Concepcion, Jenny P.

Lorenzo, Marianne Angelina R.

Nalupta, Jamie Patrice A.

EXPERIMENT 5
COLOR REACTIONS OF PROTEINS AND AMINO ACIDS

ABSTRACT
Proteins, due to the presence of peptide bonds and amino acid residues react to different kinds
of reagents to form colored products or precipitates. Different color reaction tests were
performed. In the Ninhydrin reaction, amino acids containing α-amino acid group reacts with
ninhydrin solution forming a violet-blue colour. Glycine was used in the test and yielded a
positive result. Meanwhile proline and hydroxyproline, containing the amino group create
yellow-coloured products, though it was not performed. Biuret test is used to determine the
presence of peptide bonds in protein. Ideally, in the presence of peptide or protein, the biuret
reagent with is a solution of CuSO4, NaOH and sodium potassium tartrate changed color from
blue to purple. However, a negative result occurred with glycine. In the Xanthoprotiec test, the
aromatic rings of tyrosine and tryptophan under the effect of nitric acid form yellow-coloured
nitro derivatives but yielded negative result for phenylalanine and histidine. Hopkins-Cole
reaction is a specific test for detecting tryptophan, the only amino acid containing an indole
group. The indole moiety of tryptophan reacts with glyoxilic acid in the presence of concentrated
sulfuric acid to give a purple ring on the junction of the two fluids. However, the test yielded a
negative result. Lead acetate reaction is a specific test for sulfur-containing amino acids.
Cysteine yielded positive reaction. Moreover, pieces of hair (straight and curly) were also tested
and yielded a positive result on both samples because the hair has cysteine. Lastly, Sakaguchi
reaction was used to detect the presence of the amino acid arginine with a functional group
guanidine. The result yielded a positive result of red-orange complex. Several tests had
negative results primarily due to improper handling of solutions that caused contaminations and
decreased potency. It is therefore recommended that proper preparation and procedure be
done to achieve accurate results.

INTRODUCTION
Proteins are the most abundant organic molecules of the living system. Proteins on
complete hydrolysis yield L-α amino acids, the polymer of proteins (Satyanarayana, 2013).

The Ninhydrin reaction test is one of the most general qualitative tests for proteins.
Ninhydrin (triketohydrindene hydrate) is an oxidating agent which leads to the oxidative deamination of alpha-
amino groups. When reacting with free amines from the amino acid, blue or purple color is produced. The
protein when heated in boiling with ninhydrin gives an intense blue or purple colored complex.
This is illustrated by the reaction:
 When ninhydrin reacts with amino acids, the reaction also releases CO 2. Clinically, a ninhydrin solution is
commonly used by forensic investigators in the analysis of latent fingerprints on porous surfaces such as paper.
Amino acid containing fingermarks, formed by minute sweat secretions which gather on the finger’s unique ridges,
are treated with the ninhydrin solution which turns the amino acid finger ridge patterns purple and therefore visible.
(https://fulltimes.wordpress.com/protein/).

Biuret test is often used to determine the presence of peptide bonds in protein. In an alkaline
medium, CuSO4 reacts with peptide bond nitrogen of peptides of proteins to form violet colored
complex (Shivaraja et al. ). The intensity of the color, and hence the absorption at 540 nm, is directly proportional to
the protein concentration, according to the Beer-Lambert law. In spite of its name, the reagent does not in fact
contain biuret ((H2N-CO-)2NH). The test is so named because it also gives a positive reaction to the peptide bonds
in the biuret molecule. (https://fulltimes.wordpress.com/protein/).

Xanthoproteic test is a test that detects the presence of a phenyl group in aromatic amino acids.
The aromatic ring is nitrated upon addition of concentrated nitric acid resulting to a yellow nitro-
derivative. If the pH of the solution is increased, a salt of the derivative is formed giving an orangish
solution.

Hopkins-Cole Reaction is a specific test for detecting tryptophan, the only amino acid
containing an indole group. The indole moiety of tryptophan reacts with glyoxilic acid in the
presence of concentrated sulfuric acid to give a purple colored product. Once the tryptophan is
free, it reacts with the glyoxylic acid to form the violet ring.

Lead acetate reaction is a specific test for sulfur-containing amino acids such as cysteine
[ CITATION Nig07 \l 1033 ]. Sulfur present in cysteine is converted to sodium sulfide by boiling with 20%
NaOH, as shown by the following reaction:
 +

The sodium sulfide (Na2S) was detected by the precipitation of lead sulfide (PbS) when lead
acetate was added:

Moreso, human hair has a higher content of cysteine than that of other species and it is
called -keratin (Chatterjea, 2012). The presence of cysteine contributes to the strength of the
hair shaft by binding to the adjacent sulfides (disulfide bond) of the cysteine amino acids in a
keratin helix (Nelson & Cox, 2013).

On heating conditions under alkaline solution, the sulfur of the amino acids combined with lead
acetate that formed black lead sulfide [ CITATION Nig07 \l 1033 ].

Sakaguchi reaction is a chemical test used to detect the presence of the amino acid
arginine in both free form and in arginine-containing proteins. Its functional group is guanidine
which is a strong base and dissolves in polar solvents. The guanidine reacts with α-naphtol and
sodium hypobromite which is an oxidizing agent to yield a red to orange-colored complex

The objective of
this experiment is to

4. What precautions must be observed in testing for proteins using the biuret test in
solutions salted out with (NH4)2SO4 or MgCl2? Why?
5. Are the Xanthoproetic and Millon-Nasse tests satisfactory for use in the urinary examination
for protein? Why?

6. Why does the bromine water test give negative results to protein containing tryptophan?

Satyanaranaya, U. (2013). Biochemistry. Elsevier India Private Limited. India