OUR LADY OF FATIMA UNIVERSITY

College of Arts and Sciences

Natural Science Department
SY 2022 to 2023

ACTIVITY 3: QUALITATIVE TEST ON PROTEINS

Concept:
Proteins are polymers of amino acids linked together by peptide bonds. Proteins are important
biological molecules with many functions. There are transport proteins (hemoglobin is an example),
storage proteins, structural proteins, proteins for muscular contraction, and others. Proteins that
catalyze biological reactions are called enzymes. All proteins are very large molecules with high
molecular weights that range from 6500 to 205,000 or more grams per mole.

An amino acid is a molecule that contains an amino group and a carboxyl group in the same
molecule. Amino acids found in proteins are α-amino acids. This means the amino group (NH2- or
NH3+-) is attached to the alpha carbon--this is the carbon next to the carboxyl group. There are 20
amino acids that differ from each other only in the identity of the side chain attached to the alpha-
carbon. The amino acid side chains can be classified based on whether they are nonpolar, polar, acidic,
or basic. The general structure of an amino acid is shown below. In the following structure, R represents
the side chain.

Since proteins are biological molecules, they are usually found in a neutral solution that is
buffered between pH 7.0 and 7.4. In this situation, the acidic and basic groups on the amino acids will be
ionized. Most amino acids exist as “zwitterions” (dipolar ions) at pH 7. The structure of a zwitterion is
shown below. Because of the charges, amino acids are water-soluble.

Amino acids can be linked together – the amino group on one amino acid can react with the
carboxyl group on another amino acid, forming an amide. Therefore, the bonds linking together the
amino acid residues are amide bonds, but in proteins they are called peptide bonds. Shown on the next
page is the structure of a dipeptide (the prefix "di" means two--there are two amino acids in a
dipeptide). A typical protein molecule contains around 100 amino acids joined by peptide bonds.

There are several aspects to describe structure of a protein. The primary structure of a protein is
the sequence of amino acids. This sequence determines the overall shape of the protein. The secondary
structure consists of regular and repeating structures held together by hydrogen bonds between the
C=O and the N-H groups along the backbone of the molecule. The alpha helix and beta sheet are
examples of secondary structure. The tertiary structure refers to the overall folding of the entire
polypeptide chain and is determined by interactions between the side chains off the amino acids. These
interactions include hydrogen bonding, hydrophobic interactions, salt bridges, and disulfide bridges.
Quaternary structure involves the same types of interactions as tertiary structure, but the interactions
occur between the side chains on different polypeptide chains. These interactions hold together different
subunits of a protein.

If the overall folding of a protein is changed, the protein is called denatured. A denatured protein is
no longer biological active. Some common denaturing agents include heat, organic solvents, acids,
bases, agitation, detergents, and heavy metal ions. It is important to note that denaturation affects the
secondary, tertiary, and quaternary structure of a protein, but does not affect the primary structure.
Sometimes the denaturation is reversible, and the protein can be renatured (which reestablishes its
biological activity).
Procedure: (Qualitative Test for Proteins)

1. Biuret Test
a. Add 1mL of 10% NaOH to 3mL of each of the protein suspensions (albumin, casein,
gelatin – in 3 separate test tubes) and mix.
b. Add a drop of 0.01M CuSO4 to each and mix. If no definite color develops, add another
drop or two of the CuSO4 solution.
Note: A purplish violet color indicates positive test result

2. Ninhydrin Test
a. Add 5 drops of 0.1% Ninhydrin solution to 2mL each of the 3 protein suspensions.
b. Heat in boiling water bath for 10 minutes. Observe and record the results.

3. Xanthoproteic Test
a. Slowly add 1mL of conc. HNO3 to 3mL of each of the protein suspensions.
b. Place in water bath for about 30 seconds and place each test tube in a rack to cool the
solutions.
c. Add slowly, drop by drop, saturated NaOH to each test tube until solutions are alkaline
(or basic-test with red litmus paper)

4. Millon’s Test
a. Add 5 drops of fresh Millon’s reagent to 3mL of each protein suspensions.
b. Carefully heat each mixture in boiling water bath for 5 minutes
c. Cool the tubes and note the color formed. Cation: If too much reagent is used, the color
may disappear in boiling.

5. Sakaguchi Test
a. Add 1mL of 10% NaOH and 1mL of 0.02% alpha-naphthol solution to 3mL of each of the
protein suspensions.
b. After 3 minutes, add 2-4mL of bromine water to each, A strong red color can be
stabilized by adding urea to destroy the excess hypobromite.

6. Hopkins-Cole Test
a. Add 2mL of Hopkins-Cole reagent to 3mL of each of the 3 protein suspensions.
b. Mix. Incline each test tube and with a dropper ass slowly about 1mL conc. H 2SO4, down
the side of the tube so that two layers will form. Do not stir.
c. Let stand for 1 to 2 minutes and note the color formed in between the layers.

7. Sulfur Reaction or Lead Acetate Test

a. Add 5mL of 5% NaOH and a few crystals of Pb(Ac), (lead (II) acetate) to 3mL of each
protein suspensions.
b. Heat in boiling water bath for 5 to 10 minutes with occasional mixing of the contents of
the tube. Describe the color change.

Chemicals and Equipment

1. Test tube
2. Test tub rack
3. Dropper
4. Beaker
5. Water Bath
6. Hot plate
7. NaOH
8. H2SO4
9. Ninhydrin
10. HNO3
11. Millons rgt
12. a-Napthol
13. Bromine water
14. Urea
15. Hopkin's cole rgt
16. Lead Acetate

OUR LADY OF FATIMA UNIVERSITY

College of Arts and Sciences

Score
 Natural Science Department
SY 2022 to 2023
Name: Date:
Name of Instructor: Section:
ACTIVITY 3: QUALITATIVE TEST ON PROTEINS
PRE-LAB REPORT
1. When milk gets sour, it curdles. What are the ‘‘curdles?’’ What brings
about the curdling of the milk?

2. Fat is removed by both an ethanol wash and a diethyl ether–ethanol

wash. Why are these solvents effective in removing the fat from the
casein?

3. The skin on your fingers can get stained in two of the tests carried out
in this experiment.

a. Why will ninhydrin cause blue-purple stains?

b. Why will concentrated nitric acid cause yellow stains?

4. Why do heavy metals, like Hg2+ and Pb2+, cause poisoning? How does
an antidote, like milk or raw egg white, work?

OUR LADY OF FATIMA UNIVERSITY

College of Arts and Sciences
Natural Science Department
SY 2022 to 2023 Score
Name: Date:
Name of Instructor: Section:

ACTIVITY 3: QUALITATIVE TEST ON PROTEINS

I. Biuret Test
Observations
Sample Trial 1 Trial 2

CASEIN

ALBUMIN

GELATIN

II. Ninhydrin Test

Observations
Sample Trial 1 Trial 2

CASEIN

ALBUMIN

GELATIN

III. Xanthoproteic Test

Observations
Sample Trial 1 Trial 2

CASEIN

ALBUMIN

GELATIN

IV. Millon’s Test

Observations
Sample Trial 1 Trial 2
 CASEIN

ALBUMIN

GELATIN

V. Sakaguchi Test
Observations
Sample Trial 1 Trial 2

CASEIN

ALBUMIN

GELATIN

VI. Hopkins-Cole Test

Observations
Sample Trial 1 Trial 2

CASEIN

ALBUMIN

GELATIN

VII. Sulfur Reaction or Lead Acetate Test

Observations
Sample Trial 1 Trial 2

CASEIN
 ALBUMIN

GELATIN

OUR LADY OF FATIMA UNIVERSITY

College of Arts and Sciences
Natural Science Department
SY 2022 to 2023 Score
Name: Date:
Name of Instructor: Section:
 ACTIVITY 3: QUALITATIVE TEST ON PROTEINS
POST-LAB REPORT
1. What is a peptide bond?

2. What is Ninhydrin test used for?

3. What are the amino acids that can be identified using xanthoproteic test?

4. What color indicates a positive results under Lead acetate test?