Materials Included In Kit

Additional Materials Required

Prelab Preparation

1. Prepare a 1% sucrose solution: Weigh 2.0 g sucrose and place in a labeled 250-mL Erlenmeyer flask. Add 200 mL DI water to the sucrose. Cap the Erlenmeyer flask and gently shake to mix the contents.
2. Prepare a large volume of very hot water for students to use to maintain the temperature of the 40 °C water bath.
3. Prepare one or more boiling water baths for use with the Benedict’s solution. Place test tube clamps near the boiling water baths.
4. Prepare the yeast just prior to the lab.
   1. In a chemical fume hood, add one package (7 g) of yeast to 10 mL of toluene in a mortar.
   2. Grind the mixture with the pestle until it is a uniform paste.
   3. Add DI water about 3 mL at a time to the paste until a total of 30 mL of DI water has been added. Grind the mixture with each addition of water.
   4. Transfer the yeast–toluene mixture to a labeled 250-mL beaker.
   5. Repeat steps a–d with the other two packages of yeast, adding the yeast mixture into the same 250-mL beaker.
   6. Allow the mixture to sit for 20 minutes, stirring occasionally with the glass stirring rod.

Safety Precautions

Toluene is moderately toxic by ingestion, inhalation and skin absorption. Toluene and ethyl alcohol are flammable liquids. Make sure there are no ignition sources in the area when using toluene or ethyl alcohol. Benedict’s solution is a skin and eye irritant. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Ensure the centrifuge is balanced and the lid is securely closed while spinning. Remind students to wash their hands thoroughly with soap and water before leaving the laboratory. Please review current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. Toluene should be disposed of using a licensed waste disposal company, Flinn Suggested Disposal Method #18b. All other solutions may be disposed of by flushing down the drain with an excess of water according to Flinn Suggested Disposal Method #26b. Solids may be disposed of in the normal trash, Flinn Suggested Disposal Method #26a.

Lab Hints

• Enough materials are provided in this kit for 30 students working in pairs or for 15 groups of students. This laboratory activity can reasonably be completed in one 50-minute class period. The prelaboratory assignment may be completed before coming to lab, and the Post-Lab Questions may be completed the day after the lab.
• If the maximum centrifuge time is needed, the lab may require two lab periods. A good place to stop is after step 12. Place the sucrose pellets in the freezer until the next lab period.
• For a “block schedule” class, allow the students to prepare the yeast mixture.
• Clear, plastic centrifuge tubes may become cloudy or even crack when exposed to toluene. Use glass centrifuge tubes.
• Explain to students how and why the yeast mixture was prepared. Discuss the purpose of the toluene used in this procedure.

Teacher Tips

• The protein isolated in this laboratory is an enzyme. Use the results of this laboratory to discuss the importance of enzymes used to make cheeses, to clarify juice, to catalyze reactions in the body or to cut DNA strands for DNA fingerprinting.

Answers to Prelab Questions

1. A mixture of yeast cells and toluene is spun in the centrifuge. After the centrifuge stops, the top layer of toluene is discarded, the middle layer is kept, and the bottom layer of solids is discarded. What is contained in the middle layer? What is contained in the bottom solid layer (called a pellet)?

   The middle layer contains cytoplasm and less dense cell components like proteins. The solid pellet contains the cell membrane, nucleus and organelles.

2. The middle layer is then spun again in the centrifuge. After it stops, the supernatant is discarded and the solid is kept. What is contained in the supernatant? What is contained in the layer of solid?

   The supernatant contains the water-soluble cytoplasm. The solid pellet contains the aggregated proteins.

3. What is the function of the protein sucrase? After reading the Procedure section, describe how the presence or absence of sucrase will be determined.

   Sucrase catalyzes the hydrolysis of sucrose into glucose and fructose. The presence or absence of sucrase is determined by combining sucrase and sucrose. After incubating the sample for several minutes, Benedict’s solution is added to test the sample for the presence of reducing sugars, such as glucose and fructose.

Sample Data

Answers to Questions

1. Draw a picture and describe the contents of each layer after each of the three centrifuge separations.
   {10837_Answers_Figure_4}
2. Biuret test solution is used to test for proteins. Why was Benedict’s solution used instead of biuret test solution to confirm the identity of sucrase obtained in this experiment?

   Biuret test solution would react with any protein so a positive test result would not indicate the presence of just sucrase.

3. Sucrase is not made properly in people with a rare genetic disorder called Congenital Sucrase-Isomaltase Deficiency (CSID). Affected people often take sucrase supplements just prior to eating. Why is this necessary?

   Without sucrase sucrose is not digested properly, resulting in digestive system problems. The sucrase in the supplement is able to digest the sucrose eaten during a meal so the glucose can be absorbed by the body.

Introduction

Everyone knows that muscles are composed of proteins, but did you realize that most of the more than 20,000 genes encoded in our DNA make proteins? How do you isolate and test just one type of protein?

Concepts

• Protein structure
• Supernatant
• Separation techniques

Background

Proteins were first described and named in 1838 by a Swedish scientist, Jöns Berzelius. Proteins are complex macromolecules made from amino acids joined together by peptide linkages. The exact sequence of amino acids in a protein determines the overall three-dimensional structure or shape of the protein. Protein structure can be described in four basic levels. The simplest structure is the linear sequence of amino acids that make up the polypeptide chain (see Figure 1).

This simple level of sequence is referred to as the primary structure. The primary structure is formed as a ribosome “translates” a sequence of messenger RNA (mRNA) into a chain of amino acids. Each of the 20 amino acids found in the human body has a slightly different structure. These slight differences cause the peptide chain to fold, twist and coil into a unique shape. A peptide chain may contain just one unique structural arrangement, such as only folds, or it may contain several, such as folds, twists and coils. These arrangements make up the unique secondary and tertiary structure of the protein. Finally, some peptide chains may associate with other peptide chains to form the final, quaternary structure of a protein. For example, hemoglobin consists of four negatively charged polypeptide chains (two pairs of α- and β-chains), of two slightly different types, each surrounding a positively charged iron atom (see Figure 2). Every protein has its own unique shape that is uniquely suited for a particular purpose in a living organism. After many years of research, in 1926, an American chemist named James Sumner became the first person to isolate a protein. Dr. Sumner was able to crystallize the protein urease from jack bean meal using a simple cold filtration technique still used today. Since 1926, many other methods have been developed to isolate different proteins, including precipitation using an organic solvent, salting out the protein and separation by chromatography and electrophoresis.

Water-soluble proteins found in the cytoplasm of a cell can be precipitated using an organic solvent (like ethyl alcohol) and a centrifuge. In order to isolate the protein, cells must be broken apart (lysed) and then separated in a centrifuge. The centrifuge utilizes density differences to separate more dense (solid) organelles, nucleus and cell membrane from less dense, liquid layers consisting of cytoplasm mixed with water and solvent. The liquid layer, called the supernatant, will be above the solid layer, called a pellet, in a centrifuge tube. The liquid layer is transferred to a clean centrifuge tube to which a water-miscible organic solvent, such as ethyl alcohol, is added. The ethyl alcohol interacts with the proteins in the cytoplasm causing the protein to aggregate (combine with itself). The protein aggregates are once again spun in a centrifuge to separate them from the solvent-water phase. The solid layer now contains the protein which can be further purified or tested.

Scientists use Saccharomyces cerevisiae, a type of yeast, to study genes and proteins. The entire genetic code for S. cerevisiae has been sequenced. More than 6000 genes have been identified, including the sequence for a protein called sucrase. Sucrase is an enzyme secreted by the yeast to split sucrose (a disaccharide) into two simple sugars—glucose and fructose (see Figure 3)—which can be absorbed by the yeast for its energy needs.

Experiment Overview

The purpose of this activity is to isolate and test a protein from the yeast Saccharomyces cerevisiae. Sucrase will be isolated from the cytoplasm of the yeast cells using laboratory techniques commonly used in biochemistry laboratories.

Materials

Prelab Questions

1. A mixture of yeast cells and toluene is spun in the centrifuge. After the centrifuge stops, the top layer of toluene is discarded, the middle layer is kept, and the bottom layer of solids is discarded. What is contained in the middle layer? What is contained in the bottom solid layer (called a pellet)?
2. The middle layer is then spun again in the centrifuge. After it stops, the supernatant is discarded and the solid is kept. What is contained in the supernatant? What is contained in the layer of solid?
3. What is the function of the protein sucrase? After reading the Procedure section, describe how the presence or absence of sucrase will be determined.

Safety Precautions

Toluene (in the yeast mixture) is moderately toxic by ingestion, inhalation and skin absorption. Toluene and ethyl alcohol are flammable liquids. Make sure there are no ignition sources in the area when using toluene or ethyl alcohol. Benedict’s solution is a skin and eye irritant. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Ensure the centrifuge is balanced and the lid is securely closed while spinning. Wash hands thoroughly with soap and water before leaving the laboratory. Follow all laboratory safety guidelines.

Procedure

1. Use a marker to label the following items with your group name and the appropriate label.
   1. One centrifuge tube “yeast”
   2. Two centrifuge tubes “cytoplasm”
   3. One 50-mL beaker “middle layer”
   4. One 50-mL beaker “sucrase”
   5. One test tube “control”
   6. One test tube “sucrase”
2. Use a graduated cylinder to measure 12 mL of yeast–toluene mixture solution into the “yeast” centrifuge tube.
3. Centrifuge the yeast mixture for 7 minutes. If the liquid portion is still cloudy, centrifuge for another 3 minutes. Note: Make sure the centrifuge is balanced.
4. Use a Beral-type pipet to carefully remove the top, organic solvent layer (toluene) from the centrifuge tube. Discard the toluene into the organic solvent waste container.
5. Use a clean Beral-type pipet to carefully remove the middle layer (the aqueous supernatant) and place into a 10-mL graduated cylinder. Record the volume (in mL) of the middle layer in the data table on the Protein Worksheet.
6. Pour the middle layer from the graduated cylinder into the “middle layer” beaker.
7. For every milliliter of liquid measured in step 5, add 3 mL of 95% ethyl alcohol to the beaker. For example, if 3 mL of the middle layer was recovered, add 9 mL of 95% ethyl alcohol to the middle layer liquid in the beaker. Record the volume of ethyl alcohol added in the data table on the Protein Worksheet.
8. Stir the mixture for 2 minutes using a clean glass stirring rod.
9. Allow the mixture to sit until a precipitate forms (about 2 minutes).
10. Use a 10-mL graduated cylinder to measure half of the solid and liquid into each of the “cytoplasm” centrifuge tubes.
11. Centrifuge for 7 minutes. If the liquid portion is still cloudy, centrifuge for another 3 minutes.
12. Use a Beral-type pipet to carefully remove the liquid layer from the centrifuge tube and place in the ethyl alcohol waste beaker. The solid pellet contains the protein sucrase. Note: Stop here if necessary. Place the centrifuge tubes containing the sucrase pellets in the freezer.
13. Use a clean, graduated cylinder to add 5 mL of deionized water to each centrifuge tube and stir using a clean glass stirring rod until the solid sucrase pellet has dissolved.
14. Centrifuge for 5 minutes. If the liquid portion is still cloudy, centrifuge for another 3 minutes.
15. Use a clean, Beral-type pipet to carefully remove the liquid layer (dissolved protein) and transfer it to the “sucrase” beaker.
16. Use a clean, graduated pipet to add 3 mL of 1% sucrose to both the “control” and “sucrase” test tubes.
17. Use a clean, graduated pipet to add 3 mL of deionized water to the control test tube. Use a clean glass stirring rod to mix the sample.
18. Use a clean, graduated pipet to add 3 mL of the dissolved sucrase solution (step 15) to the sucrase test tube. Use a clean glass stirring rod to mix the sample.
19. Using a test tube clamp, place the two test tubes into a 40 °C water bath for 5 minutes.
20. Remove the test tubes from the 40 °C water bath.
21. Use a clean, graduated pipet to add 2 mL of Benedict’s solution to each test tube and place the test tubes in a boiling water bath for 5–7 minutes or until a change is observed. Note: Benedict’s solution forms an orange-red precipitate in the presence of reducing sugars such as glucose and fructose. Sucrose is not a reducing sugar and should not give a precipitate. (negative test result).

Student Worksheet PDF

10837_Student1.pdf