NAMES:

Adam, Piang Samir III T. Bitte, Dannesa O. Caina, Thea Marie M.

Amilasan, Arwin Jos e D. Brillantes, Ellen Joyce L. Castillano, Kharyl Roisse C.
Barnes, Winona Ruth M. Burtado, Jovy Pauline M.
SECTION:​ BSN – 1H
DATE: ​September 18, 2019

EXPERIMENT 2B

PROTEIN DENATURATION

Objectives

● To observe the effects of several denaturing reagents on a protein sample

● To differentiate the effect of these denaturing reagents to the protein sample

Theoretical Background

Protein Denaturation involves the disruption and possible destruction of its

quaternary, tertiary, and secondary structures. Denaturation reactions are not strong

enough to break the peptide bonds, which is the primary structure and sequence of

amino acids, thus it remains the same after a denaturation process. Denaturation

disrupts the normal alpha-helix and beta pleated sheets in a protein and uncoils it into a

random shape, where it results to protein precipitation or coagulation. Furthermore,

denaturation occurs because the bonding interactions that links the protein's structure

are disrupted. These interactions could be disulfide bonds, non- polar hydrophobic

interactions, hydrogen bonds, and ionic interactions or salt bridges. In the experiment,

there are a variety of agents and conditions used that can cause protein denaturation.

These chemical agents are heat, organic solvents, heavy metal, strong mineral acids,

and alkaloidal reagents.

 Heat can supply kinetic energy to protein molecules, causing their atoms

to vibrate more rapidly. This will disrupt relatively weak forces such as hydrogen bonds

and hydrophobic interactions. A practical example of using heat is the usage in

sterilization hospital paraphernalias to denature and destroy the enzymes in bacteria.

Organic compounds such as alcohol are capable of engaging in

intermolecular hydrogen bonding with protein molecules, disrupting intramolecular

hydrogen bonding within the protein. Hydrogen bonding occurs between amide groups

in the ​secondary protein structure​. Hydrogen bonding between side chains occurs in

t​ertiary protein structure in a variety of amino acid combinations. ​In its denaturation, new

hydrogen bonds are formed instead between the new alcohol molecule and the protein

side chains. Since alcohols are capable of forming hydrogen bonds with protein

molecules, it will disorganized the hydrogen bonding within the molecule. Organic

solvents, specifically alcohol, can be used as a disinfectant where ​it is used in toiletries,

pharmaceuticals, and fuels.

Mineral acids enhance hydrophobic aggregation and thus change the

conformation of protein. There is disruption of salt bridges connected by intermolecular

forces or ionic bonds. ​Salt bridges result from the neutralization of an acid and amine on

side chains. Any combination of the various acidic or amine amino acid side chains will

have a disrupting effect. Excessive acidity can cause basic amino acids like lysine and

asparagine to lose their negative charge via protonation, the addition of a proton (H+) to

an atom, molecule, or ion, and lose the ability to participate in charge interactions.

Some of these amino acids are polar, having positively charged sides and negatively
charged sides. A change in pH simply means a change in the amount of (H+) atoms.

These hydrogen atoms are positively charged, and attract the negative side of the polar

amino acids. So, a change in the pH changes the stability of a protein structure and can

cause its denaturation. A high concentration of hydrogen ions, which signifies a low pH,

will result in more groups being protonated.

Alkaloidal Reagents causes protein atoms to vibrate more rapidly and

crystallize the protein. It combines with positively charged amino groups in proteins thus

disrupting ionic bonds. It also engages in intermolecular hydrogen bonding with protein

molecules, strengthening intramolecular hydrogen bonding within the protein. These

reagents combine with positively charged amino groups in proteins to disrupt ionic

bonds. The negative charge of these anions counteracts the positive charge of the

amino group in proteins giving a precipitate. Precipitation of proteins by alkaloidal

reagents indicates the presence of both negative and positive charges and hence the

amphoteric nature of proteins. Ultimately, alkaloidal agents affects the pH balance of the

proteins hence another reason for its denaturation. An example of its denaturation is

gastric juices causing curdling of milk during digestion where Chymosin efficiently

converts liquid milk to a semisolid like cottage cheese. This example both applied for

alkaloidal agent and strong mineral acids as both affects the pH of the protein.

Heavy metal disrupts salt bridges. These are held together by ionic

charges hence, heavy metal disrupts ionic bonding. A type of double replacement

reaction occurs where the positive and negative ions in the salt change partners with

the positive and negative ions in the new acid or base is added. Heavy metals may also
disrupt disulfide bonds or disulfide linkages because of their high affinity and attraction

for sulfur and will also lead to the denaturation of proteins. This reaction is used for its

disinfectant properties in external applications, such as Silver nitrate (AgNO​3​). It is used

as a prevention of gonorrhea infections in newborn infant’s eyes, and treatment of nose

and throat infections.

Data and Result

Denaturing Observation Precipitation Documentation

Agents

Heat The Albumin Solution was Heavy

transparent or clear but Precipitation
when it was heated, it
coagulated and turned
completely white.

Alcohol 95% Ethanol Heavy Before Stirring:

Before stirring the Precipitation
albumin solution with the
alcohol, it formed three
layers, where the second
layer is composed of the
coagulated protein. When
it was stirred, the solution
turned white.

After Stirring:
 70% Ethanol Slight Before Stirring:
Before stirring the Precipitation
albumin solution with the
alcohol, it formed three
layers, where the second
layer is composed of the
coagulated protein. When
it was mixed, the solution
became white, yet 95%
Ethanol Solution is whiter
than the 70% Ethanol
Solution. After Stirring:

Heavy Metal AgNO​3 Heavy From Left to Right

The color turned into Precipitation (AgNO​3​, CuSO​4​, ​ BaCl​
​ 2​)
white and has formed
particles.

CuSO​4 Slight
The color turned blue and Precipitation
has also formed some
particles.

BaCl​2 No
It remained transparent Precipitation
and there were no
particles formed.
 Strong H​2​SO​4 Heavy From Left to Right
Mineral The precipitation was Precipitation (H​2​SO​4​,HNO​2​)
Acids clearly observed. There
was a separation of
solution and reagents. It
turned white, where the
solution solidified

HNO​2 Heavy
The precipitation was Precipitation
clearly observed. After
mixing the chemicals and
egg combined, it turned
yellow.

Alkaloidal Picric Acid Heavy From Left to Right

Reagents Color turned into yellow Precipitation (Trichloroacetic Acid,
and albumin solution Tannic Acid, Picric Acid)
coagulated

Tannic Acid Slight

Color turned into brown Precipitation
and solid particles are
slightly seen

Trichloroacetic Acid Heavy

Color turned into opaque Precipitation
white and albumin
solution coagulated
Data Analysis

There are various ways to denature proteins. In this experiment, the

agents that will be used as denaturants are heat, alcohol, heavy metals, strong mineral

acids, and alkaloidal reagents. The albumin solution will be used as the primary subject

for protein.

In the experiment, the Albumin Solution was transparent before heating.

Then when it was heated, the albumin solution started to shake or vibrate violently and

its distinct transparency slowly changed. After heating, it coagulated and turned

completely white hence, changing its characteristics. The albumin solution, which is rich

in protein, changed because the heat disrupted its hydrogen bonds. Hydrogen bonds

plays a significant role in protein folding, which determines the functionality of a protein.

Also, it disrupted the non-polar hydrophobic interactions, which is the driving force

responsible for the folding of proteins. Heat naturally increased the kinetic energy of

molecules and this nature is similar to protein denaturation. Due to the increase rate of

molecular movement, the protein started to violently vibrate, where it will lose its

three-dimensional conformation, hence coagulation occurred when molecules stick to

each other and formed a dense network, making it insoluble.

In the protein denaturation using organic solvents such as alcohol, the

researchers used two different percentage of alcohol. The usage of 95% ethanol

resulted in heavy precipitation where the coagulation is more distinct than the 70%

ethanol. This means the percentage of alcohol is directly proportional to the amount of

proteins precipitated. Alcohol denatures proteins by disrupting the side chain

intramolecular hydrogen bonding. New hydrogen bonds are formed instead between the

new alcohol molecule and the protein side chains. Since alcohols are capable of

forming hydrogen bonds with protein molecules, this will disorganized the hydrogen

bonding within the molecule. The difference between the two percentage of ethanol is

that the 95% alcohol coagulated prominently than the 70% alcohol because it

precipitated protein in contact. The alcohol will go through the cell wall of the organism

in all direction, coagulating the protein just inside the cell wall. The ring of the

coagulated protein would then stop the alcohol from penetrating farther from the cell,

and no more coagulation would take place. At this time the cell would become inactive

but not dead hence under the favorable conditions, the cell would then begin to function

again. The 70 percent of alcohol also coagulated the protein, but at a slower rate. It

penetrated all the way through the cell before coagulation can block it then, the entire

cell is coagulated and the organism dies. This is the reason why the 70% alcohol is

favorable to a stronger solution thus making an effective disinfectant. In correlation with

heat, the alcohol reacts slower than heat because heat generally relied on the

movement of molecules or the kinetic energy, while alcohol was in room temperature.

In the experiment, the researchers used three different heavy metals to

test how the chemical agent can coagulate the sample protein. First, Silver nitrate

(AgNO​3​) is what is known as an “acidic salt”. It is an acidic salt because the acid of

silver ion significantly larger than the base of the nitrate ion. The denaturation of egg

albumin protein in heavy metal by Silver nitrate is when the 2% of egg albumin solution

was mixed with 1ml of 10% Silver nitrate, the color turned into white and has formed
particles. This means that the bond were free to move apart and so disruption

happened. After that, the researchers tested its solubility by getting a small portion of

the sample and added 5 ml of distilled water. The particles were still visible so it resulted

to heavy precipitation. On the other hand, ​the action of copper sulfate (CuSO​4​) in protein

denaturation derives from its ability to disrupt disulfide Cystine bonds, binding to

individual sulfhydryl groups. When the albumin solution was mixed with the reagent

(CuSO​4​) the color turned into baby blue and the particles arose. When this happens, the

sections of the protein which were held in close proximity by the Cystine bond are free

to move apart, which usually allows water molecules to enter the core of the protein,

which leads to further disruptions. After testing the solubility of a small portion of the

precipitate in 5 ml water, it was already diluted and turned into almost gray. Generally,

Copper sulfate denatured egg albumin by disrupting Cystine bonds in the various

albumin proteins, which are most albumins. Then, the 1 ml of Barium chloride was

added to 2.0ml of our albumin solution. As the researchers was adding the reagent, the

solution only got more clearer than it was before. There were no particles or clumps

formed however, that was the initial observation. After 5 minutes of setting the solution

aside, the researchers somehow expected to gain some changes in the solution, but

there were none. Later on, the researchers decant the supernatant liquid and was

added to 5ml of water to test its solubility and precipitation. As observed after mixing the

solution, there were still no evident or visible changes in it unlike using the other two

chemicals, which are AgNO​3 that had coagulated and had white particles and CuSO​4

that turned blue in color. The differences between the three reagents is their salt
concentration. The Silver nitrate has the most salt among the reagents, next the Copper

sulfate and the least is the Barium chloride. This is why Silver nitrate has the most

coagulation or precipitation. Also, Silver nitrate has a high affinity of sulfur thus,

disrupting the disulfide linkages.

The effect of strong mineral acids in protein denaturation was tested

through using two reagents, sulfuric acid and nitric acid, using the sample of egg

albumin solution. The tertiary structure of egg white proteins is held together by

hydrogen bonding and hydrophobic interactions. Adding strong acid to the protein

sample disrupts the intermolecular forces, and the tertiary structure is lost. The loss in

protein structure is called denaturation where the proteins precipitate, becoming

insoluble. The first test combining reagent was sulfuric acid and albumin solution. In this

process the sulfuric acid showed precipitation where it turned white and the solution

solidified. The second test is the nitric acid and albumin solution. When nitric acid is

added to a sample and the mixture is heated, a yellow solution will result if the sample

contains tyrosine or tryptophan. Both reagents gave a heavy precipitation because both

had a high scale acidic pH. Sulfuric acid has 2.73 pH level while the nitric acid has 3.01

pH level. Their differences is that Sulfuric Acid is more acidic than nitric acid, hence

theoretically sulfuric acid has more solidified proteins than nitric acid. It is evident in the

experiment that the sulfuric acid had more coagulation than nitric acid despite both

giving a heavy precipitation.

When the structures of native proteins are altered by chemical or physical

means, the protein molecules tend to agglomerate and precipitate and the protein
becomes denatured. Alkaloidal reagents are acids that can combine with alkaloids.

Certain acidic reagents such as Picric acid, Tannic acid, Trichloroacetic acid, and Picric

acid are combined with protein to form insoluble proteins. In the experiment, the 5 ml

egg white was mixed to a 45 ml of water to then create an egg albumin solution. Then,

separated to three test tube containing 2.0 ml albumin solution. Each test tube were

added 1ml picric acid, 1ml of tannic acid and 1ml of trichloroacetic acid. In picric acid,

when it was dropped into the albumin solution, it turns yellow and the protein then

coagulated half-way through the albumin solution, and when the test tubes were shaken

gently, it slowly coagulated fully and hardened heavily. Same with the second test tube

with trichloroacetic acid. In the third test tube, the 2.0ml of albumin solution is added 1ml

of tannic acid, when the alkaloidal reagent was poured its color spread within the

albumin solution but not completely. After the solution was shake properly it covered the

entire albumin solution that turns brown in color. Some particles of the solution

coagulated and are mostly visible at the sides of the test tube and most of them turned

to a brown liquid infused with the albumin solution. It does not precipitated heavily

because its solubility is high compared to the other acids, reason why it precipitated

slightly and most of the acid dissolve and formed with the solution. Acids can disrupt the

structures of protein depending on their acidity strength. The differences of the three

reagents are their characteristics as a strong acid. A pKa gives details of their

dissociation of an acid in an aqueous solution. It indicates if the solution is a weak or

strong acids, where if the pKa is high, it is most likely a weak acid and vice versa. If the

solution has a strong acidity, it is most likely had an acidic pH or a lower pH. Picric acid
has 0.38 pKa units, the Tannic Acid has 6 pKa units and trichloroacetic acid has 0.7

pKa units. With this comparison, picric acid is the strongest acid and tannic acid is the

weakest among the three reagents. This is the reason why tannic acid only gave a slight

precipitation and the other two acids gave a heavy precipitation.

Conclusion

Protein denaturation is the disruption of the secondary, tertiary, and

quaternary structure of the proteins except its primary structure, its amino acid

sequence. There are several denaturing agents and these are: heat, organic solvent in

the form of alcohol, strong mineral acids, heavy metals, and alkaloidal reagents. Firstly,

the heat made the sample protein insoluble due to its increase of kinetic energy thus

breaking hydrogen bonds. Then, the alcohol also disrupt hydrogen bonding, where the

higher concentrated solution gave a heavier coagulation. Alcohol precipitates slower

than heat because it only uses room temperature. Next, the heavy metal disrupt the salt

bridges and disulfide linkages, where the reagents salt concentration must be

considered to effectively denature proteins. The strong mineral acids also disrupt the

ionic bonds, in which the protein is denatured through the change of the pH level. Also,

alkaloidal agents changes the pH level of protein thus, distorting the ionic bonds,

relatively strong hydrogen bonding and dispersion forces. A formation of suspended

colloids also manifested, where it shows shows a precipitate of a partial hydrolysate of

egg albumin and proteins. Therefore, the different ways of denaturing proteins helps the

researchers to understand the importance of the protein structure and its effect on its
functionality. It also visualize how proteins are denatured and how an external factor

can suddenly change its structure.

References

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