Experiment No.

4
Color Reaction of Proteins

Ian Malapitan, Zerimar Dela Cruz Year/Section: BSN-1E

Date Performed: 10/15/2020 Date Submitted: 10/16/2020

Objective:
To determine whether a sample contains proteins or protein breakdown products
based on certain functional groups which are present.

Materials/Apparatus: Albumin, casein, gelatin, phenol, test tubes, test tube

racks, graduated cylinders, erlenmayer flasks, stirring rods, droppers, water bath,
Conc. HNO3, NaOH, Hopkins-Cole reagent, Conc. H2SO4, CuSO4, Ninhydrin
solution,

Procedures:
samples to be tested:
1. albumin 3. gelatin
2. casein 4. phenol

A. Xanthoproteic Acid Test

Add 1 mL of conc. HNO 3 to 2 mL of the sample. Mix and note the appearance
of any heavy, white precipitate. Warm the mixture carefully, noting any change to
a yellow-colored solution. Cool the mixture in a stream of cold water and carefully
add 10% NaOH. Note 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 the sample.
Incline the tube at an angle and carefully add a small amount of conc. H2SO4 so
that it drains to one side and forms a separate lower layer in the tube. A positive
result consists of the appearance of a violet ring at the interface without bringing
about extensive solution of the two layers in each other.

C. Biuret Test
Thoroughly mix 2 mL of 10% NaOH with 2 mL of the sample. Add one drop of
0.1% CuSO 4 solution. Mix thoroughly and note if a pink or violet color develops.
This is the positive result. If this is not observed, add up to 10 more drops of
CuSO 4, mixing the solution after each drop.

D. Ninhydrin Test
Add 4-6 drops of freshly prepared 0.1% ninhydrin solution to 2 mL of the
sample. Mix thoroughly and heat to boiling. Cool and observe if the color turns to
intense blue or violet. This is the positive result.
Results and Observations:

For Xanthoproteic Acid Test

Samples Albumin Casein Gelatin Phenol
White
Present Present None None
precipitate
Color
Yellow Light Yellow None Orange
Change

For Hopkins-Cole Test

Samples Albumin Casein Gelatin Phenol
Violet ring at
Present Present None None
the interface

For Biuret Test

Samples Albumin Casein Gelatin Phenol
Color
Violet Blue-Violet Violet Blue
Change

For Ninhydrin Test

Samples Albumin Casein Gelatin Phenol
Color
Dark Blue Dark Blue Dark Blue None
Change
Discussion and Conclusion:

All the laboratory experiments performed and observed are ways on how to
identify whether a sample contains proteins based on the functional groups
present in the sample/solution. Detection for proteins are based on the change of
color and how the samples react with their respective reagents and with the other
solutions/acids that are involved. Overall, this laboratory activity is for us to better
understand proteins, the amino acids that make up proteins.

For Xanthoproteic Acid Test

The Xanthoproteic Acid Test is used to detect aromatic amino acids in a

protein solution/sample (Bangash, 2020). Examples of aromatic amino acids are
tryptophan, tyrosine, and phenylalanine.

In this test, the group detected is a Benzene ring on which there are amino
groups (tryptophan) or hydroxyl groups (tyrosine) are easily nitrated to give
yellow-colored aromatic nitro compounds, which are given the general term
xanthoproteic acid. Concentrated HNO3 reacts with the aromatic rings that are
derivatives of benzene giving the characteristic nitration reaction. Amino acids
tyrosine and tryptophan contain activated benzene rings which are easily nitrated
to yellow colored compounds.

If there is a benzene ring present in the sample, it will change to yellow or

yellow-orange in color which is indicative of a positive reaction. Albumin, casein,
and phenol are positive since they turned to yellow while gelatin is negative
because there was no change in color.

For Hopkins-Cole Test

According to Kumar (n.d.), Hopkins-Cole Test is specifically for detecting

tryptohphan which is the only amino acid containing an indole group. The indole
ring reacts with glyoxylic acid in the presence of a strong acid to form a violet
cyclic product. The Hopkins-Cole reagent only reacts with proteins containing
tryptophan. The protein solution is hydrolyzed by the concentrated sulphuric acid
at the solution interface. Once the tryptophan is free, it reacts with the glyoxylic
acid (Hopkins-Cole Reagent) to form the violet product. However, it is also
important to note that the chemistry of this test is not yet well understood.

The formation of a violet ring at the junction of the two liquids indicates the
presence of tryptophan. This explains that only albumin and casein are positive
since both samples produced a violet ring, while gelatin and phenol are negative
due to the absence of a violet ring.
For Biuret Test
In this test, the group detected is any component (protein or otherwise) which
contains two or more of the following groups: amide, amino, -C(NH 2 )=NH,
-CH(OH)CH 2 NH 2. The name of the test is derived from the specific compound
biuret: NH 2 CONHCONH 2. This is the most general test for proteins.

A Biuret test is a chemical test used to determine the presence of a peptide

bond in a substance. It is based on the biuret reaction in which a peptide structure
containing at least two peptide links produces a violet color when treated with
alkaline copper sulfate (Aryal,2020). According to Aryal (2020), the Biuret reagent
is a solution composed of sodium hydroxide (NaOH) or potassium hydroxide
(KOH), hydrated copper (II) sulfate, and potassium sodium tartrate.
Sodium hydroxide and Potassium hydroxide provide the alkaline medium and
potassium sodium tartrate is added to chelate and thus stabilize the cupric ions in
the solution or to maintain their solubility in alkaline solution.

The development of a purple/violet colored complex is a positive indicator for

the biuret test. Only phenol is negative since it did not turn violet while albumin,
casein, and gelatin are positive because they all turned violet.

For Ninhydrin Test

A ninhydrin test is a general test performed by all amino acids. The test is
performed as a result of the reaction between the amino group of free amino acid
and ninhydrin, this means that this test depends upon the presence of a-amino
groups. Ninhydrin is the powerful oxidising agent. It causes oxidative
decarboxylation of amino acids. Once heated with ninhydrin, amino acids are
oxidatively decarboxylated, producing carbon dioxide (CO2), ammonia (NH3),
and an aldehyde. Reduced ninhydrin then reacts with the liberated ammonia and
a blue or purple coloured complex is produced (Bangash, 2020).

If the sample changed to violet or blue in color, it indicates the presence of

a-amino group. Albumin, casein, and gelatin are positive since they all changed
in color (dark blue), while phenol did not.
References:

https://medicalstudyzone.com/xanthoproteic-test/

https://www.biologydiscussion.com/proteins/qualitative-and-quantitative-tests-for-
amino-acids-and-proteins/13065

https://medicalstudyzone.com/hopkins-cole-test/#:~:text=PROCEDURE%20of%
20Hopkins%20Cole%20test%3A%201%20Add%201,down%20along%20the%2
0side%20of%20the%20test%20tube.

https://microbenotes.com/biuret-test-for-protein/#:~:text=A%20Biuret%20test%2
0is%20a%20chemical%20test%20used,violet%20color%20when%20treated%2
0with%20alkaline%20copper%20sulfate.

https://medicalstudyzone.com/ninhydrin-test/