Color Reactions of Casein Protein and Basic Hydrolysate

J.C. Garbosa, A.T. Garcia, R.S. Garcia, R.C. Gomez, D.A. Gonong

Group # 4, 2CMT, Faculty of Pharmacy, UST

Abstract
Proteins are probably the most important class of biochemical molecules, although of course
lipids and carbohydrates are also essential for life. Casein is a protein that is found in milk which
is used independently in many foods as a binding agent. The general objective of this
experiment is to isolate the protein Casein from skimmed milk and to analyze the chemical
groups responsoble for color reactions and also to explain the principles involved in each test.
Biuret Test, Ninhydrin Test, Xanthoproteic Test, Millon’s Test, Hopkins-Cole Test, Sakaguchi
Test, Nitroprusside Test, Fohl’s Test and the Test for Amides were all done to a certain portion
of the isolated Casein. Basic hydrolysis was done to the other portion of Casein. In addition to
this, the different tests performed on the first portion of Casein were also done to the Casein
that underwent basic hydrolysis. Various results were obtained and some differences were
noted between the color reactions of the isolated Casein and the color reactions of the basic
hydrolysates from the isolated Casein.

I. Introduction
Amino acids have a variety of chemically reactive groups. The reactions for side chains, a-
amino, and a-carboxyl groups can be used to characterize both free amino acids and proteins.

Biuret Test is used for detecting the presence of peptide bonds. It relies on the reduction of
copper(II) ions to copper(I), the latter form a complex with the nitrogens of the peptide bonds in
an alkaline solution. A violet color indicates the presence of proteins. It is possible to use the
biuret reaction to determine the concentration of proteins because (for most proteins) peptide
bonds occur with approximately the same frequency per gram of material. The intensity of the
color, and hence the absorption at 540 nm, is directly proportional to the protein concentration,
according to Lambert-Beer's law.

Ninhydrin is a chemical substance mostly used in biochemical laboratories as a reagent for

amino acids, which are small molecules that form proteins, as well as in forensics to detect
finger prints and faint blood stains, Ninhydrin react with amino acids, producing a colored
solution. This protocol is used to detect the presence of amino acids in certain substances by
using a solution of alcohol, ninhydrim and water.
Xanthoproteic Test is a test for the detection of proteins in which concentrated nitric acid
reacts with the proteins to form a yellow color that is intensified to orange-yellow by the
addition of alkali—called also xanthoproteic reaction

Millon's test is given by any compound containing a phenolic hydroxy group. Consequently, any
protein containing tyrosine will give a positive test of a pink to dark-red colour. The Millon
reagent is a solution of mercuric and mercurous ions in nitric and nitrous acids.

The hopkins-cole test determines the presence of amino acid tryptophan. Some chemists no
longer will use the hopkins-cole tst test becasuse it is not completely understood in terms of
chemistry. The tryptophan that the hopkins-col e test determines can be defined as an indole
nucleus. The Tryptophan creates the violet ring where the two layers meet.

The Sakaguchi Test is used to test for a certain amino acid and proteins. The amino acid that is
detected in this test is arginine. The sample to be tested is treated with alpha-naphthol and
sodium hypochlorite. A positive result yields a reddish wine color when arginine is present.

Nitroprusside test is a test wherein sodium cyanide is added first to urine and let stand for
approximately 10 minutes. In this time disulfide bonds will be broken by the released cyanide.
The destruction of disulfide bonds liberates cysteine from cystine and homocysteine from
homocystine. Next sodium nitroprusside is added to the solution and it reacts with the newly
freed sulfhydryl groups. The test will turn a red/purple color if the test is positive indicating that
there were significant amounts of amino acid in the urine (aminoaciduria).

Fohl’s reaction is used for determination of S-containing amino acids. A black or brown color
indicates the presence of the S-containing amino acids.

Bacteria need proteins as their source for essential amino acids. Proteins are large molecules
that cannot be brought directly into the bacterial cell. They need to be degraded into their
component parts.

Hydrolysis is a chemical reaction during which one or more water molecules are split into

hydrogenand hydroxide ions which may go on to participate in further reactions.

II. Materials and Methods

For the Alkaline Hydrolysis of Casein, 10 mL of 4M NaOH was added to 0.5 grams of the isolated
protein in a hard glass test tube. It was aslo labeled afterwards. The tube was stoppered with
cotton and was then submitted to the instructor for autoclaving (15 psi for 5 hours). The
appearance of the mixtured after autoclaving was then noted. Ten mL of distilled water was
added. The mixture was then transferred into a 250-mL beaker. The mixture was neutralized
using 1M HCl. This neutralized mixture was then used for the characterization tests.

For each of the color reaction test, .5g of the isolated pure Casein protein was mixed with 1mL
of distilled water, while .5mL of the hydrolysate was used.

In the Biuret test, 20 drops of 2.5M NaOH was added to the samples and it was mixed well.
After this, 2-3 drops of 0.1M CuSO4. The test tube was shaken and the color of the solution was
noted.

As for the Ninhydrin test, 6-10 drops of 0.1% Ninhydrin solution was added into the diluted
samples. The tube was the heated in a boiling water bath and the appearance of a blue-violet
coloration was noted.

Ten drops of concentrated HNO3 was slowly added to the diluted samples. The solution was
then mixed and was noted of the color. After this, 10 drops of NaOH was slowly added, mixed
and the color of the solutionw as noted.

For the Millon’s test, 5 drops of the reagent was added to the diluted samples and the change
in color was noted.

Twenty drops of Hopkins-Cole reagent was slowly added for the Hoplins-Cole test. It was then
mixed well. While the test tube was inclined, 20 drops of concentrated H 2SO4 was slowly added.
The mixture was not shaken and the color of the interface was noted.

As for the Sakaguchi test, 10 drops of 10% NaOH and 10 drops of 0.02% Naphthol solution was
added to the samples. It was mixed and let stand for 3 minutes. After 3 minutes, 3 drops of 2%
NaOBr was added and the color produced was noted.

For the Nitroprusside test, 0.5mL of 3M NaOH was added to 0.5mL of the sample. After this,
0.25mL of 2% nitroprusside solution was added and the formation of a red solution was noted.

Five drops of 30% NaOH and 2 drops of 5% (CH3COO)2Pb was added to the sample for the Fohl’s
test. The test tube was then placed in a water bath. The appearance of a dark(brown or black)
sediment was noted.
For the test for Amides, 1 mL of 20% NaOH was added to 10 drops of the sample. The tube was
then placed in a water bath. A test for the evolution of gas during heating was done by placing a
moistened red litmus paper over the mouth of the tube. The result was then noted.

III. Results and Discussions

Figure 3.1 isolated casein

Table 3.1 shows the results obtained from the various color reactions

Color Reaction Intact protein (Casein) Basic Hydrolysate

Biuret purple/ violet solution light blue solution
Ninhydrin pale blue-violet solution violet ring
Xanthoproteic pale yellow solution dark yellow solution
Millon’s white solution pale yellow solution
Hopkins-Cole white ring clear yellow solution
Sakaguchi colorless solution pale brown solution
Nitroprusside yellow solution yellow solution
Fohl’s black precipitate, brown solution pale yellow solution
Test for white basic solution(red->blue litmus white basic solution(red->blue litmus
Amides paper) paper)

The Biuret test for the intact protein was positive indicating the presence of a peptide bond. As
for the hydrolysate, the test was positive though the result was not as intense as of the isolated
protein’s results.
As for the Ninhydrin test, since a positive result should yield a purple complex, it was negative
for the isolated casein and it was positive for the hydrolysate. This indicated the presence of
either ammonia or primary or secondary amines.

As for Xanthoproteic test, both had positive results indicating the presence of an aromatic ring
but based on the intensity of the yellow color, it was observed that the hydrolysate has more
aromatic rings in it.

For Millon’s test, since a brown precipitate or a brick red color is the indicator to confirm the
presence of either Tryptophan or Tyrosine, it was negative for both the samples.

Hopkins-Cole test only reacts with proteins containing Tryptophan. Its indicator is the violet
cyclic product. Since both the hydrolysate and the casein had no violet color, the test for both
of them was negative.

For the Sakaguchi test, a wine red color indicates the presence of Arginine. Since in the casein,
only a colorless solution was obtained, it was negetive. But for the hydrolysate, since it had a
pale brown color, it is fine to say that it was positive for this test.

The Nitroprusside test only reacts with Cysteine to yield a red complex. Since for both the
samples, it turned to yellow solutions, the test was negative to either.

As for the Fohl’s test, it was positive for the Casein indicating that it has an S-containing amino
acid. It turned out to be negative for the hydrolysate.

For both the 2 samples, it was positive in the test for Amides because of the initially red litmus
paper which was placed over the mouth of the tube turned to blue.

As for the conclusion, the casein and the basic hydrolysate had different reactions for Biuret
test, Ninhydrin test, Sakaguchi test, while it had the same result in Xanthoproteic test, Millon’s
test, Hopkins-Cole test, Nitroprusside test, Fohl’s test and the test for Amides.

IV. References

http://www.elmhurst.edu/~chm/vchembook/565proteins.html

http://www.wisegeek.com/what-is-casein.htm

http://www.worldlingo.com/ma/enwiki/en/

http://www.ehow.com
http://www.chemistry.mcmaster.ca/~chem2o6/labmanual/expt11/2o6exp11.html

http://www.reference.com/motif/Science/

http://www.cerlabs.com/experiments/10875404480.pdf