BSN 1F 10/30/21

Agbayani, Althea Aubrey

Acosta, Jessica
Alunday, Keylen
Agurin, Ruth
Balaccua, Shaine Adrian
Basacoy, Azure
Bogabil, Lyka Jade
Bueza, Jo-ainah
Butac, Cristel Butac

Illustrated Laboratory Activity 4: Qualitative Test for Proteins

Directions:
1. The student will simulate the conduct of laboratory procedures that will be performed
without the actual procedure itself.
2. The student will answer the guide questions at the end of the procedure. All
accomplished laboratory reports shall be submitted to the subject teacher on a
designated schedule of submission.
3. For every test procedure conducted in the actual laboratory, rationale and procedure
will be provided in this illustrated laboratory activity for reference purposes.
4. For reference purposes, the student is directed to watch this video link:
https://www.youtube.com/watch?v=6YPWipP-Qe8 (Note: This video link is needed in
order to fill out the observation tables for each test procedures.)
Qualitative Test for Proteins
1. Biuret Test:
The Biuret Test positively identifies the presence of proteins (not less than two
peptides). The reaction in this test involves the complex formation of the proteins with
Cu2+ ions in a strongly alkaline solution.
Procedure:
To 2 mL protein solution, add 5-6 drops of dilute CuSO4 (Fehling’s solution A diluted
1/10 with water) Add 3 mL 40% NaOH solution.
Observe the color change. Fill out the table below.

Test Substance Color Test Result Inference

Casein LIGHT PURPLE The protein is positive or
present.
Albumin LIGHT BLUE Protein is absent.
Gelatin PURPLE Protein is present.

Questions:
Write the reaction(s) involved in Biuret’s Test.
 When a protein reacts with copper (II) sulfate (blue), the positive test is the
formation of a violet colored complex.
2. Ninhydrin Test:
Ninhydrin (triketohydrindene hydrate) is a chemical used to detect ammonia or primary
and secondary amines. Amino acids also react with ninhydrin at pH=4. The reduction
product obtained from ninhydrin then reacts with NH3 and excess ninhydrin to yield a
blue colored substance. This reaction provides an extremely sensitive test for amino
acids.
Procedure:
To 1 mL amino acid solution add 5 drops of 0.2% ninhydrine solution in acetone.
Boil over a water bath for 2 min. Allow to cool and observe the color change. Fill out
the table below.

Test Substance Color Test Result Inference

Casein PURPLE The appearance of purple
colour solution confirms the
presence of proteins
meaning the test is
positive.
Albumin PURPLE It appears a purple color
that the test is positive
confirms the presence of
proteins.
Gelatin PURPLE It appears in purple color
which means the test is
positive and it confirms the
presence of proteins.

Questions:
Write the reaction(s) involved in Ninhydrin Test.
 The ninhydrin test is a chemical method for determining whether a substance
contains amines or -amino acids. The presence of ammonia, primary/secondary
amines, or amino acids in the analyte is indicated by the development of a purple
color.

3. Xanthoproteic Test:
Some amino acids contain aromatic groups that are derivatives of benzene. These
aromatic groups can undergo reactions that are characteristics of benzene and benzene
derivatives. One such reaction is the nitration of a benzene ring with nitric acid. The
amino acids that have activated benzene ring can readily undergo nitration. This
nitration reaction, in the presence of activated benzene ring, forms yellow product.
Procedure:
To 2 mL amino acid solution in a boiling test tube, add equal volume of concentrated
HNO3. Heat over a flame for 2 min and observe the color. Now COOL THOROUGHLY
under the tap and CAUTIOSLY run in sufficient 40% NaOH to make the solution
strongly alkaline. Observe the color of the nitro derivativative of aromatic nucleus.
Fill out the table below.

Test Substance Color Test Result Inference

Casein ORANGE The test is positive this
confirms the presence of
aromatic groups.
Albumin ORANGE The test is positive this
confirms the presence of
aromatic groups.
Gelatin YELLOW The test is positive because
the color yellow, shows the
presence of aromatic
groups.

Questions:
Write the reaction(s) involved in Xanthoproteic Test.
 A nitration reaction is used in the Xanthoproteic test to determine the presence
of proteins in a solution. When a sample is treated with hot concentrated nitric
acid, it reacts with aromatic amino acids like phenylalanine, tyrosine, and
tryptophan to produce Xanthoprotein, a yellow-colored product.

Define “activated benzene ring”, briefly.

 Activated benzene ring is the substituents on the ring are groups that donate
electrons.

Do all the amino acids with aromatic side chains give positive result? Why?
 Yes, because a positive test is when the solution appears dark yellow or orange
in color.

4. Millon’s Test:
Millon’s test is specific to phenol containing structures (tyrosine is the only common
phenolic amino acid). Millon’s reagent is concentrated HNO3, in which mercury is
dissolved. As a result of the reaction a red precipitate or a red solution is
considered as positive test. A yellow precipitate of HgO is NOT a positive reaction but
usually indicates that the solution is too alkaline.
Procedure:
To 2 mL amino acid solution in a test tube, add 1-2 drops of Millon2s reagent.
Warm the tube in a boiling water bath for 10 min.
Fill out the table below.

Test Substance Color Test Result Inference

Casein PINK/REDDISH PURPLE It is positive, because of
the result ranging to pink
 and reddish purple.
Albumin WHITE/RED When the albumin is
heated, a red solution
precipitate forms, indicating
the presence of tyrosine
(positive result).
Gelatin LAVENDER Sulfur of gelatin present as
methionine did not react in
this test, yielding in
negative result.

Questions:
Write the reaction(s) involved in Millon’s Test.
 Metallic mercury is dissolved in nitric acid and then diluted with water to make
the reagent. The test nitrates the phenol group in tyrosine's side chain, giving in
a red color or precipitate when combined with Hg(I) or Hg(II) ions.

You have phenol, tyrosine, cysteine and β-naphtol in separate test tubes. By using
which test(s) would you find the tyrosine containing test tube? Explain, briefly.
 Millon's test is an analytical method for detecting tyrosine, the only amino acid
with a phenol group. Millon's test is specific for tyrosine but not protein because
it detects the phenolic group found in other substances.

5. Sakaguchi Test:
The Sakaguchi reagent is used to test for a certain amino acid and proteins. The amino
acid that is detected in this test is arginine. Since arginine has a guanidine group in its
side chain, it gives a red color with α-naphthol in the presence of an oxidizing agent like
bromine solution. Apply this test to arginine.
Procedure:
1 mL NaOH and 3 mL arginine solution is mixed and 2 drops of α-naphthol is added.
Mix thoroughly and add 4-5 drops of bromine solution. Observe the color change.
Fill out the table below.

Test Substance Color Test Result Inference

Casein RED The formation of red color
indicates presence of
arginine or a guaniginium
compound.
Albumin RED The formation of red color
indicates presence of
arginine or a guaniginium
compound.
Gelatin RED The formation of red color
indicates presence of
 arginine or a guaniginium
compound.

Questions:
Define and give the structure of guanidine.
 It is derived from guanine that in the form of its hydrochloride acts as a
parasympathetic stimulant and is used especially to denature proteins, and its
structure is CH5N3.

6. Hopkin’s Cole Test:

The indole group of tryptophan reacts with glyoxylic acid (glacial acetic acid, which has
been exposed to light, always contains glyoxylic acid CHOCOOH as an impurity) in the
presence of concentrated H2SO4 to give a purple color.
Procedure:
To a few mL of glacial acetic acid containing glyoxylic acid, add 1-2 drops of the amino
acid solution.
Pour 1-2 mL H2SO4 down the side of the sloping test tube to form a layer underneath
the acetic acid. Observe the color change.
Fill out the table below.

Test Substance Color Test Result Inference

Casein YELLOW/BROWNISH There is no appearance of a
purple/violet ring between
the two layers due to
 absence of tryptophan.
Albumin YELLOW/BROWNISH There is no appearance of a
purple ring between the
two layers due to absence
of tryptophan.
Gelatin PLAIN/WHITE There is no appearance of a
purple ring between the
two layers due to absence
of tryptophan.

Questions:
Write the reation(s) involved in Hopkin’s Cole Test.
 The reactions involved are Tryptophan with Glyoxylic acid, to Condensation
product with H2O, and to Violet-coloured compound with H2O.

What is the role of H2SO4 in this test? Explain, briefly.

 The H2SO4 added to the reagent helps to stabilize the glyoxylic acid and prevent
its decomposition and the release of carbon dioxide.

7. Sulfur Reaction or Lead Acetate Test:

The sulfur-containing amino acid such as cysteine, cysteine, and methionine
(sulfhydryl/thiol group) reacts with lead acetate under alkaline conditions to form a
brown precipitate. These sulfur-containing amino acids are degraded in strongly alkaline
media to release sulfide ion (S2- ) in the form of H2S (hydrogen sulfide). The sulfide ions
can react with lead (II) acetate to form a brownish-black precipitate.
Procedure:
Add 5 mL 5% NaOH and few crystals of Pb(Ac)2 Lead (II) Acetate to 3 mL to each
protein suspensions.
Heat in boiling water for 5-10 minutes with occasional mixing the contents in the tubes.
Fill out the table below.

Test Substance Color Test Result Inference

Casein YELLOW The result is negative
because no black
precipitate formed.
Albumin YELLOW The result is same with
casein because no black
precipitate formed, yellow
color appears so it is
negative.
Gelatin BLACK The result is positive
because the black
precipitate present.
Questions:
Write reaction(s) involved in the Lead Acetate Test.
 A biochemical test for the identification of amino acids such as cysteine and
cystine is the lead sulfide test (or lead acetate test). The test is designed to
detect sulfur-containing amino acids, the S-S group in cysteine, and the S-H
group in cystine.

Laboratory Guide Questions:

1. Explain why casein precipitates when acetic acid is added to it.
 When acetic acid is added, it denatures the protein, causing it to precipitate.
Because casein's isoelectric point is higher than that of neutral milk, acid must be
added to lower the pH. Because the protein is charge neutral when it is
isoelectric, it is no longer soluble in water.

2. Cite some examples of denaturation of protein encountered in everyday life.

 Some proteins become denatured when food is heated. This is why cooked meat
becomes solid and boiled eggs become hard. Egg whites, which are mostly egg
albumins in water, are a notable case of denaturing in proteins. Egg whites are
visible and liquid when they are fresh from the eggs.

3. Why is milk or raw egg used as antidote in cases of heavy metal poisoning?
 Raw egg or milk is used as an antidote in cases of heavy metal ion poisoning to
coat the lining of the mucus membranes. It acts as a protective covering to delay
the absorption of the heavy metals. Heavy metal salts denature proteins in a
similar way as acids and bases do. Hg+2, Pb+2, Ag+1, Tl+1, Cd+2 and other
metals with high atomic weights are commonly found in heavy metal salts. Salts
destroy salt bridges in proteins because they are ionic. When a heavy metal salt
reacts with a protein, the result is usually an insoluble metal protein salt. A
protein, such as milk or egg whites, can be given as an antidote to cause the
toxic salt to precipitate.