromolecules contain different amounts of energy due to variations in size and configurations of C, H, and O. Cells utilize this energy when macromolecules are broken down in different metabolic reactions. When macromolecules are combusted, their energy is released as heat. This heat is measured in a unit we know is calories. A calorie (note lowercase c) is the amount of heat required to raise the temperature of 1 mL of water by 1 C, at normal temperature and pressure. A Calorie is 1000 calories, also known as a kilocalorie (kcal). In general, carbohydrates produce an average of 3.9 kcal per gram (kcal/g), compared with a 3.5 kcal/g for proteins. Lipids, on average, produce about 0.3 kcal/g.

Seeds contain cells with large quantities of stored macromolecules that can be used as energy sources during seed germination and initial seedling growth. However, not all seeds store all macromolecule types in the same amounts. Seeds of some species contain higher amounts of carbohydrates, while other seeds contain large amounts of proteins or lipids. Therefore, seeds of different species contain different kcal/g since macromolecules are present in different ratios.

One purpose of this lab is to estimate the amounts of energy (kcal/g) yielded by different seeds or seed-derived products that are composed primarily of carbohydrates, lipids, or proteins. You will first use qualitative tests to determine which of the seed products are primarily composed of carbohydrates, lipids, and proteins. Then you will estimate the kcal/g for the three selected seed products. From these data, you will evaluate various food products made from seeds in terms of their relative macromolecule content.

Activity 4.1: Qualitative Determination of Major Macromolecules

In this activity, you will use substances of known compositions as standard for comparison. Each standard food product is composed almost entirely of a single kind of macromolecule. You will test those first to see how the test works. Then, you will apply the same qualitative tests to the three unknowns used in this lab. “Unknown” implies that you, presumably, do not know which general kind of macromolecule occupies the bulk of each food product.

As you conduct these tests, described here, enter the results as a “+” or “0” sign in Table 4.1. Conduct both macroscopic (easily visible) and microscopic (need magnification) tests as appropriate for each substance.

Testing for Protein

Macroscopic Test: The Bradford protein assay indicates protein concentration in a substance. The test uses a chemical that changes color in the presence of water-soluble protein. Substances with high concentration of protein will turn a bright blue, while substances tacking protein will remain a greenish brown color. There will be a demonstration rack of the Bradford protein assay for you to observe.

Testing for Starch

Microscopic Test: The reagent I2KI is used as an indicator for the presence of starch, a complex carbohydrate. I2Kl will stain starch grains within cells a blue-black or purple color.

Crush and then smear a small amount of each substance on a grass slide and add a drop of I2KI. After a minute, remove the excess stain using a Kimwipe, add a drop of water, and place a coverslip on top. Look for the distinctive dark staining of starch grains under the microscope at 100X or 400X magnification.

Testing for Lipid

Macroscopic Test: The smash test is used as a macroscopic test for lipid content. When a substance with a high lipid content is “smashed” on a clean paper, it will leave a clear or translucent greasy spot.

Put a small amount of each substance on a ceramic plate and firmly press down it with a clean scrap of white paper. Observe whether or not there is a translucent spot on the paper afterward.

Microscopic Test: Sudan is a fat-soluble dye that indicates the presence of lipids within cells by staining them orange or red.

Crush or smear a small amount of each substance on a glass slide and mi with a drop of water. Add a drop of Sudan IV and wait 2-3 minutes before adding a coverslip. Look for the presence of pink-to-range-stained droplets under the microscope at 100X or 400X magnification.

Activity 4.2: Experimental Determination of kcal/gram

In this activity, your lab instructor will direct you in the steps necessary to use a calorimeter to determine the approximate between plant-derived products in the amount of calories they will yield.

Safety Precautions for This Activity

A. Avoid touching hot objects and open flames.

B. Do not heat empty test tubes. They may shatter.

C. Keep long hair and dangling objects away from flames.

The objective of the next procedure is to measure the change in water temperature in a test tube, before and after you have burned a food sample under the tube.

A. Procedure (per pair of students)

  1. Obtain and weigh food samples. Each sample should weigh between 0.4 and 0.5 grams. Record the weights in Table 4.2
  2. Add 15 mL of cold tap water to the test tube provided.
  3. Clamp the test tube with the provided clamp and place in the top opening of the metal can on the ceramic plate to avoid heat damage to the bench top.
  4. Use the thermometer to measure initial temperature of the water and record that value.
  5. Place the flame source on the ceramic plate next to the can and light the wick.
  6. Place the oateos on the charred needle provided. Quickly light the oateos by holding them in the flame and immediately move the needle so that the oateos burn directly under the bottom of the test tube. Continue burning until all the oateos burn directly under the bottom of the test tube. Continue burning until all oateos are completely charred. Relight if needed.
  7. As the oateos are burning, monitor the temperature change by holding the thermometer in the water so that it does not touch the bottom of the tube. (Remember we want to measure the temperature of the water, not the glass)
  8. Record the highest temperature achieved during the burn.
  9. Discard the hot water into the waste beaker, rinse the tube with cold water, and refill with 15 mL of fresh cold water. repeat using walnut, then again with tofu.