Experiment # 4

COLOR REACTIONS OF PROTEINS

Group Number:  1                                                                                     Year/Section: BSN 1-B                   

Date performed: October 8, 2020         Date Submitted: October 8, 2020

Group Leader: Jamiel James Arceno

Members: Star Arricivita

Seth Basio

OBJECTIVE:  To determine whether a sample contains proteins or protein

breakdown products based on certain functional groups which are
present.

APPARATUS AND MATERIALS:

Materials • Sulfuric Acid

• Albumin • Copper Sulfate Solution

• Phenol • Ninhydrin’s Solution

• Casein Apparatuses

• Glycerol • Test tubes

• Nitric Acid • Test tube rack

• Sodium Hydroxide • Droppers

• Hopkins-Cole Reagent • Graduated cylinder

PROCEDURE:

Samples to be tested:

1. Albumin
2. Casein
3. Gelatin
4. Phenol

A. XANTHOPROTEIC ACID TEST

B. HOPKINS-COLE TEST

C. BIURET TEST
D.  NINHYDRIN TEST

RESULTS AND OBSERVATIONS:

TEST RESULTS AND OBSERVATIONS

For albumin, when it is added with nitric acid, the liquid becomes white. The color changed
into yellow (due to nitrated side chains of phenylalanine and tyrosine which are present in
the protein) upon heating. Afterwards, the addition of sodium hydroxide an orange ring is
formed at the top of the liquid; which gives albumin a positive response in the
xanthroproteic acid test.

For casein, it also turned into a clear yellowish liquid after being added by nitric acid. There
is no heavy white precipitate formed. There is a presence of a deep yellow ring on the top of
the liquid, which is the xanthroprotein. After being added by sodium hydroxide, the color
became a murkier, faded yellow. The response of casein to the test is a positive.
XANTHOPROTEIC
ACID TEST
For gelatin, the liquid is clear at first but soon turned into a clear yellowish liquid after being
added by nitric acid, which looks quite similar to casein. No white precipitate is formed. A
presence of a deep yellow ring also formed at the top of the liquid. After being added by
sodium hydroxide, the color of the liquid turned into a semi-clear yellow liquid. It has a
positive result, which means proteins are present in gelatin.

For phenol, the color changed from transparent to an amber brown color after the addition
of nitric acid. There is no presence of a yellow ring on the top of the liquid. After the
addition of sodium hydroxide, the color of the liquid remains the same. Therefore, there is
no presence of proteins in phenols.
For albumin, there is a violet ring that separates the white liquid at the top and the clear
liquid at the bottom. This indicates a positive reaction from the test.

For casein, there is also a violet ring that separates the white liquid at the top and the clear,
liquid at the bottom of the test tube. This also indicates a positive reaction from the test.
HOPKINS-COLE
For gelatin, the liquid remains the same after the addition of glyoxilic acid and sulfuric acid.
TEST
There is no violet ring that separates the liquid. This indicates a negative reaction from the
test.

For phenol, the liquid also remains the same after the addition of glycoxilic acid and sulfuric
acid. There is also no violet ring present that separates the liquid. This also indicates a
negative reaction from the test.
BIURET TEST
NINHYDRIN TEST

CONCLUSION:

Xanthoproteic acid test is a test that detects the presence of benzene rings or amino acids
containing aromatic groups namely tyrosine, tryptophan and phenylalanine. Their presence is identified
when the sample changes to a color ranging from yellow to orange due to the xanthoproteic acid that is
formed when the aromatic benzene ring undergoes nitration after adding concentrated nitric acid then
warming the sample in a water bath. In the experiment, out of the four samples, three samples (phenol,
albumin, casein) turned yellow or showed a positive reaction and only gelatin had no reaction indicating
that there are no benzene rings present in it.

Hopkins-Cole test is a test that detects the presence of tryptophan, an aromatic amino acid in
the sample. A positive reaction is indicated by a purple ring due to the indole group of tryptophan
reacting with glyoxylic acid in the presence of concentrated sulfuric acid. The ring appears in the
interface of the two liquids after gradually adding concentrated sulfuric acid. In the experiment, once
again only three samples (phenol, albumin, casein) showed a positive reaction implying the presence of
tryptophan and gelatin once again, had no reaction; hence, the absence of tryptophan in the gelatin.

Biuret test is a test that detects the presence of peptide bonds. It is based on the biuret reaction
in which a peptide structures containing at least two peptide links or two or more of the following
groups: amide and amino, produces a violet color when treated with alkaline copper sulfate. The purple
color indicates a positive reaction. The colored coordination complex is formed between Cu2+ ion and
carbonyl oxygen and amide nitrogen of the peptide bond. Once this complex has been formed, the
solution turns from blue to purple. In the experiment, three of the samples (albumin, casein, and
gelatin) showed a positive reaction confirming the presence of two or more peptide bonds. However,
phenol had no reaction and stayed the same.

Ninhydrin test is a test that detects the presence of alpha amino groups in the sample. A deep
blue color indicates the presence of ammonia, primary or secondary amines, or amino acids in the
analyte. The amino acid undergoes oxidative deamination, resulting in the liberation of carbon dioxide,
ammonia, and an aldehyde along with hydrindantin (which is a reduced form of ninhydrin) after it
undergoes a chemical reaction with ninhydrin, which acts an oxidizing agent. Then the ammonia goes on
to react with another ninhydrin molecule to form diketohydrin which is responsible for the deep blue
color. In the experiment, three of the samples (albumin, casein, and gelatin) showed a positive reaction
confirming the presence of alpha amino groups in the samples. However, phenol had no reaction and
stayed the same indicating the absence of the alpha amino groups.

QUESTIONS:

1. How will you distinguish aromatic amino acids from non aromatic amino acids?

Aromatic amino acids are amino acids that have an aromatic ring in the side-chain. Amino acids
are biologically important organic compounds that contain amine (-NH2) and carboxylic acid (-COOH)
functional groups, with a side-chain (-R) specific to each amino acid. Therefore, we can say that a non
aromatic amino acid does not have an aromatic ring in the side chain.

2. Why is Biuret Test used as a general test for the detection of proteins?

   The biuret test, also known as Piotrowski's test, is a chemical test used for detecting the
presence of peptide bonds. Since a peptide is two or more amino acids joined together by peptide
bonds; a polypeptide is a chain of many amino acids; and a protein contains one or more polypeptides.
Therefore, proteins are long chains of amino acids held together by peptide bonds, allowing the biuret
test to determine the presence of a protein due to its relation with a peptide bond.

3. Will all amino acids be detected using Ninhydrin Test? Why?

Free amino groups will react with the ninhydrin reagent to yield a purple solution. Almost all
amino acids contain a free amino group (except proline and hydroxyproline). Therefore, non polar
amino acids will not be detected using ninhydrin test, due to the absence of a free -NH2 group that
reacts with ninhydrin the forms the blue-purple complex.