Denaturation of Proteins

Chasstene Shynne C. Ragmac

Hannah Jean V. Esteban

Instructor

Biochemistry Laboratory

CHY 47.1

TTh 1:00 – 4:00 PM

April 20, 2023

 Denaturation of Proteins

I. Abstract

Proteins are essential for humans to survive. Protein denaturation may temporarily or
permanently disrupt its structure. Disruption of structure will cause the proteins to lose its
function. An example of a complete protein is egg white (albumin). The experiment
investigated the different methods of denaturing the protein found in egg white (albumin);
and it aims understand how the structure of proteins are disrupted under these conditions. A
control group was isolated from the test group. In a triplicate sample, egg white (albumin)
was denatured with different denaturing agents: heat, acid (vinegar), and polar organic
solvent (rubbing alcohol). The denaturants were mixed separately on each test sample and
was observed and compared to the control. Results showed that all the samples have
coagulation, all the denaturants were effective, and all the samples were denatured. Based
on the result, an inference was made that denaturation occurs caused by physical and
chemical factors and that the secondary, tertiary, and quaternary bonds responsible for
maintaining the protein structure were disrupted. Lastly, each of the denaturing agents has
its own specific rate of reaction and has caused different effects on the appearance, texture,
and solubility of the egg white (albumin) because there are different bonds that were
disrupted as well.

II. Introduction

Proteins are amino acid polymers that are essential for all living things. It has
primary, secondary, tertiary, and quaternary structures. Proteins are essential for humans to
survive as it gives energy, helps repair cell, transport molecules, respond to signals, and it is
an important factor in DNA replication process (Campbell, Farrell, & McDougal, 2016). Thus,
if proteins will lose its structure, it will not function properly. Accordingly, an egg is a
complete protein and it has a rich supply of minerals and vitamins (Onyenweaku, et. al,
2022). As defined by Whitehead, Teschke, and Alexandrescu (2022), egg whites (albumin)
are globular proteins, a lengthy, twisted, folded, and coiled less spherical native
conformation. It has a series of weak chemical bonds that maintain the protein structure.
The bonds or forces that help maintain the structure of proteins are the following: hydrogen
bonding, electrostatic/ionic interaction, hydrophobic interaction, and disulfide bonds.
Changing the conformation of the protein and disrupting the bonds that maintain its structure
is called denaturation. It temporarily or permanently disrupts the structure and function of
proteins. When protein is exposed to denaturing agents such as heat, acids/bases, and
alcohols, the structure and bonds will be disrupted causing the proteins to unfold, uncoil, and
change from its native conformation into its denatured state (Munoz-Enano, et. al, 2022). For
instance, heating will break the hydrogen bonding, acids and bases disrupt salt linkages,
application of polar organic solvents such as alcohol will disrupt its hydrogen bonding, heavy
metals will disrupt the disulfide bonds, extreme whipping and agitation will expand the
protein and destroy all its cross linkages. Denaturation usually disrupts the secondary,
tertiary, and quaternary structure of proteins but not the primary structure (Khan, et. al,
2023). Meanwhile, proteins are usually water-soluble but when in its denatured state, the
protein accumulates coagulation and is usually water-insoluble in nature.

To verify or validate the above concept, the experiment was conducted with the
following objectives: to investigate the different methods of denaturing the protein found in
egg white (albumin); and to understand how the structure of proteins are disrupted under
these conditions.

III. Methodology

A. Chemicals, Apparatus, and Materials

The materials used in the experiment are as follows: egg albumin, boiled water,
rubbing alcohol, vinegar, transparent containers, permanent marker, tape and tablespoon.

B. Procedure

In the preparation of egg albumin sample, egg whites and yolks were separated. The
egg whites were placed in a transparent container and the yolks were discarded. In the
preparation of control, one tablespoon egg albumin was placed on a transparent container
labelled as “Control” and was set aside at room temperature. In the denaturation of protein
by heat, one tablespoon egg albumin was placed on a transparent container labelled “A”.
Two tablespoons of boiling water was added to the sample placed on the container and
swirled gently. In the denaturation of protein by acid, one tablespoon egg albumin was
placed on a transparent container labelled “B”. One tablespoon of vinegar was added to the
sample placed on the container and swirled gently. In the denaturation of protein by alcohol,
one tablespoon egg albumin was placed on a transparent container labelled “C”. One
tablespoon of rubbing alcohol was added to the sample placed on the container and swirled
gently. Each container was observed and compared with the control. Observations were
written and recorded.

IV. Results and Discussion

This experiment describes the different ways of denaturing protein. Denaturation is a

process that refers to the disruption of bonds such as hydrogen bonding, electrostatic
attraction, hydrophobic interaction, and disulfide bonds that alters the three-dimensional
shape of proteins affecting its native conformation and losing its function (Faruk, Peng, &
Sosnick, 2023). The hydrogen bonds, ionic attraction, hydrophobic interaction as well as the
disulfide bonds are essential in the folding of peptide bonds, formation of protein structure,
and its function. In the process of protein deformation, the hydrophobic components of the
protein gets unravelled and exposed with the denaturants along with other protein molecules
causing coagulation which leads the denatured form of protein to become insoluble in water.
In this experiment, there are three different types of denaturing agents used: heat, vinegar,
and rubbing alcohol while egg white (albumin) was used as the protein sample.

Table 1. Denaturation of Proteins using Different Denaturants

Sample Observation Remark

No occurrence of
Control No coagulation
denaturation
Sample A (Heat) Coagulation Occurrence of denaturation
Sample B (Vinegar) Coagulation Occurrence of denaturation
Sample C (Rubbing Alcohol) Coagulation Occurrence of denaturation

The results of the experiment are summarized in Table 1. Result shows that the
control has no coagulation observed because no denaturing agent was added to it and
therefore there was no occurrence of denaturation. Sample treated with heat has
coagulation, inferring that there was an occurrence of denaturation. On the other hand, the
sample treated with vinegar has coagulation suggesting that denaturation occurred. Lastly,
the sample treated with alcohol has coagulation observed and therefore denaturation has
happened.

The control group was the untreated sample identical to each test group which was
used to determine whether there are changes that occurred during the denaturation process
of different test samples subjected to heat, vinegar, and rubbing alcohol. According to Chu
and Robledo (2022) the control group is essential in minimizing uncertainties and
marginalizing percentage error of the experimental data.

On the process of denaturing the egg albumin protein by heat, when boiling water
was added to the sample, the protein was denatured which causes coagulation. This was
made possible because the protein structure was disrupted by heat. As the temperature
increases, the kinetic energy also increases causing rapid and violent vibration of molecules
that disrupted the hydrogen bonds and hydrophobic interaction of the protein sample
(Joeres, et al., 2022). Thus the protein lose its structure and function as various parts were
destabilized.

On the other hand, the process of denaturing egg albumin protein was made possibly
because of vinegar which has an acidic entity because of the presence of acetic acid. When
vinegar was added to the sample, coagulation occurred due to the decrease in pH signifying
that the protein was denatured because salt linkages and electrostatic interaction was
disrupted by the ionic property of acetic acid present in vinegar. The disruption happened on
the R-groups of amino acids in the protein structure which causes the unfolding and
straightening of peptide bonds (Ning, et. al, 2022). Electrostatic interaction in an egg albumin
protein structure is important in protein folding, stability, flexibility, and function, when it was
disrupted by the acetic acid in vinegar, the protein was denatured.

Lastly, the egg albumin protein was denatured when alcohol was added to the
sample because there was coagulation that indicates the successful disruption of the
proteins structure and function. This is supported with the statement of Senarat,
Phaechamud, and Narakornwit (2022) which states that the molecules of alcohol compete
with the residues of amino acid in forming new hydrogen bonds, causing the side chains to
be distant from each other and thus breaking the proteins tertiary structure causing the egg
albumin protein to coagulate rapidly. Theoretically, alcohols are polar organic solvents which
has the ability to denature protein by disrupting the hydrogen bonds in the side chains
causing new hydrogen bonds to be formed between the protein side chains and new alcohol
molecule.

Among the three denaturants, alcohol reacted fast and produced the largest amount
of coagulation, followed by the use of heat which reacted moderately, and lastly vinegar
reacted slowly causing few amounts of proteins to coagulate.

V. Conclusion

Denaturation of proteins occur when acted upon physical and/or chemical factors as
it disrupts the bonds or forces that keep proteins in their secondary, tertiary, and
quaternary structures. Heat, pH level, and the polarity of solvents are examples of the
factors that denatures the protein structure. Based on the experiment conducted, the
structure of the protein was altered because the parameters were beyond its normal state.
In the experiment, the addition of boiled water, vinegar and alcohol separately in each
containers leads to the denaturation of protein in egg white (albumin) causing coagulation.
The objectives of the experiment were met and it proved that the denaturing agents affects
the proteins of egg albumin, its appearance, texture, and solubility. Each of the denaturing
agents that was used has different effects in the appearance and texture of the protein.
VI. References

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978-1-305-96113-5

Chaiya, P., Senarat, S., Phaechamud, T., & Narakornwit, W. (2022). In vitro anti-
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Chu, M. M. & Robledo, D. A. (2022). An investigation on the effects of varying temperatures

on gelatin denaturation in response to enzymatic reactions from fruit extracts.
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Faruk, N. F., Peng, X., & Sosnick, T. R. (2023). Factors that control the force needed to
unfold a membrane protein in silico depend on the mode of denaturation.
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Joeres, E., Schölzel, H., Drusch, S., Töpfl, S., Heinz, V., & Terjung, N. (2022). Ohmic vs.
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Munoz-Enano, J., Peytral-Rieu, O., Velez, P., Dubuc, D., Grenier, K., & Martin, F. (2022).
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Ning, X., Wang, L., Jin, S., Fu, X., Sun, X., & Cao, J. (2022). A strategy for sample
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Onyenweaku, E. O., Akah, L. U., Kesa, H., Alawa, D. A., Ebai, P. A., Kalu, U. U., Ajigo, I., &
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Protein Science, 31(5). https://doi.org/10.1002/pro.4321

Documentation

Figure 1. Control group with the three test samples.

Figure 2. Control Figure 3. Effect of heat.

Figure 4. Effect of acid (vinegar). Figure 5. Effect rubbing alcohol.