Essential Protein and Enzyme

Introduction

Biochemistry is a diverse scientific discipline encompassing chemical processes of living things. Essential life processes that may be understood using biochemical principles include cell signaling, energy and metabolism and genetics. Each of these demonstrations teaches students about the biochemistry occurring in their own bodies.

Use this set of engaging demonstrations to help students understand major principles of protein and enzyme biochemistry.

1. pH and Protein Solubility—Any changes in the pH of a protein’s environment will cause observable changes in the solubility of the protein. This demonstration looks at the reversible solubility behavior of casein, the main protein in milk, as hydrochloric acid or sodium hydroxide is added to the protein.
2. Digestive Enzymes at Work—Observe the biochemistry of digestion and learn how the body transforms the food we eat into essential nutrients needed to carry out cell processes and growth.
3. The Floating Catalyst—An enzyme may make a chemical reaction occur a million times faster than it would in the absence of a catalyst. Examine the reaction of catalase, which catalyzes the decomposition of hydrogen peroxide in plant and animal tissues.

This kit also includes the Flinn ChemTopic™: Biochemistry—The Molecules of Life. This book contains four experiments and five demonstrations that allow students to explore the relationship between the biological world and the chemical world. Students examine the structures and properties of key classes of biological molecules.

Materials Included In Kit

Additional Materials Required

Experiment Overview

pH and Protein Solubility

Any change in the pH of a protein’s environment will cause observable changes in the solubility of the protein. Solubility changes, in turn, reflect changes in the three-dimensional structure of the protein. The effect of pH on protein solubility explains why most enzymes function well at an optimum pH, and why their activity decreases substantially at pH values other than the optimum. This demonstration examines the effect of pH on the solubility and structure of casein, a milk protein.

Digestive Enzymes at Work

People must eat to live but how does the body transform food into the essential nutrients (peptides, amino acids, fatty acids and glucose) needed to carry out cell processes and growth? This demonstration introduces the biochemistry of digestion.

The Floating Catalyst

Almost all chemical reactions that take place in living organisms are catalyzed by enzymes—nature’s catalysts. A typical enzyme may make a chemical reaction occur about one million times faster than it would without a catalyst. This demonstration looks at the reaction of catalase, which catalyzes the decomposition of hydrogen peroxide in plant and animal tissue.

Materials

Safety Precautions

Hydrochloric acid solution is a corrosive liquid, is toxic by ingestion and inhalation and is an eye and skin irritant. Sodium hydroxide solution is corrosive and is especially dangerous to the eyes. Universal indicator is a flammable liquid—keep away from flames and heat. Avoid contact of all chemicals with eyes and skin. Biuret test solution contains copper(II) sulfate and sodium hydroxide and is a corrosive liquid. It is moderately toxic by ingestion and is dangerous to skin and eyes. Iodine solution contains iodine and potassium iodide and is an eye and skin irritant; it will stain skin and clothing. Avoid contact of all chemicals with eyes and skin. Hydrogen peroxide is a strong oxidizing agent and may be irritating to eyes and skin. Avoid contact of all chemicals with eyes and skin. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory. Follow all laboratory safety guidelines. 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. The casein solution may be stored at basic pH for several months. Alternatively, the solution may be rinsed down the drain with excess water according to Flinn Suggested Disposal Method #26b. Excess biuret test solution may be neutralized with acid according to Flinn Suggested Disposal Method #10. Excess hydrochloric acid may be neutralized with base according to Flinn Suggested Disposal Method #24b. Excess iodine solution may be reduced with sodium thiosulfate solution according to Flinn Suggested Disposal Method #12a.

Prelab Preparation

Digestive Enzymes at Work

Prepare the following solutions up to five days in advance of the lab.

• Use 100 mL of DI water to prepare a 1% albumin (protein) solution. Add 100 mL of the DI water to 1 g of albumin. Gently mix and refrigerate.
• To prepare starch solution, boil 100 mL of DI water. Add a small amount of the boiling DI water to 0.5 g of starch. Mix well, forming a paste. Continue to add 10 mL of boiling water to the beaker or flask until the entire 100 mL of boiling water has been added. Allow the solution to slowly cool to room temperature or refrigerate.
• Prepare 100 mL of 0.01 M hydrochloric acid from 1 M hydrochloric acid. Fill a 100-mL volumetric flask about half full with distilled or deionized water. Using a graduated pipet measure 1 mL of 1 M HCl and add it to the flask and swirl. Dilute to 100-mL with DI water.

Prepare the following solution the day of the lab.

• Prepare a 1% pepsin solution by adding 50 mL of 0.01 M hydrochloric acid to 0.5 g of pepsin. Mix well. The solution should have a pH of 1.5 to 2.5.
• Use 100 mL of DI water to prepare 1% amylase solution. Add 100 mL DI water to 1 g of amylase. Mix well.

The Floating Catalyst

Prepare the catalase solution.

1. Fill a 100-mL volumetric flask about half-full with distilled or deionized water.
2. Measure 0.01 g of catalase and add it to the volumetric flask. Swirl until dissolved.
3. Fill with DI water to the 100-mL mark and mix.
4. Test the activity of the enzyme solution using a 2% hydrogen peroxide solution before performing the demonstration. Adjust the concentration as needed to obtain convenient “floating” times (neither too fast nor too slow).

Procedure

pH and Protein Solubility

1. Add 25 mL of 0.1 M sodium hydroxide and a stirring bar to a 600-mL beaker or flask and place the beaker on a magnetic stirrer. Add 225 mL of distilled or deionized water and stir at moderate speed. Add 1 g of casein and stir to dissolve. (The solution will be slightly cloudy or translucent.)
2. (Optional) Add 1–2 mL of universal indicator to observe pH changes, if desired. Consult the universal indicator color chart for pH values.
3. With rapid stirring, add 2 M hydrochloric acid in 0.5-mL increments using a graduated, Beral-type pipet. (The solution will turn cloudy, but will clear up again as the hydrochloric acid is dispersed. After 1–2 mL of acid has been added, the cloudiness will reach a maximum—this is the isoelectric point. The pH at the isoelectric point is 4–5.)
4. Once the isoelectric point has been reached, pause just long enough to record observations (15–20 seconds.) Continue adding 2 M hydrochloric acid in 0.5-mL increments with stirring until the solution is clear again. (The cloudiness will fade and the precipitate will redissolve after the addition of another 2–3 mL of acid, when the pH drops below the isoelectric point, pH ≤2.)
5. Continue to stir the solution. Reverse the process by adding 2 M sodium hydroxide in 0.5-mL increments using a clean, graduated, Beral-type pipet. (After 2–3 mL of sodium hydroxide has been added, the protein will precipitate out again at the isoelectric point.)
6. Continue adding sodium hydroxide in 0.5-mL increments with stirring until the solution is clear again. (The solution will clear up after an additional 1–2 mL of sodium hydroxide has been added and the pH >10–12.)
7. The process may be repeated using the same casein solution. This will give time to explain the observations.

Digestive Enzymes at Work

Part A. Protein Digestion

1. Add 50 mL of the 1% albumin solution to each of two 500-mL beakers.
2. Add 50 mL of DI water to one of the two beakers. Mix well.
3. Add 50 mL of the 1% pepsin solution to the second beaker. Mix well.
4. Wait 2 minutes before adding 10 mL of biuret test solution to each beaker. Mix well.
5. Observe the color and appearance of the resulting solution in each beaker and record observations in the data table on the worksheet. Note: Biuret test solution is bluish-purple in the presence of proteins and polypeptides and lavender pink in the presence of amino acids.

Part B. Carbohydrate Digestion

6. Add 50 mL of the 1% starch solution to each of two 500-mL beakers.
7. Add about 5 drops of iodine to each beaker.
8. Add 50 mL of DI water to one of the two beakers. Mix well.
9. Add 50 mL of the 1% amylase solution to the second beaker. Mix well.
10. Observe the color and appearance of the resulting solution in each beaker and record observations in the data table on the worksheet. Note: Dark blue-black color indicating a positive starch test will fade in the cup containing the amylase solution as the enzyme digests the starch into sugars.

The Floating Catalyst

1. Label four 600-mL beakers A–D.
2. Prepare a series of hydrogen peroxide solutions at different concentrations as shown in Table 1. Measure and add the appropriate amounts of 3% hydrogen peroxide and distilled water into each beaker and stir to mix. Use a 100-mL or 500-mL graduated cylinder to measure the liquid volumes as needed.

3. Pour 20–30 mL of catalase solution into a wide, shallow container such as an evaporating dish or a Petri dish.
4. Immerse four pieces of filter paper in the catalase solution and soak the filter paper for 2–3 minutes.
5. Using forceps or tongs, remove the filter paper from the catalase solution and gently blot dry on a paper towel.
6. Using forceps or tongs, submerge one piece of filter paper in the bottom of the hydrogen peroxide solution in Beaker A. Release the filter paper and immediately start timing. (The solution will start bubbling at the surface of the filter paper and the filter paper will gradually float to the surface of the hydrogen peroxide solution.)
7. Measure and record the time in seconds when the center of the filter paper touches the surface of the solution.
8. Repeat steps 6 and 7 three more times in Beakers B, C and D. Use a fresh piece of catalase-soaked filter paper for each.
9. (Optional) Repeat the demonstration using fresh pieces of catalase-soaked filter paper and average the reaction times at each concentration. It is not necessary to prepare fresh hydrogen peroxide solutions—the solutions in the beakers may be used several times.
10. Compare the reaction times for the four concentrations of hydrogen peroxide. How does the concentration of hydrogen peroxide affect the rate of the catalase reaction?

Student Worksheet PDF

11090_Student1.pdf

Teacher Tips

• For Digestive Enzymes at Work, the biuret test solution does not contain the compound biuret. Biuret is the simplest compound that gives a positive test result with biuret test solution.

• For an advanced class you may want to use the Digestive Enzymes at Work activity to introduce the terms substrate, active site, cofactors, coenzymes and the induced fit theory.
• In order to help students understand that amylase is found in saliva, have each student chew on an unsalted, unsweetened saltine type cracker until the cracker tastes sweet. The sweetness is due to the amylase hydrolyzing the starch into glucose and other mono- and disaccharides. Complete the Digestive Enzymes at Work activity in a food-appropriate area.
• In The Floating Catalyst, the time required for the filter paper to float to the surface depends on the activity of the catalase solution, which is related to its concentration and to the purity or activity of the enzyme itself. The shelf life of many enzymes is poor. Store enzymes in the refrigerator and use them within one year of purchase.
• The enzyme catalase may be extracted from living tissue. Cut small sections (about 1 cm³) of potato or beef liver, mash or grind them, and soak the pulp in 50 mL of ice-cold distilled water for 10 minutes. Strain the extract through cheesecloth and test its activity in 2% hydrogen peroxide. Dilute the extract, if necessary, to obtain convenient reaction times.
• Catalysts cause slow reactions to occur more quickly by lowering the activation energy necessary for the reaction to occur. A ski lift is an analogy for a catalyst. If the reaction is “skiing,” then the skier must first get to the top of the ski hill. One option is for skiers to climb to the top and once they reach the top, enjoy the potential energy they earned as they ski back down the hill. The ski lift allows many more skiers to reach the top of the hill very quickly without the skiers expending much energy. Once at the top, they still enjoy the same energy release as they ski down the ski hill. The reaction can occur many, many more times and the ski lift (the catalyst) is not changed during the process.

Sample Data

Demonstration 1—pH and Protein Solubility

Demonstration 2—Digestive Enzymes at Work

Demonstration 3—The Floating Catalyst

Answers to Questions

Demonstration 1—pH and Protein Solubility

1. Casein has both acidic side chains and basic side chains. At a high (basic) pH, ionization occurs in the acidic chains. At a very low (acidic) pH, protonation occurs in the basic chains. Do you think casein is most soluble with a net charge that is positive, negative, or around zero? Why?

Casein is most soluble when it has either a highly positive or highly negative charge. Bases ionize its acidic chains, resulting in a negative charge, and acids protonate its basic chains, resulting in a positive charge. During the demonstration, there was the least amount of solid in the solution at the pH extremes. Therefore, casein is the least soluble when the net charge is zero.

2. A protein’s isoelectric point is the pH at which the protein has a net charge of zero. Approximate the isoelectric point of casein.

The isoelectric point of casein is probably around 4–5, because that is when the solution is cloudiest during the demonstration.

Demonstration 2—Digestive Enzymes at Work

3. Compare and contrast the observations of the biuret test results. Describe the evidence, if any, for the digestion of protein using pepsin.

The beaker containing protein solution, water, and biuret test solution is the negative control. The solution is a cloudy, blue-purple color indicating this cup is positive for polypeptides. The beaker containing protein solution, pepsin and biuret is a clear, pink-purple color. Pepsin digests the albumin protein leaving peptides which gives a pink-purple biuret test.

4. The pepsin solution was prepared using 0.01 M hydrochloric acid in order to optimize the pepsin enzyme. Why was this necessary?

Pepsin is active in the acidic environment of the stomach. A basic or neutral pH would inactivate the enzyme.

5. Compare and contrast the iodine test results for starch and starch/amylase. Explain the test results based on the activity of amylase.

The beaker containing the starch, water and iodine solution is the control sample yielding a positive iodine test for starch. The remaining beaker contains starch, amylase and iodine. It has a negative iodine test because the amylase has hydrolyzed the starch to glucose.

Demonstration 3—The Floating Catalyst

6. What is the purpose of catalase in the human body?

It prevents hydrogen peroxide from accumulating at dangerous levels within the body.

7. Create a graph comparing the Average Reaction Rate to the Concentration of H₂O₂ (%).

Student answers will reflect data obtained during the class demonstration.

8. Examine the graph from Question 7. How does the reaction rate change at high versus low concentrations of hydrogen peroxide?

At low concentrations of hydrogen peroxide the rate of reaction increases almost linearly as the concentration increases. At higher concentrations of hydrogen peroxide the relationship changes as the reaction rate becomes more gradual.

Discussion

pH and Protein Solubility

Casein is the principal protein in milk (80% of the total protein content). Casein is a phosphoprotein—it contains a large number of phosphate groups attached to the amino acid side chains in its polypeptide structure. The negatively charged phosphate groups are balanced by positively charged calcium ions and are responsible for the high nutritional calcium content in milk. Casein is almost completely insoluble in water at neutral pH (pH = 7).

Casein, like other proteins, is an ionic species containing amino groups and carboxyl groups on its terminal amino acids. It also contains a variety of other acidic and basic groups on the side chains of its non-terminal amino acids. The effect of pH on the solubility of casein reflects the ionization of the acidic and basic groups in its structure.

At high pH, casein will have a net negative charge due to ionization of all the acidic side chains (—CO₂^–) in its structure. Because casein is ionized at high pH values, it is soluble in dilute sodium hydroxide solution.

At low pH, casein will have a net positive charge due to protonation of all basic side chains (—NH₃⁺) in its structure. Because casein is ionized at low pH values, casein is also soluble in strongly acidic solutions.

At intermediate pH values, casein will contain roughly equal numbers of positively and negatively charged groups and the protein will have a net charge of zero. Casein is insoluble in neutral solutions because it is not charged under these conditions.

The solubility of a protein is usually at a minimum at its isoelectric point. The isoelectric point is defined as the pH at which a protein has a net charge of zero. For casein, due to the attached phosphate groups, the isoelectric point is close to pH = 4.

Digestive Enzymes at Work

Digestion begins in the mouth. The food mixes with saliva while the teeth grind the food. Saliva provides the first chemical treatment of the food. Saliva is composed of a neutral pH mixture of water, mucus, proteins, mineral salts and the enzyme amylase. Amylase begins the breakdown of starch, a carbohydrate, into glucose. Glucose is the sugar used during cellular respiration as a source of energy.

In the stomach gastric juices containing mucus, hydrochloric acid, pepsinogen and small amounts of other enzymes continue the process of digestion. Hydrochloric acid acts to denature (uncoil) the proteins in food and activates pepsinogen, the inactive precursor of the enzyme pepsin. Glucose, alcohol, fat-soluble drugs, some salts and small amounts of water are absorbed through the walls of the stomach directly into the bloodstream for transport to the liver, where they are metabolized or sent on to other cells in the body.

Once in the small intestine, the remaining food combines with enzymes from the pancreas and epithelial cells of the small intestine and with bile salts from the liver. The digestion of carbohydrates into glucose and other simple sugars is completed in the small intestine by the enzymes sucrase, maltase, lactase and pancreatic amylase. The resulting sugars are absorbed through the mucous lining of the small intestine into the bloodstream for transport to the liver.

The Floating Catalyst

A catalyst is a substance that increases the rate of a chemical reaction without itself being consumed during the reaction. Decomposition of hydrogen peroxide to produce water and oxygen gas (Equation 1) is energetically favorable but very slow in the absence of a catalyst.

In nature, this reaction is catalyzed by the enzyme catalase. This is an important reaction within cells. Hydrogen peroxide is generated as a by-product of metabolic processes and the catalase enzyme prevents the accumulation of dangerous levels of this toxic chemical. The rate of the catalase reaction can be determined by measuring the time required for the enzyme-soaked filter paper disk to rise to the surface in a solution of hydrogen peroxide. Oxygen bubbles form on the filter paper and cause it to float. The rate of the reaction is inversely related to the reaction time. See Table 2 for sample data and results.

At low concentrations of hydrogen peroxide, the rate of the reaction increases almost linearly as the concentration increases. At higher concentrations of hydrogen peroxide, the enzyme-catalyzed reaction behaves differently than a typical chemical reaction—the rate increase becomes more gradual. Eventually, the rate of the enzyme-catalyzed reaction would be expected to level off or reach a maximum value.