Testing Biomolecules

Introduction:

The most common organic compounds found in living organisms are lipids, carbohydrates, proteins, and nucleic acids.  Common foods, which often consist of plant materials or substances derived from animals, are also combinations of these organic compounds.  Substance called indicators can be used to test for the presence of organic compounds.  An indicator is a substance that changes color in the presence of a particular compound.  In this investigation, you will use several indicators to test for the presence of lipids, carbohydrates, and proteins in various foods.

Problem:  What are the major types of organic compounds in some common foods?

Pre-lab Discussion:

1. What is an indicator?  How are indicators used in this experiment?
2. What is the purpose of using distilled water as one of your test substances?
3. What is the controlled variable for each group of testing?
4. What is the purpose of washing the equipment thoroughly?
5. What is the IV? DV?
6. You have added Sudan IV stain to each substance.  What change indicates the presence of lipids?

Make a data table with the following information: food samples (IV); prediction of biomolcs present in food; results from benedicts; results from biurets; results from lugols; results from grease spot test

Substances (food samples) being tested:

1. honey solution
2. egg white solution
3. corn oil
4. lettuce solution
5. gelatin solution
6. melted butter
7. potato solution
8. apple juice
9. distilled water
10. whole milk
11. non-fat milk

Part A:  Testing for Carbohydrates: Monosaccharides

Glucose, commonly called “blood sugar,” and fructose, “fruit sugar,” are two of the most common monosaccharides.  Ribose, an important component of nucleic acids, is also a monosaccharide and will be discussed during our study of DNA and RNA.  Monosaccharides can be identified in a substance using an indicator called Benedict’s solution.  When Benedict’s is heated in the presence of a monosaccharide, the color changes from blue to green to yellow to reddish-orange, depending on the amount of monosaccharide present.

 Procedure:  You can start step 1 and then proceed to another test while the water heats up.

1. Turn on the hot plate to high and fill a beaker half full of tap water.  Place the beaker on the hot plate. 
2. While this is heating, add 2 dropper-full of each sample to a test tube.  Add 10 drops of Benedict’s solution to each test tube. 
3. Place the test tubes in the hot-water bath.  Heat the test tubes until the indicator changes color OR for 1 minute max if it doesn't seem to be changing color. 
4. With a test tube holder, remove the tubes from the hot-water bath and place them back in the test tube rack. 
5. Record any changes in your data table.

Part B: Testing for Carbohydrates: Polysaccharides

Monosaccharides may join together to form long chains called polysaccharides that may be either straight or branched.  Starch is an example of a polysaccharide formed entirely of glucose monosaccharides.  Polysaccharides can be tested for using the indicator Lugol’s reagent (iodine/potassium iodine).  Lugol’s will change color from yellow-brown to blue-black-purple in the presence of starch.

Procedure:

1. Add 1/2 dropper full of each solution to a well in the well tray. 
2. Add 1 drop of Lugol’s to each sample. 
3. Record any changes in your data table.

Part C: Testing for Lipids

Lipids can be generally categorized into two subgroups: saturated fats and unsaturated fats.  In saturated fats, the carbon chains are bonded together with only single covalent bonds and the remaining electrons are bonded to hydrogens.  This structure keeps the hydrocarbon tails relatively straight, so they readily align with each other, pack closely together, and thus tend to be solid at room temperature.  Saturated fats are most common in animals.  Unsaturated fats has double bonds between the carbons and therefore do not have the maximum number of links to hydrogens.  These regions of double bonding allow kinks to form in the hydrocarbon tails, keeping them from packing closely together.  For this reason, unsaturated fats are usually liquid at room temperature.  Unsaturated fats are common in plants.  Because fats are nonpolar, we will test for their presence using Sudan IV, a nonpolar dye that will readily mix with lipids but form small bubbles or separate with non-fats.  Lipids can also be tested for using the “sack lunch” method: the lipid is rubbed into a brown bag.  If a grease spot appears (the brown bag appears translucent), then a lipid is present in the food.

Procedure: Sudan IV

1. Add o1/2 dropper full of each solution to a clean well in the well tray.
2. Add 3-5 drops of Sudan IV to each sample.
3. Look for small bubbles or separation.
4. Record results in data table.

Procedure: Grease spot test (sack lunch):

1. Cut a piece of a paper lunch sack and make a grid (using a pencil, not a pen) for all the food samples you will test.  Label each square with a different food sample.  Write your lab table and period.
2. Add 4-5 drops each solution to the appropriate square on the lunch bag.
3. Rub the solution until a “wet” spot appears on the paper. 
4. Set the paper aside overnight.  Check the next day.
5. Hold the brown paper up to a bright light.
6. Record if the paper is translucent or not in data table.

Part D: Testing for Proteins

Proteins are made up of one or more polypeptides, which are linear polymers of monomers called amino acids.  Amino acids derive their name from the amino group and the carboxyl group (which is acidic).  Polypeptides are formed when amino acids are joined together by peptide bonds between the amino group of one amino acid and the carboxyl group of another amino acid.  Proteins can be tested for using Biuret’s reagent, which reacts with the peptide bond between amino acids in the polypeptide.  Biuret’s reagent turns from blue to purple-violet if peptides bonds are present.

Procedure:

1. Add 1/2 dropper full of each solution to a clean well in the well tray.
2. Add 3-5 drops of Biuret’s reagent to each sample.
3. Record any changes in color.

Lab Write up requirements (20pts total):

Title: Must be descriptive (includes the IV and DV of the experiment) 1 point

Results:        Data Table of class results:  (9 points total)

                            a. includes a descriptive title (scientific...not cutsey <sorry all you English buffs!>). (2 points)

                            b.  column headings include the name of the indicator and what the indicator detects in parenthesis... example: Benedict's Solution (monosaccharides)... (5 points)

                        c. Key for explaining the symbols you might use... example "+" = positive for biomolc  (2 points)

Post Lab Questions:  make sure you pick one version of questions to answer and provide any citations requested for full credit. Also remember incomplete sentences, sentences starting with "it" or "because", or sentences using pronouns will not receive full credit.

Answer questions  1-5 to earn a C on this section: (up to 5 points), answer questions 6-9 to earn a B (up to 8 points), to earn an A (up to 11 points answer 6-10).

1. Why is a monosaccharide called a monomer?
2. Why is a polysaccharide called a polymer?
3. List which chemical detects which molecule.
4. A lipid refers to three different types of substances, a _____________, a _____________ and __________.
5. Why would water be a good substance for a control in this lab activity?
6. People with diabetes are instructed to avoid foods that are rich in carbohydrates.  How could your observations in this investigation help you decide whether a food should be served to a person with diabetes?  (explain the relationship between diabetes and monosaccharides and their connection to this lab activity...include citations to references) (2 points)
7. What conclusion could you make if a positive test for any of the organic compounds occurred in the distilled water?  (what is in distilled water normally... what could the source of this "positive" test come from during the lab testing process). (2 points)
8. Why can’t Biuret’s reagent be used to test for amino acids? (what does Biuret's actually detect on a  protein, what is the relationship between an amino acid and a protein? cite your sources at the end of your answer..) (2 points)
9. The leaves of many plants are coated with a waxy substance that causes them to shed water.  How would you expect this substance to react in the Sudan IV test?  ( what type of biomolecule is detected by Sudan's what is the relationship to wax... what is the name of the waxy substance... cite your sources at the end of your answer.) (2 points)
10. In winter, plants exchange the saturated lipids in their cell membranes for unsaturated lipids.  Unsaturated lipids are “bent” and keep the membranes more fluid, or pliable, because they cannot be stacked closely together.  Of what advantage would this be for green stemmed (herbaceous) plants that live through the winter?  (Hint: what happens to bacon grease or the grease on top of chicken soup when you put it in the refrigerator?) ( Which is a liquid at room temperature, a solid at room temperature.. why would a plant want to switch the types of fatty acids in the winter... what could happen if the cell membrane only had saturated fatty acids during the winter, or only unsaturated fatty acids in the summer... don't forget to cite your sources properly at the end of your answer.)_(3 points)

Helpful articles to answer #10 article #1  http://tea.armadaproject.org/atwood/12.2.1998.html  (Especially see section F)