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:

• Sulfuric Acid
Materials • Copper Sulfate Solution
• Albumin • Ninhydrin’s Solution
• Phenol
• 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

Add 1 mL of Nitric Acid to each of the 2mL samples (Ratio 1:2). Mix and note the
appearance of any heavy, white precipitate. Warm the mixture carefully in a water bath, noting a
change in color in the samples (yellow). Cool the mixture in a stream of cold water and carefully
add 10% Sodium Hydroxide. If the color deepens to orange or yellow orange. This is the
positive result.

B. HOPKINS-COLE TEST

Add 2 mL of Hopkins-Cole reagent (or glyoxilic acid) to 2 mL of each of the samples

(Ratio 1:2).Incline the tube at an angle thencarefully add a small amount of concentrated
Sulfuric Acid so that it drains on one side and forms a separate lower layer in the tube. If a
purple ring forms in the interface of the two liquids, the result is positive.

C. BIURET TEST
 Add 2 mL of 10% Sodium Hydroxideto 2 mL of each of the samples. Add one drop of
0.1% Copper sulfate solution. Mix thoroughly and note if there is a change in color, either pink
or purple, then the result is positive. If this is not observed, add up to 10 more drops of Copper
sulfate solution, mixing the solution after each drop and continue noting the change in color.

D. NINHYDRIN TEST

Add 4-6 drops of 0.1% Ninhydrin solution to 2 mL of each of the samples. Mix thoroughly
andheat by placing the samples in a 100-degree water bath. Cool and observe. If the color turns
to intense blue or violet, it indicates that the result is positive.

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.
HOPKINS-COLE For albumin, there is a violet ring that separates the white liquid at the top and the
TEST 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.

For gelatin, the liquid remains the same after the addition of glyoxilic acid and
sulfuric acid. 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.
After mixing 10% NaOH and 0.1% of CuSO 4 in Albumin, Casein, and Gelatin, it
produced a pink-purple color which indicates that all three are proteins or have
amide or amino groups in their molecular structure. After mixing the 10% NaOH
BIURET TEST
and 0.1% of CuSO 4 in Phenol however, it did not produce a pink-purple color,
which indicates that Phenol does not have amide or amino groups in its molecular
structure.
After mixing the Ninhydrin solution with Albumin, Casein, and Gelatin, it produced
a positive result which showed that the 3 solution turn into a blue or violet color
which indicates that Albumin, Casein, and Gelatin have α-amino groups present
NINHYDRIN TEST
in them. After mixing with Phenol however, it did not result in a blue or purple
because Phenol is part of the Hydroxyl group, which means that it does not
contain α-amino groups.

CONCLUSION:

A. XANTHOPROTEIC ACID TEST

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.

B. HOPKINS-COLE TEST

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.

C. BIURET TEST

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.

D. NINHYDRIN TEST

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 NinhydrinTest? 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.