LABORATORY EXERCISE 5:

QUALITATIVE ANALYSIS OF PROTEINS

DISCUSSION:
Proteins are diverse molecules and the most important biomolecule in the body. They are the main
constituents of the living cells and serve many functions as catalyst to control the rate of biochemical
reactions, regulators for various body functions, transport of gases and nutrients and as essential part of the
defense mechanism of the body. It is a complex of nitrogenous organic compounds which when hydrolyzed
produces α-amino acids. These amino acids are amphoteric molecules with an amino group (-NH2) and
carboxylic group (-COOH). Amino acids have side chain (R group) which gives the identity of the amino
acid and dictates if it is a polar and non-polar molecule. The general formula is RCH(NH2)COOH.

R-group represents the side chain and differetiates the amino acids from each other. Most of the
qualitative tests for prtotein depend on the presence of certain characteristics of chemical groups of specific
linkages in the R-chain. These groups react with a variety of reagents forming colored products. Since not all
proteins contain the same amino acids, the different color reactionds will yield products varying in color
intensity in accordance with the nature and amount of the groups contained in a particular protein.

Protein formation starts when two or more amino acids polymerize by peptide bond formation.
Peptide bond is formed by a hydrolysis reaction where the amino end of one amino acid interacts with the
carboxylic end of another amino acid thus forming a carbamino compound and water. Polypeptide
formation is the backbone for the formation of protein structure. A primary structure is only the sequences
of amino acids or the polypeptide itself. The secondary structures commonly exist in α-helix and β-pleated
sheets which are being stabilized by hydrogen bonding. Tertiary protein structure is where the biological
function of proetin starts. It is regulated by different types of interactions specifically formation of salt
bridges, hydrophobic interaction, disulfide linkages and hydrogen bonding. The quaternary structure of
proteinsoccurs when sevaral protein units combined together to form a more complex type of proteins.

Denaturation is the process of unfolding and rearrangement of the secondary and tertiary structure
of protein causing to loose its biological function. Proteins are coagulated by heat, strong acids, alcohols,
some metallic ions like Hg2+, Pb2+ and Ag+, and alkaloidal precipitating agents like tannic acid and picric
acid. Proteins do not dissolve in water rather they form colloidal suspensions. These colloidal properties are
exhibite by proteins which hinders them passing through a semi-permeable membrane.

OBJECTIVES:
At the end of the exercise, the students must be able to:

1. Compare and contrast the different classes of protein based on their chemical and physical
properties.
2. Demonstrate and explain the denaturation process and the factors affecting it.
3. Analyze the amino acids and proteins present in egg and other food and relate them to its biological
importance.

29|LABORATORY MANUAL IN BIOCHEMISTRY FOR MEDICAL LABORATORY SCIENCE

MATERIALS/APPARATUS:
Egg albumin solution 0.1% Ninhydrin 0.1% NaNO2 Sat. Picric acid
Egg albumin powder 1% phenylalanine Hopkins-Cole rgt. Test Tubes
1% gelatin conc. HNO3 95% ethanol Beaker
10% NaOH conc. NH4OH 5% Pb(C2H3O2)2 stirring rod
1% CuSO4 conc. H2SO4 1% HgCl2 pipet
0.2% urea Millons reagent 1% Pb(C2H3O2)2 dropper
0.2% glycine 5% phenol 10% tannic acid bunsen burner

PROCEDURE:
I. BURNING TEST FOR PROTEINS
Place a small amount of egg albumin on a evaporating dish and apply heat gently.

II. PREPARATION OF EGG ALBUMIN SAMPLE

Separate the white from the yolk of an egg. Place 150 ml of distilled water in a 250 ml beaker. Add
the egg white to the water while stirring the mixture. Observe the appearance of the mixture. Filter
the mixture and use the filtrate for the following procedures.

III. COLOR TESTS FOR AMINO ACIDS AND PROTEINS

A. BIURET TEST: Prepare 3 test tubes; on test tube 1 place 2 ml of egg albumin solution, on test tube
2 place 2 ml of 0.2% urea, on test tube 3 place 2 ml of 0.2% glycine solution. Label each test tube
properly. Add 3 ml 0f 10% NaOH, then add 2 drops of 10% CuSO4 to each test tube. Observe for
color change. Record your results in Table 1.

B. NINHYDRIN TEST: Place 1 mL each of egg albumin solution, ammonia water, o.2% urea and
0.2% glycine in separately labeled test tubes, add 0.5 mL of 0.1% freshly prepared ninhydrin reagent
into each test tube. Mix the solution and boil over water bath. Allow to cool and observe the color
produced. Record your results in Table 2.

C. XANTHOPROTEIC TEST: Into 3 separate labeled test tubes; place 1 ml of egg albumin, 1%
gelatin and 1% phenylalanine, respectively. Add 5 drops of conc. HNO3 to each test tube. Mix
thoroughly. Observe the formation of precipitate. Apply heat to the mixture. Observe the color
change of the solution. Cool and make solution slightly basic by adding NH4OH solution. Observe
the changes. Record your results in Table 3.

D. MILLON’S TEST: Place 1 mL each of the following solutions in separately labeled test tubes: egg
albumin solution, gelatin, phenylalanine, and phenol. Add 4 drops of Millon’s reagent into each test
tube. Heat in boiling water bath for 10 minutes, and then allow it to cool by placing the tube in
running water. Then add 4 drops of freshly prepared 0.1% NaNO2 and warm gently. Note any
change in the color of the precipitate/solution. Record your observations in Table 4.

E. HOPKIN’S-COLE TEST: Prepare two test tubes; on test tube 1 place 1 ml of egg albumin solution,
on test tube 2 place 1 ml of 5% gelatin solution. Label each test tube properly. Add 5 drops of

30|LABORATORY MANUAL IN BIOCHEMISTRY FOR MEDICAL LABORATORY SCIENCE

 Hopkins-Cole Reagent. Incline the test tube and carefully add 1 ml of concentrated H2SO4 on the
side of the test tube. DO NOT SHAKE. Observe the color formed at the junction of the 2 liquids.
Record your observations in Table 5.

F. LEAD ACETATE TEST: Prepare two test tubes; on test tube 1 place 1 ml of egg albumin solution,
on test tube 2 put a pinch of gelatin. Label each test tube properly. Add 5 drops of 10 % NaOH and 3
drops of 5% Lead Acetate solution into each of the test tubes. Shake and heat in boiling water bath.
Describe the color of the precipitate formed. Record your observations in Table 6.

IV. PROTEIN DENATURATION TEST

A. COAGULATION BY HEAT, ACIDS AND ALCOHOL
1. HEAT: Put a pinch of powdered egg albumin in each of 2 test tubes labeled no. 1 and no. 2. To
test tube no.1, add 2 mL distilled water then place both test tubes in a boiling water bath for 10
minutes, with constant shaking. Remove the test tubes, cool to room temperature and then add 2
mL of distilled water to test tube no.2. Filter the solutions and test both filtrates with Biuret
reagent. Compare the results and record it in table 7.

2. (a) INORGANIC ACIDS: Prepare 2 test tubes containing 1 mL each of egg albumin solution.
To test tube no.1, add conc. HCl, dropwise until a precipitate is formed, counting the number of
drops of the acid for precipitation to occur. Then add an excess of HCl and take note whether it
will increase or dissolve the precipitate formed. Repeat the same procedure in test tube no.2, but
this time use conc. H2SO4. Record your observations in Table 7.

(b) HELLER’S TEST: Place 1 mL of egg albumin in a test tube. Hold the test tube in an inclined
position, and then add 4mL conc. HNO3 slowly along the side of the test tube. DO NOT
SHAKE. Record your observations in Table 7.

3. ALCOHOLS: Place 1 mL each of egg albumin solution, add 5mL of 95% ethanol in each test
tube, mix thoroughly. Observe the formation of precipitate. Filter off the precipitate and test the
solubility of this precipitate in water. Record your observations in Table 7.

B. PRECIPITATION OF PROTEINS
1. BY HEAVY METALS: Place 2 ml of egg albumin in each of two test tubes. Add the following
reagents to the test tube drop by drop with shaking:
Test Tube 1: 1% Lead Acetate Test Tube 2: 1% HgCl2
Count the number of drops needed to produce a precipitate. Centrifuge the test tubes if cloudiness is
observed. Then add an excess of the reagent. Note whether the precipitate is increase or dissolved by
an excess reagent. Record your observation in Table 8.

2. ALKALOIDAL REAGENTS: Place 3 mL of egg albumin solution into two test tubes. In test
tube 1, add 2 mL of tannic acid solution, and in test tube 2, add 2 mL of picric acid solution.
Describe the protein solution in each test tube after addition of alkaloidal reagents. Record your
observations in Table 9.

31|LABORATORY MANUAL IN BIOCHEMISTRY FOR MEDICAL LABORATORY SCIENCE

 LABORATORY EXERCISE 5:
QUALITATIVE ANALYSIS OF PROTEINS
Name: ______________________________ Yr./Sec:_______ Group No.: ____ Date: _______

LABORATORY PERFORMANCE RUBRICS

CRITERIA SCORE 5 4 3 2 1
(1) Able to 10 minutes late 15 minutes late 15 minutes More than 15
submit data. in accomplishing in accomplishing late in BOTH minutes late
one of the two one of the tasks tasks. in either task.
Timeliness (2) Return tasks. OR 5-10 minutes
clean materials late in both tasks.
before the end of
session.
(1) Proper One of the Both criteria
disposal is criteria is not not followed.
Cleanliness followed. followed.
(1) Working area
is clean.
Complete Has missing
Materials
materials. materials.
All tubes are Criterion not
Labels correctly followed.
labeled.
All steps were One to two More than
done properly steps not done two steps not
Following the
without the need properly. done
Procedure to repeat/modify properly.
the procedures.
The student was The student had The student
able to answer all a hard time failed to
Evaluation of the questions answering the answer all the
Results asked by the questions asked questions
preceptor. by the preceptor. asked by the
preceptor.

Evaluated by:

_____________________________________________
Name and Signature of the Laboratory Preceptor

_________________
Date of Evaluation

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RESULTS & OBSERVATIONS:
1. Describe the results for burning tests of proteins:
_____________________________________________________________________________________

2. Describe the solution made by mixing water and egg white and name this kind of mixture.
_____________________________________________________________________________________
_____________________________________________________________________________________

TABLE 1: RESULTS FOR BIURET TEST

SAMPLE OBSERVATIONS
Egg Albumin
0.2% Urea
0.2% Glycine

3. What chemical structure in the protein molecule is responsible for a positive biuret test?
_____________________________________________________________________________________
_____________________________________________________________________________________

4. Which of the above substances show positive results with Biuret test? Account for the difference
observed.
_____________________________________________________________________________________
_____________________________________________________________________________________
5. Will sample amino acids give positive biuret test? Explain.
_____________________________________________________________________________________
_____________________________________________________________________________________
_____________________________________________________________________________________

TABLE 2: RESULTS FOR NINHYDRIN TEST

SAMPLE OBSERVATIONS
Egg Albumin
Ammonia water
0.2% Urea
0.2% Glycine

6. What group in amino acids or proteins is responsible for the ninhydrin reaction?
_____________________________________________________________________________________
_____________________________________________________________________________________

7. What other substance may yield positive result? Why?

_____________________________________________________________________________________
_____________________________________________________________________________________

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 TABLE 3: RESULTS FOR XANTHOPROTEIC TEST
SAMPLE OBSERVATIONS
Egg Albumin
1% Gelatin
1% Phenylalanine

TABLE 4: RESULTS FOR MILLON’S TEST

SAMPLE OBSERVATIONS
Egg Albumin
1% Gelatin
1% Phenylalanine
5% Phenol
8. What is responsible for the change?
_____________________________________________________________________________________
_____________________________________________________________________________________

9. What amino acid gives positive Millon’s Test? Write its formula.

_____________________________________________________________________________________

TABLE 5: RESULTS FOR HOPKINS-COLE TEST

SAMPLE OBSERVATION AT THE JUNCTION OF TWO LIQUIDS
EGG ALBUMIN
5% GELATIN

1. What is responsible for this color reaction?

_____________________________________________________________________________________
_____________________________________________________________________________________

2. Name the amino acid responsible for this test and write its formula.

_____________________________________________________________________________________

TABLE 6: RESULTS FOR LEAD ACETATE TEST

EGG ALBUMIN
GELATIN

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 TABLE 7: RESULTS FOR COAGULATION BY HEAT, ACIDS AND ALCOHOL

Coagulation by Heat

Coagulation Acids

Heller’s Ring Test

Coagulation by Alcohol

TABLE 8: RESULTS FOR PRECIPITATION WITH HEAY METALS

TEST TUBE WITH NO. OF DROPS OBSERVATION
1% Lead Acetate
1% HgCl2

TABLE 9: RESULTS FOR PRECIPITATION WITH ORGANIC ACIDS

TEST TUBE WITH OBSERVATION
Picric Acid
Tannic Acid

STUDY GUIDE EXERCISES:

1. What is the principle of Biuret Test? What is the importance of this test in studying the
hydrolysis of proteins?

2. Define zwitterions? Do they interfere with the precipitation test and color reactions?

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 3. What is egg and milk are used as antidotes for heavy metal poisoning? State its action.

4. Explain the use of tannic acid and picric acid in the treatment of burns.

5. Why are Xanthoproteic and Millon’s Tests unsatisfactory for use in urinary examination of
proteins?

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