Name: Date Performed:

Course and Year: Group #: 1 Date Submitted:

Exercise No. 6

Proteins

OBJECTIVES:

At the end of the experiment, student should be able to:

1. Identify the structural patterns of proteins.

2. Use the isoelectric point of casein in milk to isolate the protein

3. Use chemical tests to identify amino acids and proteins

4. Observe the denaturation of proteins

RESULTS:

A. Separation of protein (casein) from milk

Erlenmeyer flask 82.1433 g

e-flask with milk 132.8872 g

Mass of milk 50.7439 g

Folded filter paper 1.2321 g

Casein 4.6983 g

% casein 9.2588
 B. Biuret test for proteins

Sample Observation

glycine Blue Amino acid

alanine Blue Amino acid

glutamine Blue Amino acid

aspartic acid Blue Amino acid

albumin Mixture of blue and purple Protein

casein Blue Protein

C. Ninhydrin test

Sample Observation

glycine Blue-violet

alanine Blue-violet

glutamine Blue-violet
 aspartic acid Blue-violet

albumin Two-phase: blue-violet

casein Blue-violet

tyrosine Two-phase- blue-violet

D. Xanthoproteic test

Sample

Glutamine Yellowish Does not contain aromatic

Aspartic acid Colorless Does not contain aromatic

Albumin Egg-white color Contain aromatic ring

Casein Colorless but has yellowish Contain aromatic ring

Valine White Does not contain aromatic

E. Sulfur test

Sample Observation

Alanine White

Albumin Yellowish ppt on top; brown ppt in the

Casein White

Valine White ppt with brownish ppt in the bottom

Lysine White

F. Denaturation of proteins

Denaturation agent Observation

Heat Coagulation of protein occur

Heavy metal: AgNO3 Coagulation of protein occur

Heavy metal: PbAc2 Coagulation of protein occur

Strong acid Coagulation of protein occur

Alcohol Coagulation of protein occur at the top of

A. Separation of protein (casein) from milk

The quantitative measurement of the protein content in milk was conducted

by causing a chemical reaction between the milk and an acid, resulting in the

formation of two distinct substances known as "curd" and "whey." "Curd" refers to

casein, a protein that undergoes precipitation from milk, while "whey" denotes the

residual liquid component of the milk solution. The observed percentage of casein in

milk was determined to be 9.2588%. The pH of non-fat milk typically falls between

6.7 to 6.8. When 10% acetic acid is added to the milk solution, the pH is reduced,

causing it to reach the isoelectric point of casein, around pH 4.5 to 4.7. The isoelectric

point (pI) of casein or any protein refers to the specific pH value at which the protein

molecule attains electrical neutrality, resulting in a zero net charge. Due to the

absence of repelling forces between the molecules and the high molecular weight of

the protein, the protein molecules prefer to precipitate out of solution, as their

charges are neutral. This is the primary reason for the observed precipitation of casein

from the solution in the experiment.

B. Biuret test for proteins

The biuret test is a chemical test that can be used to see if an analyte has peptide

bonds or not. As a result, the biuret test may be used to figure out how much protein is in

the analyte. In this test, the presence of peptides induces the copper (II) ion to form pale

purple (or mauve) coordination complexes (when the solution is sufficiently alkaline)

(Vedantu, n.d.).
 The experiment results in a negative result due to the presence of glycine, glutamine,

alanine, and aspartic acid, all classified as amino acids. Albumin is distinguished by a

mixture of blue and purple colors, which yields a positive result. It is a protein composed

of two or more peptides. In contrast, it is expected that casein would produce a positive

result in the biuret test. However, the observed negative result in the casein sample may

be due to unexpected factors, such as the potential presence of contaminants within the

solution.

C. Ninhydrin test

Ninhydrin test is a chemical test performed to detect the presence of ammonia,

primary/secondary amines, or amino acids. This test involves the addition of ninhydrin

reagent to the test sample that results in the formation of deep blue color, often termed

as Ruhemann’s purple, in the presence of an amino group (Sapkota, Microbe Notes, 2022).

In the experiment, it was seen that all the samples consisted of amines, which,

therefore, led to the manifestation of a cheerful blue-violet color during the test. The

observed blue-violet color on the top and white color on the bottom of the albumin and

tyrosine samples may be due to the reaction of the ninhydrin reagent. Alternatively, it is

possible that the samples (Albumin and Tyrosine) were contaminated or denatured,

leading to alterations in their native structure.

D. Xanthoproteic test

Xanthoproteic test is a biochemical test for the detection of amino acids containing

phenolic or indolic groups like phenylalanine, tyrosine, and tryptophan (aromatic amino

acids). The test is named Xanthoproteic test due to the formation of a yellow precipitate

of xanthoproteic acid. The term ‘Xantho’ refers to ‘yellow’, so the test is often termed as
the Yellow Protein Test. The test gives a positive result for amino acids containing benzene

rings or other aromatic groups. The test is a qualitative test that provides information only

on the presence or absence of the amino acids (Sapkota, Microbe Notes, 2022).

In the experiment, tyrosine has an amino-containing aromatic ring. Thus, it reacts to

xanthoproteic reagent and produces a color of orange. Albumin and casein, on the other

hand, contain tyrosine in the structure, thus producing an egg-white color in albumin, and

casein produces a yellow particle at the bottom of the test tube. Albumin and casein can

be nitrated with nitric acid, and this nitrated aromatic ring will react to NaOH, producing

an egg-white color for albumin and a yellow particle for casein. In glutamine, it produces

a yellowish color, though glutamine does not contain an aromatic ring; however, it has an

amide in the side chain, which reacts to the nitric acid in the xanthoproteic test.

E. Sulfur test

Lead sulfide test (or Lead acetate test) is a biochemical test for the detection of amino

acids like cysteine and cystine. The test is a specific test for the detection of amino acids

containing sulfur, S-S group in cysteine, and S-H group in cystine. The test is also called a

lead acetate test as the reagent for the test is lead acetate. Even though the test is specific

for the detection of sulfur-containing amino acids, methionine doesn’t give a positive

result in this test (Sapkota, Microbe Notes, 2022).

Based on the experiment, it indicates that alanine, casein, and lysine produced

negative results, indicating the absence of sulfur inside these molecules. A change in the

precipitate was seen in the substance albumin and valine. Mainly, albumin produced a

precipitate consisting of three distinct phases: a yellowish layer on the top part, a brown

layer in the center, and a white layer at the bottom. On the other hand, valine produces
a biphasic precipitate, which has a white layer on the top part and a brown layer on the

bottom. Therefore, albumin and valine produced a positive result, indicating the presence

of sulfur.

F. Denaturation of proteins

Denaturation is the process of the modification of the molecular structure of a protein.

This involves the breakdown of bonds such as hydrogen bonds and covalent bonds

(disulfide bonds (S-S). Proteins generally have a compact globular shape or are “folded”

instead of having random structures. Additionally, denaturation can be reversible or

irreversible (ChemTalk, n.d.).

Denaturation of proteins is one of the phenomenon that results in the disturbance of

stability and structure of the protein. The chemistry of proteins has always been

important owing to the abundance of these biomolecules in the living system. The

fundamental blocks of our body structure and their functioning require protein (Byjus,

n.d.).

The process of protein denaturation occurs when heat is applied, leading to the

disruption of the hydrogen bonds, ionic bonds, and disulfide bonds that contribute to the

protein's native conformation. This process could cause protein denaturation and

promote protein molecule aggregation. Heavy metals, such as silver ions (Ag+) and lead

ions (Pb2+), cause protein denaturation by their interaction with the amino acids present

in the protein. As a comparison, it is seen that silver ions can interact with the sulfur atoms

present in cysteine residues. In contrast, lead ions exhibit an affinity for the phosphate

groups in phospholipid molecules. Proteins undergo denaturation when exposed to

strong acids, which cause changes in the pH of the surrounding fluid. Proteins exhibit
 maximum stability at their isoelectric point, a pH value at which the overall charge of the

protein becomes neutral. The change in the pH of the solution causes a corresponding

change in the net charge of the protein. The observation of albumin coagulation at the

top of the solution can be due to the decreased solubility of unfolded albumin molecules

in alcohol compared to water. Thus, the albumin molecules that have undergone

unfolding appear on the top of the solution and mix to form a precipitate.

CONCLUSION:

The experiment conducted was successful in quantitatively measuring the protein

content in milk using the acid precipitation method. The observed percentage of casein in

milk was 9.2588%. The biuret test results showed a negative result for casein, possibly due to

unexpected factors, such as the potential presence of contaminants within the solution. The

ninhydrin test results showed that all the samples consisted of amines, which led to the

manifestation of a cheerful blue-violet color during the test. The observed blue-violet color

on the top and white color on the bottom of the albumin and tyrosine samples may be due

to the reaction of the ninhydrin reagent or the contamination/denaturation of the samples.

The xanthoproteic test results showed that tyrosine produced an orange color due to the

presence of an amino-containing aromatic ring. Albumin and casein, on the other hand,

produced an egg-white color and a yellow particle, respectively, due to the presence of

tyrosine in their structure. Glutamine produced a yellowish color due to the presence of an

amide in the side chain. The lead acetate test showed that alanine, casein, and lysine

produced negative results, indicating the absence of sulfur inside these molecules. A change

in the precipitate was seen in the substance albumin and valine, indicating the presence of

sulfur. The experiment also demonstrated the process of protein denaturation, which occurs
when heat, heavy metals, or strong acids are applied to proteins. Denaturation disrupts the

native conformation of the protein molecule and promotes aggregation.

REFERENCES:

Byjus. (n.d.). Retrieved from [Link]

ChemTalk. (n.d.). Retrieved from [Link]

Sapkota, A. (2022, July 27). Microbe Notes. Retrieved from

Sapkota, A. (2022, May 19). Microbe Notes. Retrieved from

Sapkota, A. (2022, June 23). Microbe Notes. Retrieved from [Link]

Vedantu. (n.d.). Retrieved from [Link]