ILOILO DOCTORS COLLEGE

MC2 (laboratory)
Biochemistry
BSN1-G

Class schedule: 2:30 -5:30 PM (Tue & Thurs)

Professor: Florencia C. Jarder

EXPERIMENT No: 5
CHEMISTRY OF PROTEIN
TITLE

Date Performed : October 17, 2023 Date Submitted : November 16, 2023

Group # : Group 2

Group Members:

1. Allanah Francesca Sister

2. Dea Margaret Guadalupe
3. Angel Mae Pillado
4. Frietzy Alcalde
5. Apple Mae Villanueva
6. Daphnee Chrystee Diamante
7. Chariz Barcenas
8. Evarie Castillon
9. Gwen Marivie Lustre
10. Beanna Ladera

I. Objective:
At the end of the experiment, the students should be able to:

1. Enumerate and explain the different color reaction tests used in the laboratory to
identify proteins based on their properties.
2. Point out the typical chemical groups present in proteins and the chemical rest.
3. Point out different ways by which chemical properties of proteins may be used for
practical purposes.

II. Introduction:
Proteins are large molecules that play many critical roles in the body. They do most of the work
in cells and are required for the structure, function and regulation of the body's tissue and organs.

Proteins are made up of hundreds or thousands of smaller units called amino acids, which are
attached to one another in a long chain. There are 20 different amino acids, which are combined to
make protein. The sequence of amino acids determines each protein sequence's 3-dimensional
structure and its specific function. Amino acids are coded by combination of three DNA building blocks
(nucleotide) sequences of genes.

III. Procedure:
A. Preparation of casein from milk
1. Mix 50 ml evaporated milk with 50 ml water in a beaker.

2. Heat the mixture to 400C for 10 minutes.

 3. After 10 minutes add 1 dropwise with constant stirring 4 ml of acetic acid, until a flocculent
precipitate forms.

4. Let it stand for 5 minutes, then add 3 ml of 1M sodium acetate solution, stir.

5. Set aside for 5 minutes, then centrifuge to separate the supernatant liquid from the precipitate.

6. The precipitate is your mixture A.

7. Mix the precipitate with 50 ml water and stir.

8. Allow the precipitate to settle and decant the supernatant liquid.

9. Add 10 ml ethanol to the precipitate, stir until it is well mixed, then centrifuge again.

10. Pour off the supernatant liquid and add 1 ml ether to the precipitate to reconstitute.

11. Spread the mixture on a piece of filter paper then air dry. This is now your Casein.

12. Use 30 ml 0.1N NaOH to dissolve the air-dried Casein in preparation for the following test.

B. Color Reaction Tests

Using mixture A collected from the previous procedure, do the following color reaction test for
proteins simultaneously running a similar test using 1% Egg Albumin as control solution.

1. Biuret Test

Pour 1 ml of the precipitate in the test tube and add 2 ml of 10% NaOH. Mix well and add 4
drops of 1 ml 1% CuSO4. Do the same procedure for 1 % Egg Albumin. Record the result.

2. Ehrlich’s Diazo Test

To 1 ml mixture A adds an equal volume of 0.5% HNO3. Set aside for 2 minutes, then add 1 ml
concentrated HCl. Make the solution alkaline, using litmus paper, by adding 10% Sodium Carbonate
dropwise. Repeat the same procedure for 1% Egg Albumin. Note the result.

3. Hopkins-Cole Test

Pour 1 ml of the mixture in a test tube and add 0.5 ml Hopkins-Cole reagent. Shake. Then
incline the test tube and add 1 ml H2SO4 by letting it run on the side of the test tube until two layers
become visible. Note the color at the junction of the two liquids. Repeat using 1% Egg Albumin.

4. Ninhydrin Test

Using 0.1N HCl to neutralize 1 ml of mixture A, add 5 drops of freshly prepared 1% Ninhydrin
reagent. Heat in a water bath for 1 minute. Observe the produced purple color. Repeat using 1% Egg
Albumin. Note the result.

5. Millon’s Test

Pour 1 ml of the mixture A in a test tube and add 5 drops of Millon’s reagent. Heat in a water
bath for at least 3 minutes. Note the result. Repat using 1% of Egg Albumin. Note the result.

6. Sulfur Test

Place 1 ml of the mixture A in a tube then add 1 ml of 3M NaOH. Heat gently in a water bath for
15 minutes. Cool and acidify (test with litmus paper) by adding dropwise 3M HCl until the blue litmus
paper turns red. Place a lead acetate impregnated paper (cut 1.4-inch x 0.5-inch filter paper the moisten
with lead acetate over the mouth of the test tube. Boil the contents gently. Observe the color change
in the lead acetate paper. Record the result and run the same test using 1% Egg Albumin.

7. Xanthoproteic Test

Place 1 ml of Mixture A then carefully add 5 drops of concentrated Nitric acid. Heat the solution
in a water bath for 1 minute. Cool and add dropwise of 10% NaOH until the solution turns red litmus
paper to blue (Alkaline). Notice the change in color. Repeat the procedure using 1% Egg Albumin.

Denaturation

1. Place 3 ml of the mixture A in a test tube and heat gently until the solution boils. Remove from
heat and add 7 ml of ethyl alcohol. Shake and note the result.

2. Prepare 3 test tubes and label 1,2,3. Note the formation of precipitates.

Test tube #1` 1 ml Mixture A + 5% Lead Acetate

Test tube # 2 1 ml Mixture A + 5% Mercuric Chloride

Test tube # 3 1 ml Mixture A + 5% Ferric Chloride

3. Repeat the procedure using 1% Egg Albumin

Test tube # 1 1ml Egg Albumin + 5% Lead Acetate

Test tube # 2 1ml Egg Albumin + 5 % Mercuric Chloride

Test tube # 3 1ml Egg Albumin + 5 Ferric Chloride

IV. Reagents and Materials:

Table 1: . Color Reaction Test
Comparison of Result

1% Egg Albumin
Casein
Testing
Reagents
Observation Results Observation Results

Biuret Test The color While the color The violet or

The solution change in the is still purple, it purple color is
turns violet or Biuret test, resembles a positive
purple. The from blue to milky purple in result,
violet or purple purple or comparison to indicating the
color indicates violet, occurs a very clear presence of
the presence of due to a purple in the peptide bonds
peptide bonds, complexation absence of egg in the protein.
suggesting the reaction albumin. This color
presence of between the change
proteins or copper ions in signifies that
peptides in the the Biuret the Biuret
 reagent and reagent has
tested solution. the peptide reacted with
bonds in the protein,
proteins or specifically
peptides. The with the
reaction peptide bonds,
involves forming a
coordination of complex that
the copper ions results in the
with the observed color.
nitrogen atoms
of the peptide
bonds.

Ehrlich’s Test The change to Adding egg The change to

The addition of a "milky white" albumin to a "dirty white"
0.5% HNO3 to color after mixture A will color when
mixture A will these steps introduce mixing egg
likely acidify indicates the proteins into albumin with
the solution. formation of a the solution. mixture A,
After setting it precipitate in Proteins, like followed by
aside for 2 the solution. egg albumin, subsequent
minutes, the This milky are sensitive to treatment with
addition of appearance pH changes concentrated
concentrated suggests the and undergo HCl and
HCl will further presence of denaturation in Sodium
increase the suspended acidic or Carbonate,
acidity of the particles that alkaline might suggest
solution or scatter light, conditions.Afte the formation
introduce commonly r setting aside of a precipitate
additional associated with the mixture for or the
chemical the formation 2 minutes, the alteration of
species of insoluble addition of substances in
depending on compounds or concentrated the
the substances in HCl will likely solution.Egg
components in the create an acidic albumin is a
mixture A. This solution.The environment. protein, and
might induce addition of This acidic proteins are
various 0.5% HNO3 condition sensitive to
reactions or likely initiated might lead to changes in pH.
changes in the changes in the the The addition of
solution's chemical denaturation concentrated
chemical composition of or alteration of HCl likely
composition.Fi mixture A. the protein induced an
nally, making Subsequently, structure acidic
the solution the addition of present in the environment,
alkaline by concentrated egg which can
adding 10% HCl increased albumin.When cause
Sodium the acidity of 10% Sodium denaturation
Carbonate the solution or Carbonate is or alteration of
dropwise using introduced added the protein
litmus paper as new chemical dropwise to structure in
an indicator species, make the egg
could potentially solution albumin.The
neutralize the leading to the alkaline using subsequent
excess acid and formation of litmus paper as addition of
potentially insoluble an indicator, it Sodium
cause the compounds or could Carbonate
 precipitates.Th potentially might have
formation of a e final step of neutralize the neutralized the
precipitate or a making the excess acid and excess acid and
change in color solution revert the restored the
into a milky alkaline by solution to an solution to an
white color. adding 10% alkaline state. alkaline state.
Sodium However, the However,
Carbonate denatured during this
dropwise might proteins might process, the
have further not return to denatured
induced the their original proteins might
precipitation of structure.The have formed
these color has insoluble
compounds changed into a aggregates or
due to their dirty white precipitates,
insolubility color. leading to the
under alkaline observed "dirty
conditions. white" color.

Hopkins’-Cole When 1 ml of The color Upon mixing The change in

Test the mixture change 1% Egg the lower layer
was combined indicates a Albumin with from its initial
with 0.5 ml of positive result 0.5 ml of light yellow to
Hopkins-Cole for the Hopkins-Cole a brownish-
reagent and presence of reagent and yellow shade
shaken, purines in the then adding 1 strongly
followed by the mixture. ml of H2SO4, a suggests a
addition of 1 The observed notable and specific
ml of H2SO4 difference in distinct color chemical
along the side consistency alteration, interaction or
of the test between the specifically a reaction. This
tube, a distinct upper and change to a alteration in
and observable lower layers of brownish- color indicates
color change the mixture, orange hue, a potential
occurred at the with the upper was observed chemical
interface of the layer exhibiting at the transformation
two liquid a notably boundary of within the
layers. The thicker texture the two liquid components
original light compared to layers within present in the
yellow color the lower the test tube. solution. The
transformed layer, suggests Moreover, the upper layer
into a a significant upper layer formed into a
yellowish- variation in the transformed thick and
brown hue. physical into a thick and whitish
properties or whitish consistency
composition of consistency subsequent to
these layers. mixing which
hints at a
potential
alteration in
the physical
properties of
the solution.

Ninhydrin Test Upon Tryptophan is The mixture of The result is

completing the confirmed egg albumin negative for
mixture, two positive as with 5% tryptophan,
distinct colors indicated by sodium indicated by
 are noticeable, the presence of chloride (NaCl) the absence of
separated by a the purple ring. and 0.5% a purple ring
purple ring. Hopkins-Cole and the
The top layer reagent results development
appears in color of an overall
creamy white, changes. As the black color
while the reagent
bottom layer dissolves the
exhibits a egg white, it
yellowish hue. transforms into
a deep black
color.

Millon’s Test Upon adding A positive The solution Millon's

Millon's result in turns red or a reagent reacts
reagent (a Millon's test brick-red specifically
solution indicates the precipitate with the
containing presence of forms. tyrosine
mercuric and proteins with residues in
nitric acids) to tyrosine proteins,
the sample, a residues. The forming a red
reaction occurs red color or complex. Since
with the brick-red egg albumin
tyrosine precipitate contains
residues in forms as a tyrosine, you
proteins. result of the should observe
The reaction specific the
leads to the chemical development
formation of a reaction of a red color
red-colored between the or a brick-red
complex or tyrosine precipitate.
solution. residues and
the
components of
Millon's
reagent.

Sulfur Test Upon adding A positive A black or The formation

lead(II) acetate result in a dark-colored of the black
to the sample sulfur test, precipitate precipitate is a
solution specifically the forms in the positive result,
containing test for sulfide solution after confirming the
sulfide ions, a ions, typically adding lead(II) presence of
black or dark- involves the acetate. sulfide ions
colored formation of a derived from
precipitate black or dark- sulfur-
forms. colored containing
precipitate. compounds in
One common the egg album
method for
testing the
presence of
sulfide ions is
to add lead(II)
acetate to the
solution.
 Xanthoproteic The The yellow If the egg The
Test appearance is a color observed albumin appearance of
yellow color or is due to the contains the yellow
a deepening of formation of aromatic color is a
the yellow nitro amino acids positive result,
color in the derivatives like tyrosine or indicating the
reaction during the tryptophan, a presence of
mixture reaction. yellow color aromatic
will develop in amino acids in
the reaction the egg
mixture. albumin.

V. DATA and RESULTS:

Documentation of Biuret Test:

Documentation of Ehrlich’s Test:

Documentation of Hopkins-Cole Test:

Documentation of Ninhydrin Test:

Documentation of Millon Test:

 (NO DOCUMENTATION AS WE WERE UNABLE TO COMPLETE IT DUE TO APPARATUS
DEFICIENCY)

Documentation of Sulfur Test:

(NO DOCUMENTATION AS WE WERE UNABLE TO COMPLETE IT DUE TO APPARATUS
DEFICIENCY)

Documentation of Xanthoproteic Test:

(NO DOCUMENTATION AS WE WERE UNABLE TO COMPLETE IT DUE TO APPARATUS
DEFICIENCY)

Table 2: Denaturation Test

Comparison of Results

Tests Casein 1% Egg Albumin

Observation Results Observation Results

Lead Acetate By adding a few The result of this There was a The lead acetate
test drops of lead test, based on formation of a test with 1% egg
acetate solution the observation, black lead albumin resulted
to Mixture X there was a sulfide in the formation
there was white white precipitate precipitate, of a black lead
precipitate upon adding which indicates sulfide
appeared .Mean lead acetate to the presence of precipitate,
s that there is a Mixture X, it sulfur-containing indicating the
presence of indicates the amino acids in presence of
sulfide ions in presence of the egg albumin sulfur-containing
the solution. sulfide ions in sample. amino acids in
the solution. the egg albumin
This result is sample. This
positive for the positive result
lead acetate aligns with the
test, suggesting expected
the occurrence outcome of the
of sulfide test, as the
formation during reaction of
the acid sulfur-containing
hydrolysis amino acids with
process. lead acetate
under alkaline
conditions leads
to the formation
of a brown to
black precipitate
of lead sulfide
(PbS).

Mercuric Upon the The formation of Egg albumin, The addition of

chloride test addition of a precipitate being a globular mercuric
mercuric indicates protein, is chloride might
chloride to the denaturation or relatively stable induce changes
casein solution, coagulation of in its native in the egg
 we observed casein, form. albumin
visible changes, suggesting that The solution solution.
such as the the protein has may appear A noticeable
formation of a undergone transparent or cloudiness,
white or cloudy structural slightly turbidity, or
precipitate. changes in opalescent. precipitation
response to the may occur,
mercuric indicating
chloride. denaturation of
egg albumin.

Ferric chloride The solution The formation of Egg albumin is a Similar to the
test may be opaque a complex globular protein, casein test, the
or milky due to between ferric and the solution addition of ferric
the colloidal ions and casein may be clear or chloride to the
nature of casein. may result in a slightly cloudy. egg albumin
visible color solution may
change, often induce a color
from the initial change.
milky The color change
appearance to a could be
different color distinctive,
(e.g., a deeper or indicating the
lighter shade). interaction
The color change between ferric
indicates the ions and egg
denaturation or albumin.
alteration of the The color change
casein structure signifies the
in the presence denaturation or
of ferric structural
chloride. modification of
egg albumin
caused by the
ferric chloride.
VI. DATA and RESULTS:

Documentation of Lead Acetate test:

(NO DOCUMENTATION AS WE WERE UNABLE TO COMPLETE IT DUE TO APPARATUS
DEFICIENCY)

Documentation of Mercuric chloride test:

(NO DOCUMENTATION AS WE WERE UNABLE TO COMPLETE IT DUE TO APPARATUS
DEFICIENCY)

Documentation of Ferric chloride test:

(NO DOCUMENTATION AS WE WERE UNABLE TO COMPLETE IT DUE TO APPARATUS
DEFICIENCY)

IV. Conclusion :

The laboratory experiment successfully achieved its objectives by using color reaction tests
for protein identification. We gained valuable insights into protein chemistry and the molecular
composition of proteins. The experiment also highlighted the practical application of protein
properties in biomedical application, food industry quality control measures, environmental
monitoring, and pharmaceutical. This experiment equipped students with technical skills for
protein identification and broader applications in various industries, preparing them to
contribute to advancements in healthcare and environmental science.

In the laboratory, various color reaction tests are employed to identify proteins based on
their distinctive chemical properties. These tests capitalize on the specific chemical groups
present in proteins, such as amine groups in amino acids. The Biuret test, for example, detects
the presence of peptide bonds, resulting in a violet or purple color change upon interaction
with copper ions. Millon's test is sensitive to the aromatic amino acid tyrosine, producing a red
or brick-red precipitate. The Xanthoproteic test targets aromatic amino acids like tyrosine and
tryptophan, causing a yellow coloration upon nitration. Sulfur tests, including the lead acetate
test, form black precipitates in the presence of sulfide ions, indicating the occurrence of sulfur-
containing amino acids.

The chemical properties of proteins can be harnessed for practical purposes in various
applications. In biochemistry, these properties are crucial for elucidating the structure and
function of proteins. In diagnostics, protein tests serve as markers for certain diseases. In the
food industry, protein tests ensure quality control, assessing the protein content in food
products. Moreover, in molecular biology, knowledge of protein properties guides experimental
design, protein purification, and structural analysis. By leveraging the diverse chemical
characteristics of proteins, these color reaction tests contribute significantly to both
fundamental research and practical applications across various scientific disciplines.

V. REFERENCES:
https://byjus.com/chemistry/laboratory-test-of-proteins/
https://www.biologydiscussion.com/experiments/experiments-on-proteins-
biochemistry/56403
https://study.com/learn/lesson/biuret-test-protein.html#:~:text=As%20previously%20mentio
https://www.google.com/url?sa=i&url=https%3A%2F%2Fuomustansiriyah.edu.iq%2Fmedia%2
Flectures%2F6%2F6_2020_03_15!
02_22_40_PM.pdf&psig=AOvVaw0lsQCr2POF8dJzzZvpvAQL&ust=1700223378886000&source=images
&cd=vfe&opi=89978449&ved=0CBIQjRxqFwoTCJjz6e2_yIIDFQAAAAAdAAAAABAX

VI. Contribution of each member :

Name Contribution

Angel Mae C. Pillado Hopkins Cole Test

Daphnee Chrystee Diamante,Chariz Ehrlich’s Test

Barcenas and Apple Mae Villanueva

Frietzy Alcalde and Gwen Lustre Ninhydrin Test

Allanah Francesca Sister Biuret Test

Dea Margaret Guadalupe Mercuric chloride test, and Ferric

chloride test
Evarie Joy Nicole L. Castillon Lead Acetate Test