Introduction

Our body is made up of tons and tons of protein that differs from each; some protein like the
myoglobin are used to store important molecules needed by our body; our hair are composed of
the protein keratin which is a fibrous protein that has sulfur-containing amino acids and when
denatured, it gives the hair a slick, straight hair. Understanding proteins can help us unlock and
have a better understanding of the things around us. With this, we must first identify or further
subdivide them by their classifications and how they are maid. Plants synthesize proteins from
inorganic substance present in the air and soil by undergoing different biological cycles. Animals,
like us cannot synthesize proteins on our own from such minerals like the plants. In order to obtain
them, animals digest this plants that contains proteins. While, we humans digest animals or
sometimes even plants directly as a source of proteins.

Proteins are made up of hundreds of amino acids that are connected via peptide bond and this bond
when denatured or hydrolyzed gives rise to a single amino acid. Amino acids are amphoteric which
can act as an acid due to its -COOH and basic due to is -NH2. This property of amino acids is
crucial in buffing the blood’s pH thus avoiding acidosis or alkalosis. Essential amino acids are
those that are not present in out body and it is essential for people to have a normal intake of this
animo acids (Histidine, Isoleucine, Leucine, Methionine, Phenylalanine, Threonine, Tryptophan,
and Valine). The importance of amino acids especially in the pharmaceutical practice is absurdly
high for some pharmaceutics are derived from amino acids like tyrosine where the neurotransmitter
dopamine is derived from; dopamine is not the only neurotransmitters derived from amino acid
another one is tryptophan where serotonin is derived from

Qualitative tests are used to identify the amino acids present in the protein by using the change in
color as the means of identifying wether or not the test made gave a positive result or not. In the
experiment, ten (10) qualitative test was made namely: Biuret Test, Ninhydrin Test, Xanthoproteic
Test, Millon’s Test, Hopkins-Cole Test, Sakaguchi Test, Fohl’s Test, Nitroprusside Test, Test for
amide, and Pauly’s Test.
Results

Qualitative test of hydrolyzed gluten

Qualitative Test Results

Biuret Test (+) Blue Coloration

Ninhydrin Test (+) Purple Coloration

Xanthoproteic Test (+) Yellow Coloration

Millon’s Test (-) Transparent

Hopkins-Cole Test (-) Transparent

Sakaguchi Test (+) Light pink

Fohl’s Test (+) Light Brown precipitate

Nitroprusside Test (-) Yellow Coloration

Test for Amide (+) Red to blue coloration of litmus

paper
Pauly’s Test (+) Red-Orange

Discussions

Acidic Hydrolysis of the gluten was done during the experiment and qualitative test (Biuret test,
Ninhydrin test, Xanthoproteic test, Millon’s test, Hopkins-Cole test, Sakaguchi test, Fohl’s test,
Nitroprusside test, test for Amide, and Pauly’s test) was done to the hydrolyzed samples.

Biuret Test

The experiment performed gathered a positive result of blue coloration. Biuret test is used in
identifying peptide linkage but, mono-peptide and dipeptide will not have a positive result for the
test must at least have a tri-peptide to have a positive result. Cupric ions in the Biuret reagent
chelate with the peptide bonds present of the protein in an alkaline medium which produces a pink
or violet color. The intensity of the color is directly proportional to the amount of peptide bonds
present in the protein. Figure no. is the mechanism of action of proteins with biuret reagent.
Ninhydrin Test

The Ninhydrin test that the group performed gave a positive result of a purple coloration. This test
is used to identify whether the amino acid present is an a-amino acid. A negative result (yellow
coloration) means that the amino acid present is a proline which is the only imino acid. An imino
acid differs from an amino acid in a way that the amine (-NH2) present becomes an imine (-NH);
this happens when the amino group combines with a carboxy group hence proline being an imino
acid. The reason why amino acids give a purple coloration is that the free amine (-NH2) present
reacts with the ninhydrin reagent. Fiigure no. is the mechanism of action of amino acid with the
ninhydrin reagent.

Xanthoproteic Test

The experiment performed gave a positive result (yellow coloration) to our sample (hydrolyzed
gluten). The test is used to detect aromatic acid and those amino acids are (Phenylalanine,
Tryptophan, Tyrosine, and Histidine). The principle of xanthoproteic test is the nitration of the
aromatic ring see figure no. 3. The production of a yellow colored product upon the addition of
nitric acid is a test for the presence of tyrosine or tryptophan in a protein. The addition of strong
base will deepen the color to orange. The yellow stains on the skin caused by nitric acid are the
result of the xanthoproteic reaction. Given the results of the Millon’s test and Hopkin-Cole test, it
can be concluded that the aromatic ring present in gluten is not tyrosine nor tryptophan.
Millon’s Test

The Millon’s test gave a negative result (transparent/ no color change) to our sample which is
hydrolyzed gluten. This test is used to detect phenolic ring containing amino acid by giving a
positive result of old rose to fleshy red coloration. In Millon’s test, the phenol group of tyrosine is
first nitrated by nitric acid in the test solution. Then the nitrated tyrosine complexes mercury (I)
and mercury (II) ions in the solution to form a red precipitate or a red solution, both positive results.
See figure no. 4 for the reaction mechanism. Proteins that contain tyrosine will, therefore, yield a
positive result. By comparing the results from the xanthoproteic test, this confirms that the positive
result given by the xanthoproteic test was not tyrosine but other aromatic rings.

Hopkin-Cole Test

The experiment using the Hopkin-Cole method gave a negative result (transparent/ no coloration)
to the hydrolyzed gluten sample. This test is used to identify whether or not the protein contains
the amino acid tryptophan. The indole group of tryptophan reacts with glyoxylic acid in the
presence of conc. H2SO4 to give a purple colored complex. Glyoxylic acid is prepared by
reducing Oxalic acid with magnesium powder or sodium amalgam. Tryptophan is another example
of an aromatic amino acids wherein the Xanthoproteic test was used as the means of identification.
Given the results of the Xanthoproteic test and Hopkin-Cole test, we concluded that the aromatic
ring present in the hydrolyzed gluten sample is not tryptophan.

Sakaguchi Test

The experiment performed using the Sakaguchi method garnered a positive result (pink coloration).
This test is used to detect uni or monosubstituted guanidine; the guanidine containing amino acid
is Arginine.