Materials Included In Kit

Additional Materials Required

Prelab Preparation

Before the lab, you will need to prepare the denaturing solution and the three different concentrations of sodium hydroxide and hydrochloric acid.

To prepare 300 mL of the denaturing solution, combine 180 g of guanidine hydrochloride and 0.61 g of potassium phosphate dibasic with 300 mL of distilled water. The dissolution of guanidine hydrochloride in water is strongly endothermic. Best results are achieved if this solution is prepared the day of the experiment.

To prepare 300 mL of 1 M HCl or NaOH, take 100 mL of the provided 3 M solution and dilute to 300 mL.

To prepare 300 mL of 0.1 M HCl or NaOH, take 30 mL of the 1 M solution and dilute to 300 mL.

Safety Precautions

Sodium hydroxide solution is corrosive to skin and eyes. Avoid body tissue contact. Hydrochloric acid solution is a corrosive liquid and is toxic by ingestion and inhalation. The denaturing solution contains guanidine hydrochloride, which is harmful if swallowed, causes skin irritation and severe eye irritation. Avoid contact of all chemicals with skin and eyes. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron or laboratory coat. Wash hands thoroughly with soap and water before leaving the laboratory. Please review current Safety Data Sheets for additional safety, handling and disposal information. Please follow all laboratory safety guidelines.

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. Excess hydrochloric acid may be neutralized with base according to Flinn Suggested Disposal Method #24b. Sodium hydroxide may be neutralized with acid and then poured down the drain with an excess water according to Flinn Suggested Disposal Method #10. Excess guanidine hydrochloride may be disposed of according to Flinn Suggested Disposal Method #26a, and the denaturing solution according to Flinn Suggested Disposal Method #26b.

Lab Hints

• Due to the thickness of the egg yolk, it is a good idea to cut the tip off the pipet so it has a wider nozzle.
• Please remind students that nothing that has been in the lab should ever be consumed. The chemicals that are being mixed with the eggs are hazardous.
• One egg provides more than enough material for two groups to use. However, it is recommended to allocate one egg per group since some students will struggle with separating the yolk from the whites.

Teacher Tips

• The original experiment on which this lab is based used an 8 M buffered urea solution and took eight hours to denature the egg whites. This denaturant for this lab has been changed to provide a more obvious reaction within a single lab period. However, the egg whites will not fully dissolve, even if left overnight.

Further Extensions

Online Educational Resources

Foldit, a gamified protein structure determination program: https://fold.it/portal/

Protein Data Bank, a repository of protein structures: https://www.rcsb.org/

Answers to Prelab Questions

1. Check your understanding with a drag-and-drop activity to arrange the following interactions from weakest to strongest.

Dispersion forces, Dipole-dipole interactions, Hydrogen bond

2. Identify the strongest intermolecular force in each of the molecular compounds. Drag each formula to the correct column in the chart.

3. How many nonwater atoms are present in ovalbumin?

11591

Answers to Questions

1. What did you observe for the egg whites in water?

The egg whites sink to the bottom of the water. No changes are observed.

2. What did you observe for the egg whites in the denaturing solution?

The egg whites float in the denaturing solution. After ten minutes, they take on a fluffy appearance and a concentration gradient of protein dissolving into the solution can be observed when the sample is gently swirled. At the end of the lab session, the undissolved egg whites have become translucent.

3. Propose an explanation for these observations.

Water is unable to interact strongly with the tangled proteins, so they remain as they are. The denaturing solution is able to disrupt the intermolecular forces that caused the proteins to tangle and separate them.

4. What did you observe when the yolk was added to the different concentrations of sodium hydroxide?

The egg yolk in 0.1 M solution mostly dispersed; however, a small amount of yolk was observed to be clumped together. Both the 1 M and 3 M solutions cooked the yolk, with no obvious change to the color of the solution.

5. What did you observe when the yolk was added to the different concentrations of hydrochloric acid?

The egg yolk in 0.1 M solution dispersed throughout the solution when agitated. The yolk in the 1 M solution only dispersed slightly with most of the yolk staying together. The yolk in 3 M acid took on a cooked appearance, a small amount of egg white was added along with the yolk, and it took on an opaque white appearance.

6. From your results, which is better at cooking egg yolk, acid or base?

From observations in Part 2, the conclusion is that the base is better at cooking egg yolk than the acid.

7. Propose an explanation for your answer to Question 6.

While the exact mechanism by which chemical denaturing occurs is not known, it is generally accepted that it must involve disruption of the intramolecular forces within the proteins. Hydrochloric acid is able to protonate the amino acids within the protein, disrupting the superstructure. Sodium hydroxide is able to deprotonate the amino acids, which would also disrupt the superstructure. In addition, the hydroxide groups are able to hydrogen bond, which could further explain why it is observed to be better at cooking the yolk. Another consideration is that egg yolk contains a large amount of fat, which can undergo a saponification reaction with the sodium hydroxide.

References

Tom Z. Yuan, Callum F. G. Ormonde, Stephan T. Kudlacek, Sameeran Kunche, Joshua N. Smith, William A. Brown, Kaitlin M. Pugliese, Tivoli J. Olsen, Mariam Iftikhar, Colin L. Raston and Gregory A. Weiss, ChemBioChem  2015, 16, 393–396.

Stein, P.E., Leslie, A.G., Finch, J.T., Carrell, R.W., J. Mol. Biol  1991, 221, 941–959.

A.S. Rose, A.R. Bradley, Y. Valasatava, J.M. Duarte, A. Prlić and P.W. Rose, ACM Proceedings of the 21st International Conference on Web3D Technology 2016, 185–186.

A.S. Rose and P.W. Hildebrand, Nucl Acids Res 2015, 43(W1), W576–W579.

Introduction

Chemistry and chemical processes are all around us. The very act of cooking involves promoting certain reactions in order to achieve a certain texture and taste. While most of the time heat is used when cooking it doesn’t have to be. Ceviche is a fish dish where the acids found in citrus fruits are used to cook and flavor the fish rather than heat. Sous-vide is a cooking method where food is cook for longer at lower temperatures to give greater control over the final product. In this laboratory you will learn about the chemical reactions involved during cooking as well as conduct experiments relating to the 2015 paper by Yuan et al where they reported being able to uncook egg whites.

Concepts

• Thermodynamics
• Biochemistry
• Food science
• Intramolecular forces
• Intermolecular forces

Background

The three main components of food are carbohydrates (sugars and starches), fats and proteins. Traditional cooking methods involve using heat to induce chemical reactions in these compounds. For example, sugars are caramelized, fats are rendered and proteins are denatured. It is the third of these processes, the denaturing of proteins, that is the focus of this laboratory.

Proteins are large molecules comprised of amino acids. Proteins are responsible for performing a wide range of tasks within the body, with variations in the amino acid sequence as well as the make-up of the active site determining function. In order for most proteins to function properly, they need to have a specific shape. Figure 1 shows an animated representation of ovalbumin, the major protein found in egg whites. The shape of a protein arises through the maximization of intramolecular forces. These intramolecular forces are the same types of interactions you may have previously heard referred to as intermolecular forces, such as dispersion forces, dipole-dipole interactions and hydrogen bonding. Here they are called intramolecular forces because they are occurring within a single macromolecule rather than between two different molecules.

The weakest type of interaction is the dispersion force. All molecules exhibit this type of interaction, which arises through the movement of electrons generating temporary regions of positive and negative charge (see Figure 2). Because larger atoms and molecules have more electrons, they are able to exhibit stronger dispersion forces.

Dipole-dipole interactions occur between polar regions of molecules. When two atoms share electrons unequally the result is that one end of the bond will be slightly positive and the other end slightly negative. The positive end of the bond will be attracted to the negative end of another bond (see Figure 3). Since these charged regions are always present, they are stronger than the temporary interactions that arise through dispersion force interactions.

The final interaction we will be considering in this lab is the hydrogen bond. Hydrogen bonds occur between the lone pair on a nitrogen, oxygen, or fluorine atom and a hydrogen atom, which is bound to another nitrogen, oxygen or fluorine (see Figure 4). The electrons in a bond between hydrogen and either nitrogen, oxygen, or fluorine are primarily associated with the non-hydrogen atom, resulting in a positively charged hydrogen. The electron lone pairs on nitrogen, oxygen and fluorine are relatively dense regions of negative charge. The attractive forces between the negatively charged lone pair and the positive hydrogen atom result in an interaction that is even stronger than a standard dipole-dipole attraction.

Proteins fold in order to maximize the number of positive interactions (see Figure 5). Although hydrogen atoms have been omitted for the sake of clarity, the dashed lines indicate locations of hydrogen bond interactions. When cooking, heat energy causes the protein chains to vibrate. As the temperature increases, these vibrations become stronger and eventually the proteins will shake themselves apart. When a protein comes apart, it is referred to as denatured. Once the protein is denatured, the strands will begin to tangle together and what were once intramolecular interactions become intermolecular interactions. A good way to think about what is happening is to imagine several long strips of Velcro^®. By exerting energy, you can pull the two halves of the strips apart, if you then begin to toss the separated strands around they will snag and tangle together.

To reverse this cooking process, you would need to untangle the strands (which is thermodynamically unfavorable) and the refold the protein back into its original shape (which is entropically unfavorable). Due to the thermodynamic and entropic hindrances associated with reversing the cooking process we say that it is an irreversible reaction. Yuan et al developed a technique for refolding proteins, using a combination of sheer stress and chemical denaturants. Cooked diluted egg whites were dissolved in 8 M urea overnight. Urea is able to disrupt the noncovalent bonds that are holding the tangled proteins together. Once the proteins have been separated they are spun to introduce a sheer stress. This stress will cause sub-optimally folded proteins to denature, effectively giving them as many chances as needed to refold into their original shape. It is important to note that the speed and duration of the spinning needs to be carefully controlled so as to not also unfold correctly folded protein strands.

Experiment Overview

In this laboratory, you will explore the use of chemicals as an alternative to heat when cooking as well as untangle cooked egg whites. Unfortunately, due to technical limitations, you will not be able to do the sheer stress mediated protein refolding that comprises the second half of the uncooking process.

Materials

Prelab Questions

1. Check your understanding with a drag-and-drop activity to arrange the following interactions from weakest to strongest.
2. Identify the strongest intermolecular force in each of the molecular compounds. Drag each formula to the correct column in the chart.

Head over to the Protein Data Bank website (https://www.rcsb.org/) and find the ovalbumin protein 1OVA.

3. How many nonwater atoms are present in ovalbumin?
4. Look up at least two more proteins in the protein data bank. Give each protein’s unique identifier and a brief summary of where is it found and its function.

Safety Precautions

Sodium hydroxide solution is corrosive to skin and eyes. Avoid body tissue contact. Hydrochloric acid solution is a corrosive liquid and is toxic by ingestion and inhalation. The denaturing solution contains guanidine hydrochloride, which is harmful if swallowed, causes skin irritation and severe eye irritation. Avoid contact of all chemicals with skin and eyes. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron or laboratory coat. Wash hands thoroughly with soap and water before leaving the laboratory. Please review current Safety Data Sheets for additional safety, handling and disposal information. Please follow all laboratory safety guidelines.

Procedure

Part 1. Uncooking Egg Whites

1. Crack an egg and separate the yolk and the white. Set aside the yolk for use in Part 2.
2. Place the egg white in a boiling tube.
3. Half fill the 500 mL beaker and place it on the hot plate.
4. Once the water comes to a gentle boil, place the boiling tube in the hot water bath.
5. Wait for the egg whites to become fully cooked (this can take up to 20 minutes).
6. Decant any excess fluid from the tubes, then roughly chop the egg whites with a spatula.
7. Wash the cooked egg whites twice with distilled water. The water can be discarded in the sink.
8. Dry and transfer about 1 g of cooked egg white into a test tube with a screw cap.
9. Repeat step 9 with a second test tube.
10. To the first test tube, add 10 mL of water and screw on the cap.
11. To the second test tube, add 10 mL of the denaturing solution and screw on the cap.
12. Place both test tubes into the test tube rack.
13. After 10 minutes, gently swirl each test tube and record your observations.

Part 2. Cooking with Chemicals

1. Obtain three 50 mL beakers and label them as 0.1 M, 1 M and 3 M.
2. Add 20 mL of the corresponding sodium hydroxide solution to each beaker.
3. Squirt a small amount of yolk into each beaker and record your observations.
4. Repeat steps 1–3, this time using hydrochloric acid instead of sodium hydroxide.
5. Return to your samples from Part 1. Again, swirl each sample and record your observations.
6. Consult your instructor for appropriate disposal procedures.

Questions

1. What did you observe for the egg whites in water?
2. What did you observe for the egg whites in the denaturing solution?
3. Propose an explanation for these observations.
4. What did you observe when the yolk was added to the different concentrations of sodium hydroxide?
5. What did you observe when the yolk was added to the different concentrations of hydrochloric acid?
6. From your results, which is better at cooking egg yolk, acid or base?
7. Propose an explanation for your answer to Question 6.