ACTIVITY NO.

8
COLOR REACTIONS OF PROTEINS

Introduction:

Protein is colorless in its natural state, but blue-

green proteins are created when it folds up in
different shapes. These protein structures can create
different colors when exposed to certain substances,
such as the purple protein created when the protein
reacts to light waves. This particular molecule
absorbs cyan light and reflects magenta light, so it
becomes purple when wet or dry. The resulting reds
and oranges occur due to nitrogen in the air reacting
with oxygen in water vapor.
Protein is a polymer with amino acids that are
linked together by peptide linkage as its repeating
unit. The amino acids contain carboxyl and amino
groups, and their R groups can differ greatly based on
the type of protein. There are 20 kinds of proteins
that contain carbon atoms, and they can be classified
as globular proteins or fibrous proteins based on their
structures.
Proteins maybe classified according to their
nutritional value. For example, the protein of red
meat, chicken and cow's milk may be classified in
different ways depending on whether they originate
from animal products or plant foods. However, these
proteins are not all identical and may be in different
 proportion of the total number of protein
components. We can also classify proteins whether
they are complete and partially complete. Complete
and partially complete proteins with amino acids that
occur naturally in plants, whereas protein breakdown
may occur when enzymes in plants break down the
amino acids in their environment and can cause
protein breakdown with some species of plant (Sokol,
1998).
Objectives: At the end of the activity, the student can:

1. Identify specific amino acids based on their

chemical group present.
2. Identify which sample is complete or
incomplete protein.
3. Perform qualitative tests on protein samples
4.Distinguish amino acids through qualitative tests
Reagents:
- 1 ml 10% NaOH - 2 ml Hopkin’s-Cole
- 3 drops 1% CuSO4 reagent
- 10 drops 0.1% - 1 ml sulfanilic acid
ninhydrin solution - 1 ml 0.5% NaNO2
- 5 drops conc. - 10% Na2CO3
HNO3 - 1 ml conc. NaOH
- 5 drops millon’s - 10 drops 1% lead
reagent acetate
- 2 ml conc. H2SO4 - 15 drops bromine
water
 - 1 ml n-amyl alcohol - 10 ml 2% albumin
- 1 ml 20% NaOH - blue and red litmus
- 10 drops alpha paper
naphthol
- 10 ml 2% gelatin
- conc. NH4OH

Materials:
- 10 Test tubes (20 - tripod
ml) - alcohol lam
- test tube rack, test - wire gauze
tube brush - 10 ml graduated
- test tube holder cylinder
- beaker - dropper
- BRING: 2 marbles
CHEMICAL SAFETY NOTES:
1. Do not return chemicals to their original packaging.
An incompatible mixture may accidentally be
formed.
2. Keep chemical containers closed. Dust and vapour
may escape from an open container, while gases and
suspended material may penetrate this, causing the
nature of the chemical to change. This will also
avoid unnecessary exposure.
3. Never use a wrong or an unmarked reagent. If you
are unsure about the compound, do not use it.
Instead, have it disposed of
4. Chemical bottles must not be carried by the neck of
the bottle, nor next to your body. Suitable carrying
arrangements should be employed, e.g. buckets or
trolleys.
5. When pouring from bottles, the label should always
face upward to prevent any spillage from destroying
the label.
6. Never put any chemicals in the bottle other than the
one indicated on the label.
7. Special precautions should be taken when handling
concentrated acids. Dilution of acids should be
performed by pouring the acid into water and
stirring continuously.
Schematic Diagram:
***REPEAT PROCEDURES 1-9 USING GELATIN, GLUTEN AND CASEIN INSTEAD OF
ALBUMIN

NAME: Andres, Reano Zendrix Date:_ 09-08-21

SCORE:___________
Lab. teacher: Clarissa T. Balolot___ Course& Section___________ Grp. No. 1
ACTIVITY NO. 8
COLOR REACTION OF PROTEINS
Results:

Test Albumin Gelatine Gluten Casein

Purple Purple Purple Purple

Biuret
color was color was color was color was
formed formed formed formed
(+) (+) (+) (+)
Presence Presence Presence Presence
of of of of
Ninhydrin violet/pur violet/pur violet/pur violet/pur
ple color ple color ple color ple color
(+) (+) (+) (+)
Yellow No Yellow Yellow
Xanthoproteic color (+) presence color (+) color (+)
a.With HNO3
of yellow
color (-)
Orange No Orange Orange
b. with NH4OH
color (+) presence color (+) color (+)
of Orange
color (-)
Millon’s
Red No Red Red color
color (+) presence color (+) (+)
 of Red
color (-)
Hopkin’s-Cole
Purple No purple Purple Purple
ring (+) ring (-) ring (+) ring (+)
Black Black Black
precipitat precipitat precipitat Black
Lead Acetate
e (+) e (+) e (+) precipitat
e (+)
Cherry Cherry Cherry Cherry
Pauly red color red color red color red color
(+) (+) (+) (+)
Bromine Water
Colorless Orange Colorless Colorless
(+) color (-) (+) (+)
Sakaguchi
Red color Red Red color Red color
(+) color (+) (+) (+)

Generalization:
Color reactions in the protein lab are critical because
they help distinguish which tests can identify which
amino acid in a protein. Throughout all of the qualitative
tests, it separates the amino acids. These vital tests, like
the rainbow's various hues, reveal a spectrum of color in
the types of amino acids found in protein. These types of
tests must be understood because they go through a
variety of samples that can be used in the present world.
Because of the biological understanding required to
pursue a profession in nursing, protein detection can be
beneficial.

NAME: Andres, Reano Zendrix Date:_ 09-08-21

SCORE:___________
Lab. teacher: Clarissa T. Balolot___ Course& Section___________ Grp. No. 1
 ACTIVITY NO. 8
COLOR REACTION OF PROTEINS

Question Guide:
1. Write the positive result, the chemical group responsible for the positive and the
importance of each test.

Test Positive Result Chemical Group Importance

Formation Amino Used to detect the

of purple group presence of
Biuret color proteins or the
concentration.
Form the backbone
of proteins.
Formation NH2 group Used to detect the
Ninhydrin of purple presence of the
color alpha amino
group .
Xanthoprotei
c
HNO3:Yel Aromatic This test is used to
low amino acid/ detect the presence
NH4OH:O nitro group of amino acid and
range aromatic acids.
Serve as precursors
for the synthesis of
many biologically
active compounds
that are essential
for maintaining
normal biological
 functions
Formation Phenol Used to detect the
Millon’s
of red group tyrosine containing
color proteins.
Formation Indole It is used to detect
of purple group the presence of
Hopkins-Cole
ring. tryptophan in
samples containing
proteins.
Formation Sulfuric Used to detect the
Lead Acetate of black containing presence of
precipitati amino acid hydrogen sulfide in
on a given substance.
Formation Imidazole Used to detect the
Pauly of red and presence pf
color hydroxyl tyrosine and
group histidine.
Colorless Indole It is used to
Bromine
Water
group determine the
presence of alkenes
and alkanes.
Sakaguchi
Formation Guanidine It is used to detect
of red group the presence of
color arginine in
proteins.

2. What color will be produced by proline with ninhydrin test? Why?

 The color that is produced by proline with
ninhydrin test is Yellow. This is because of the reason
that proline is a secondary amine which gives a positive
(Yellow color) reaction when tested in ninhydrin.
3. Which test can be used to show the extent of hydrolysis of proteins? Why?

The test that can be used to show the extent of

hydrolysis is the biuret test. In it, the hydrolysis of a
polypeptide into its constituent amino acids is
catalyzed by B-elimination. The enzyme responsible
for this reaction is hydrolase, B -eliminating. In order
to test whether a particular preparation exhibits
hydrolytic activity or not, the solution to be tested is
added to a tube containing sodium nitrite and sodium
sulfide. A small amount of biuret dye is also added
before the solution boils to prevent further
decomposition compounds from being formed. In
other words, this is because biuret test detects the
presence of the peptide bonds, which is a positive
result.
4. Classify proteins according to nutritional value. To which classification does albumin
gelatin, gluten and casein belong?
According to nutritional value, proteins are
classified into two classifications. That is the
complete protein and the incomplete protein.
Complete proteins are complete with all 9 essential
amino acids. Incomplete proteins are missing some of
the essential amino acids. Foods that contain
complete proteins come from animals while
incomplete protein foods come from plants. There are
many different types of vegetables that have both
complete and incomplete protein. Albumin belongs to
complete protein because it includes ten essential
amino acids. Albumins are already in milk, eggs,
meats, and vegetables. On the other hand, casein is
considered an incomplete protein because it only
contains 8 essential amino acids. Casein in foods is
usually in dairy products especially in cheese.
Another example of incomplete protein is gluten in
grains which only has 7 essential amino acids. Soya
is also an incomplete protein since it only contains 6
essential amino acids.