The samples used for the qualitative color

reactions include the intact protein, acidic

hydrolysate, basic hydrolysate, and enzymatic
hydrolysate of the isolated protein albumin found
in skimmed milk. The samples were all tested to
characterize and determine the functional groups
that they contain, depending on their reactions to
the reagents of each test.
Biuret test is a general test for proteins
and is a test for detecting peptide linkage. The
biuret reagent (copper sulfate in a strong base)
reacts with peptide bonds in proteins to form a
blue to violet complex known as the Biuret
complex. At least two peptide bonds are required
for the formation of this complex [1].

Based on the results obtained, the intact

protein showed a positive result (purple solution)
for this test since its peptide linkage is not yet
broken. This goes the same for the acidic and
enzymatic hydrolyzed samples which also
presented a positive result (purple solution) for
the Biuret test. This means that the acidic and
enzymatic hydrolysis werent able to completely
hydrolyze the sample unlike in the basic
hydrolysis which presented a clear blue solution
depicting a lower intensity of color correlating to
the presence of hydrolyzed proteins in the form
of amino acids.
Ninhydrin is a test for detecting free
alpha-amino groups. The covalently bonded
peptide bonds present in proteins are disrupted
as they are hydrolyzed. After completion of
hydrolysis, the hydrolysate or hydrolyzed protein
is what remains, containing a mixture of amino
acids that react with ninhydrin. Ninhydrin reacts
with alpha-amino acids (--NH2) in proteins to
give a purple complex as illustrated in the
reaction that follows, with the exception of
proline and hydroxy proline which both give a
yellow color due to the absence of free alphaamino acids (no NH2). Ninhydrin is most
comonly used as a forensic chemical to detect
fingerprints, as amines left over from proteins
sloughed off in fingerprints react with ninhydrin
giving a characteristic purple color [1].

The principle behind test is oxidative

deamination and decarboxylation. A positive
indication of this test would be a blue-violet
coloration in the solution. In the results obtained,
only the basic hydrolysate tested positive for the
ninhydrin test as the intact protein, acidic
hydrolysate, and enzymatic hydrolysate all
presented a colorless solution in the presence of
ninhydrin. The basic hydrolysate is the only
sample which contained free alpha-amino groups
as a result of its complete hydrolysis.
Xanthoproteic test is a test for the
presence of aromatic amino acids which include
tyrosine and tryptophan. Although phenylalanine
is considered an aromatic amino acid, it will not
yield a positive result since it is inactive. Tyrosine
and tryptophan reacts with concentrated HNO3 at
high temperature to form nitro-compounds which
are yellow in color. At alkaline pH, the color
changes to orange due to the ionizaiton of the
phenolic group [2].

In the results obtained, only the intact

protein and the acidic hydrolysate yielded a
positive result (clear yellow solution).

At alkaline pH, the color changes to orange

due to the ionization of the phenolic group.
Millon Test
Principle
This test is specific for Tyrosine.
Millons reagent (Hg/HNO3) gives
positive
results
with
proteins
containing the phenolic amino acid
tyrosine
Hopkins-Cole Test
Sakaguchi Test
Principle
Sakaguchi test is specific for Arginine
Sakaguchi test is positive for the
amino acid containing the guanidine
group in Arginine
Guanidine group present in the amino
acid reacts with alpha-Naphthol and
alkaline hypobromite to give a redcolored complex.

Nitroprusside Test (Red color)

Principle
This test is specific for proteins
containing sulfur, --SH (in cysteine and
cystine)
Gives a red-purple color called Mrner
test.

REFERENCES
[1] Biochemistry Practical Proteins and Amino
Acids. (2013) (1st ed., pp. 7-20). Kingdom of
Saudi Arabia, Ministry of Higher Education.
Retrieved from
http://www.magdyelnashar.com/new/images/pdf
/Practical.Proteins.and.Amino.Acids.Identification.
pdf
[2] Qualitative Analysis of Amino Acid (Theory) :
Biochemistry Virtual Lab I : Biotechnology and
Biomedical Engineering : Amrita Vishwa
Vidyapeetham Virtual Lab. (2011).
Vlab.amrita.edu. Retrieved 24 March 2016, from
http://vlab.amrita.edu/?
sub=3&brch=63&sim=1094&cnt=1