CHEMICAL ANALYSIS OF

PROTEINS
Activity No. 5
 OBJECTIVES
• At the end of the activity, the students
must be able to:
• 1. Mindfully carry out the procedure with
high accuracy and precision;
• 2. Identify the positive and negative
results in every chemical procedure, and;
• 3. Know the principles behind every test
conducted in this experiment
 AMINO ACIDS
• Amino acids are the basic building
blocks of protein.
• The structure of amino acid
consisting of amine group (-NH2),
carboxylic group (-COOH) and R-
group or side chain.
• The side chain differs among all 20
different naturally occurring amino
acids. These 20 naturally occurring
amino acids are the essential amino
acids, which broadly classified into
three types:
• Polar amino acids
• Non-polar amino acids
• Aromatic amino acids
• Qualitative analysis of amino
acids can define as
the analytical method which
detects the presence or absence
of amino acids in a solution on
the basis of colour
change, precipitation ,etc.
 PROTEINS
• Comes from the greek word
“Proteis”meaning first in rank.
• Are macromolecules composed of
polymers of covalently linked amino
acids that are involved in every
cellular processes.
• Are amphoteric – because of their
acid and basic amino compositions.
 FUNCTIONS
• Repair of body tissues.
• Important in blood coagulation and
immunologic function.
• For transport of metabolic
substances.
• Maintenance of osmotic pressure.
• Maintenance of blood pH.
• Biocatalysts
 CLASSIFICATION OF PROTEINS

• Simple Proteins
– Contains peptide chains which on hydrolysis yield
only amino acids.
– May be fibrous or globular
• Conjugated Proteins
– Comprise of a protein and non protein moiety.
– Metalloproteins
– Lipoproteins
– Glycoproteins
– Mucoproteins or proteoglycans
– Nucleoproteins
 REAGENTS
• 2% glycine
• 1% proline
• 2% albumin
• 5% alanine
• 1% casein
• 10% NaOH
• 0.5% CuSO4
• Concentrated HNO3
• Ninhydrin Reagent
• Millon’s Reagent
 MATERIALS/ EQUIPMENTS

• Beral pipettes
• Test Tubes with Rack
• Glass Rod
• Water bath
PROCEDURES
 A. THE BIURET TEST

1. Place 15 drops of each of the following

solutions in six clean, labeled test tubes.
a. 2% glycine
b. 1% proline
c. 2% albumin
d. 5% alanine
e. 1% casein
2. To each of the test tubes, add 5 drops of
10% NaOH solution and 2 drops of 0.5%
CuSO4 solution.
3. Mix well and record your observations.
 B. THE NINHYDRIN TEST

1. Place 15 drops of each of the following

solutions in six clean, labeled test tubes.
a. 2% glycine
b. 1% proline
c. 2% albumin
d. 5% alanine
e. 1% casein
2. To each of the test tubes, add 5 drops of
ninhydrin reagent* and heat the test tubes in a
boiling water bath for about 5 min.
3. Record your observations.
 C. THE MILLON’S TEST

1. Place 15 drops of each of the following

solutions in six clean, labeled test tubes.
a. 2% glycine
b. 1% proline
c. 2% albumin
d. 5% alanine
e. 1% casein
2. To each of the test tubes, add 5 drops of
Million’s reagent and heat the test tubes in a
boiling water bath for about 2 min.
3. Record your observations.
 D. THE XANTHOPROTEIC TEST

1. Place 15 drops of each of the following solutions

in six clean, labeled test tubes.
a. 2% glycine
b. 1% proline
c. 2% albumin
d. 5% alanine
e. 1% casein
2. To each of the test tubes, add 10 drops of
concentrated HNO3 (UNDER THE HOOD) with swirling.
3. Heat the test tubes in a boiling water bath for
about 2 min.
4. Record your observations.
 E. HOPKIN’S COLE TEST

1. Place 1 mL of each of the following solutions in six

clean, labeled test tubes.
a. 2% glycine
b. 1% proline
c. 2% albumin
d. 5% alanine
e. 1% casein
2. Add 4 drops of Hopkin’s Cole reagent in all tubes.
3. Incline the test tube in the test tube rack.
4. Carefully, allow 2 mL of conc. sulfuric acid to
slide down the side of the inclined tube so that it
will form a layer below the protein solution.
5. Take note of the color of the junction of the two
liquids.
 F. LEAD ACETATE TEST

1. Place 1 mL of each of the following solutions

in six clean, labeled test tubes.
a. 2% glycine
b. 1% cystine
c. 2% albumin
d. 5% methionine
e. 1% casein
2. Add 5 drops of 10% NaOH, and 3 drops of
5% lead acetate solution into each test tube.
3. Shake and heat in boiling water bath.
4. Describe the color of the precipitate formed.
RESULTS
 BIURET TEST

• This test will give a positive result for

compounds that contain 2 or more
peptide linkages. It will give a
distinctive purple color which is
probably due to the formation of a
complex by cupric ions with the
amino groups called Biuret. Hence,
dipeptides and amino acids like
serine and threonine do not give
positive results with this test
SAMPLE REAGENT RESULTS
2% glycine Negative

1% proline Negative

2% albumin Positive

5% alanine Negative

1% casein Positive
 NINHYDRIN TEST

• When amino acids are sprayed with

ninhydrin, it will give a blue to violet
colored result. The presence of amino
group including those found in
amines and ammonia will also give
the same result except for proline
and hydroxyproline that will give a
yellow-colored result.
SAMPLE RESULTS
REAGENT
2% glycine Negative

1% proline Positive

2% Positive
albumin
5% alanine Negative

1% casein Positive
 MILLON’S TEST

• The presence of phenol group in

amino acid like tyrosine is nitrated by
a solution of mercuric and mercurous
nitrates in concentrated nitric acid. A
white precipitate will start to form,
turning brick red on prolonged
heating due to the formation of a
mercury complex of nitrophenyl
derivatives. Addition of NaNO2 turns
the precipitate to darker pink or red.
SAMPLE REAGENT RESULTS

2% glycine Negative

1% proline Negative

2% albumin Positive

5% alanine Negative

1% casein Postive
 XANTHOPROTEIC TEST

• Nitration of amino acids that contain

benzene ring will yield the product
nitrobenzene that will give a yellow
to orange coloration. Tryptophan,
tyrosine, and phenylalanine will
produce nitrobenzene when treated
with concentrated nitric acid.
Collagen and gelatin do not give a
positive reaction.
SAMPLE REAGENT RESULTS

2% glycine Negative

1% proline Negative

2% albumin Positive

5% alanine Negative

1% casein Positive
 HOPKIN’S COLE TEST

• The aldehyde present in the reagent

will cause the formation of a blue or
violet colored ring due to the
formation of a complex between the
reagent and the indole ring of
tryptophan. Gelatin and collagen fail
to give a positive result with this test
indicating the absence of tryptophan
in these proteins.
SAMPLE REAGENT RESULTS

2% glycine Negative

1% proline Negative

2% albumin Positive

5% alanine Negative

1% casein Positive
 LEAD ACETATE TEST

• This test is specific for sulfur-

containing amino acids like cysteine
and methionine. The sulfahydryl or
disulfide groups are converted to
inorganic sulfide, Na2S, in strongly
alkaline solution. This will react
further with lead acetate to form a
brownish–black precipitate of lead
sulfide (PbS).
SAMPLE REAGENT RESULTS
2% glycine Negative

1% Cysteine Negative

2% albumin Positive
(formation of brownish–
black precipitate)
5% Methionine Positive

1% casein Positive
(formation of brownish
precipitate)