Experiment 8: Color Reaction of Proteins and Amino Acids

VI. Analysis
1. Discuss each test to detect groups in proteins and amino acids. Write equations to support
result.
Ninhydrin Test
Ninhydrin is a general test used to detect the presence of amino acids, In the pH range of
4-8. In the presence of Ninhydrin, amino acids undergo oxidative deamination liberating a few
compounds such as ammonia, CO2, an aldehyde and hydrindantin. Hydrindantin further reacts
with Ninhydrin which gives of a blue product. However, in case of imino acid like proline and
hydroxyproline, a different product having a bright yellow color is formed. Asparagine, which has
a free amide group, reacts to give a brown colored product. All was positive except for the Blank
test, since all contains amino acids.

Biuret Test
Biuret is a test for compounds containing a peptide bond. Biuret is created when urea is
heated to 180C. When Biuret is treated with dilute copper sulfate in alkaline condition, a purple
colored compound is observed. This test is also used to detect the presence of protein in
biological fluids. The purple color is due to the formation of co-ordination complex of cupric ions
with unshared electron pairs of peptide nitrogen and O2 of water. All of the amino acids as well
as the casein and albumin are positive towards this test since they all contain peptide bonds.

Xanthoproteic Test
 This test is used to detect amino acids containing aromatic nucleus. The aromatic ring
undergoes nitration to give yellow colored products. In the presence of concentrated nitric acid,
the aromatic phenyl ring is nitrated to give yellow colored nitro-derivatives. At alkaline pH, the
color changes to orange due to the ionization of the phenolic group. The amino acids and the
casein and albumin held a positive result, thus, they have aromatic nucleus.

Million- Nasse Test

This test is specific for the amino acid tyrosine and the protein containing this amino acid.
Tyrosine when reacted with acidified mercuric sulphate solution gives yellow precipitate of
mercury-amino acid complex. On addition of sodioum nitrate solution and heating, the yellow
complex of mercury-amino acid complex converts to mercury phenolate which is in red color.
Tyrosine was the only one who had a positive test which indicates that casein and albumin most
likely does not contain tyrosine.

Hopkins- Cole Test

This test is specific test for detecting tryptophan. The indole group of tryptophan reacts
with glyoxylic acid in the presence of concentrated H2SO4 to give a purple colored
complex. Glyoxylic acid is prepared by reducing Oxalic acid with magnesium powder or sodium
amalgam. Both casein and albumin contain tryptophan so naturally it has a positive test result as
well as the amino acid, tryptophan.

Bromine Water Test

 This test is used to determine the presence of double bonds among carbon atoms. These
test will let us distinguish alkanes from alkenes and determine the presence of such. If mixed with
an alkene bromine water will become colorless because of its reaction with the alkene. Thus,
from the result observed both casein and albumin contain a carbon to carbon double bond or an
alkene.

Pauly Test
This test is specific for the detection of tyrosine or histidine. The reagent used for this
test contains sulphanilic acid dissolved in hydrochloric acid. Sulphanilic acid upon diazotization in
the presence of sodium nitrite and hydrochloric acid results in the formation a diazonium salt.
The diazonium salt formed couples with either tyrosine or histidine in alkaline medium to give a
red coloured chromogen (azo dye). This test specifically is used for locating tyrosine and histidine
which is why these amino acids had a positive test result along with casein and albumin which
contains these amino acids.

Lead Acetate Test

This test is specific for sulphur containing amino acids (Cysteine, Cystine and Methionine).
Upon boiling the Sulphur with amino acids, the Sulphur is split off from the amino acids and
become metallic sulphides (K2S or Na2S). By adding lead acetate, they are oxidized to form black
colored lead sulphides. Cysteine indicates a positive test result along with albumin and casein
which contains traces of sulfur. Also, our hair had a positive test result for it has sulfur as its
building block.

Sakaguchi Test
 This test is used to determine the presence of arginine in compounds or proteins. The
reagent sakaguchi comprises of 1-Naphtol and a drop of sodium hypobromite. The red complex
comes from the reaction of the sakaguchi reagent and the guanidine group of arginine. Since
Arginine is tested it has a positive test result as well as casein which is a dairy product which
contains arginine.

Conclusion
Color Reaction among Proteins and Amino Acids are only possible due to the tests
performed in this specific experiment. We have understood the importance of understanding the
factors, processes and test result, as well as its significance towards determining the contents of
specific compounds and substances. These set of experiments are also highly utilized to
specifically determine which amino acids are present and proteins observed in a specific test
sample.

References:
https://www.onlinebiologynotes.com/ninhydrin-test-principle-requirements-procedure-and-result/
https://www.onlinebiologynotes.com/biuret-test-principle-requirements-procedure-and-result/
https://www.onlinebiologynotes.com/xanthoproteic-test-objective-principle-reagents-procedure-and-
result/
http://vlab.amrita.edu/?sub=3&brch=63&sim=1094&cnt=1
https://www.onlinebiologynotes.com/paulys-test-objective-principle-reagents-procedure-and-result/
http://ecoursesonline.iasri.res.in/mod/page/view.php?id=53516