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Worksheet - Experiment 4 Color Reactions of Proteins

This document describes 6 different color reaction tests performed on proteins: 1. Biuret test detects peptide bonds and forms a purple color with copper. 2. Ninhydrin test detects amino groups and forms a purple color. It cannot detect whole proteins due to steric hindrance. 3. Xanthoproteic test detects aromatic amino acids, forming a yellow then orange color with nitric acid and alkali. 4. Millon's test detects tyrosine, forming a red color. 5. Adamkiewicz test detects tryptophan, forming a purple ring between layers with glyoxylic acid and sulfuric acid. 6. Lead acetate test detects sulfur amino acids, forming

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170 views3 pages

Worksheet - Experiment 4 Color Reactions of Proteins

This document describes 6 different color reaction tests performed on proteins: 1. Biuret test detects peptide bonds and forms a purple color with copper. 2. Ninhydrin test detects amino groups and forms a purple color. It cannot detect whole proteins due to steric hindrance. 3. Xanthoproteic test detects aromatic amino acids, forming a yellow then orange color with nitric acid and alkali. 4. Millon's test detects tyrosine, forming a red color. 5. Adamkiewicz test detects tryptophan, forming a purple ring between layers with glyoxylic acid and sulfuric acid. 6. Lead acetate test detects sulfur amino acids, forming

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Worksheet CHEM1163L

Biochemistry Laboratory

Experiment 4
Color Reactions of Proteins

Names: __Iona Fran J. De Los Santos and Kyla F. Fernandez___________ Schedule ________________

Write your correct observations and inferences/conclusions for each of the following:

A1. Biuret Test


Reagent’s NaOH and CuSO4
Composition
Observations: The color of the mixture turned purple indicating that the test is positive
due to the presence of proteins.
Inferences: What is the principle behind this test?
-The principle behind this test is to determine the presence of a peptide
bond in a substance. This chemical test is based on the biuret reaction in
which a peptide structure having at least two peptide links produces a
violet color. The color is formed between Cu2+ ion and carbonyl oxygen
(>C=O) and amide nitrogen (=NH) of the peptide bond. Once this complex
has been formed, the solution turns into a blue color to purple. The
intensity of the color depends on the number of the peptide-copper
complexes.

A2. Ninhydrin Test


Reagent’s Ninhydrin dissolved in ethanol
Composition
Observations: A purple-colored complex was present in the sample which it indicates
that the test is positive due to the presence of amino groups and amines.
Inferences: What is the principle behind this test?
-The principle behind this test is to verify the presence of an amino group.
When the two molecules of ninhydrin (2,2- dihydroxyindane-1,3-dione)
react with a free alpha-amino acid, a deep purple or blue color known as
Ruhemann’s purple will produce.

Can ninhydrin test distinguish amino acids and proteins? Can this test be
used to detect the presence of proteins in a sample? Elaborate your
answers.
-Ninhydrin can only distinguish amino acids and cannot detect the
presence of proteins. It is because the steric hindrance limits the
ninhydrin from reaching the α-amino groups therefore, the test is not
effective enough as Biuret test in terms of detecting the proteins of a
given sample.

A3. Xanthoproteic Test

Reagent’s Conc. HNO3


Composition
Observations: The color of the sample has a combination of yellow and orange colors

Inferences: What is the principle behind this test?


Worksheet CHEM1163L
Biochemistry Laboratory

-The principle behind this test is based on the nitrated aromatic groups in
the amino acids that were heated with concentrated HNO3 in order to
have an intensely yellow-colored nitro derivative. On the addition of
alkali, however, the residue turns orange because of the salt of the
tautomeric form of the nitro compound was able to produce.

What type of amino acid will give a positive result in Xanthoproteic test?
-The type of amino acid that will give a positive result in Xanthoproteic
test is aromatic amino acid.

A4. Millon’s Test

Reagent’s Millon’s Reagent consisting of mercuric nitrate and mercurous nitrate


Composition that is dissolved in concentrated nitric acid.
Observations: Millon’s reagent is added into the test tube containing egg albumin. The
mixture was then heated for a couple of seconds. When the test tube was
taken out it shows a yellowish color rather than a Brick Red color
showing that it has a negative reaction meaning that there is no Tyrosine.

Inferences: What is the principle behind this test?


-The principle behind this test is that compounds that contain
hydroxybenzene radical react with the reagent to form a red complex,
therefore the test is specific for the amino acid containing tyrosine and
the protein containing this amino acid.

What type of amino acid will give a positive result in Millon’s Test?
-The type of amino acid that will give a positive result would be Tyrosine
and Phenol Solution which will give of a Brick Red color.

A5. Adamkiewicz Test

Reagent’s Adamkiewicz Reagent: glyoxylic acid(prepared by reducing oxalic acid


Composition with magnesium powder or sodium amalgam)
Observations: The glacial acetic acid is added to the test tube containing egg albumin
and shaken to combine, after combining the two concentrated Sulfuric
Acid is then added to the mixture where it is then left to set for a few
minutes, and after 10 minutes a purple ring starts to show meaning that it
is positive with the presence of tryptophan.

Inferences: What is the principle behind this test?


-The indole group of tryptophan reacts with glyoxylic acid in the presence
of H2SO4 giving a purple color, which looks more like purple rings
showing between the layers.

What type of amino acid will give a positive result in Adamkiewicz Test?
-The presence of Tryptophan showing purple rings between two layers
means that it is positive.

What is the role of concentrated sulfuric acid?


Worksheet CHEM1163L
Biochemistry Laboratory

-With the addition of the concentrated sulfuric acid to the mixture it


combines with the protein and the glyoxylic acid helping produce a
purple color helping prove that it is positive.

A6. Lead acetate Test

Reagent’s Lead Acetate is the test used.


Composition
Observations: After adding NaOH to the test tube containing egg albumin and shaking
vigorously it is put in a hot water bath after it is taken our of the boiling
water it is being cooled down by running water, after cooling down a few
crystals of Pb(OAc)2 to 2-3 mL of the protein suspension is added and
after a few second a brown precipitate starts to form making it positive
for the Lead acetate Test.

Inferences: What is the principle behind this test?


-Sulfur containing amino acid will react with the lead acetate under
alkaline conditions forming a brown precipitate.

What type of amino acid will give a positive result in this test?
-Sulphur containing amino acid such as Cysteine, Cystine, and Methionine.

ACTan 1st Sem SY 2021-2022


ETBacolod and JBRabor 1st Sem SY 2020-2021

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