Isolation and Qualitative Test of Proteins

EXPERIMENT NO. 3

ABSTRACT
Chemical analysis can either be qualitative or quantitative. In qualitative analysis, we only identify the
substances present in a given sample. We are no longer concerned with the quantity of each substance.
The main objective of this experiment is to isolate the protein casein from skimmed milk. In order to analyze
the chemical groups responsible for the color reactions and to also explain the principle involved in each
tests. We have Biuret, Ninhydrin, Xanthoproteic, Millon’s, Hopkin’s-Cole, Sakaguchi, Nitroprusside, and
Fohl’s test were all done to the first portion of the isolated casein and also to the other part which
underwent acidic hydrolysis. Various results were obtained and some differences were noted between the
color reactions of the intact casein and the color reactions of the acidic hydrolysates from the isolated
casein.
II. INTRODUCTION produces red solution, and folh's test produces
red solution.
Proteins are large organic compounds made of
amino acids arranged in a linear chain and joined
together by peptide bonds between the carboxyl
and amino groups of adjacent amino acid III. METHODOLOGY
residues. A number of qualitative color reactions Materials
have been devised which are useful for detection
of proteins. These tests are to be used with the (2) 250mL Beaker Litmus Paper
knowledge that they test for specific chemical pH meter Filter Paper
groupings on the protein structure. Casein is a pH Indicator (5) 10mL Test Tubes
protein that is found in milk and used (2) 5mL Pipet Hot Plate
independently in many foods as a binding agent.
It is part of a group called phosphoproteins, a
Reagents
collection of proteins bound to something
containing phosphoric acid. Casein is a salt, Skimmed Milk 0.1M HCl
meaning it has no net ionic charge, of the Hopkins-Cole Reagent 10% NaOH
element calcium. It is not susceptible to Millon’s Reagent 0.02% Naphthol
denaturing because of its structure. Our group Ninhydrin Solution 2% NaOBr
was assigned to isolate casein from skim milk 0.01M CuSO4 Conc. H2SO4
through isoelectric precipitation with acetic acid. 2.5M NaOH Conc. HNO3
After precipitating casein, several samples were BradFord Reagents Conc. NaOH
used for qualitative color reactions. And after Bovine Serum Albumin Milk
which we did the acidic hydrolysis of the isolated
casein for biuret test produces violet solution,
ninhydrin test produces blue/ violet solution,
xanthoproteic test produces yellow solution,
millon's test produces red color, hopkins-cole test
produces violet ring solution, sakaguchi test
produces red solution, nitroprusside test
Procedure
A. Isoelectric Precipitation of Casein
1. 20mL Skimmed milk in 250mL Beaker.

4. Decant and discard the supernatant.

2. Slowly add HCl from a buret with constant

mixing and heating. (check if pH is about 4.6)

5. Wash the precipitate with water

6. Dry the precipitates
B. Qualitative Tests
Test tubes before observing colors

3. Allow to stand until Casein settles.

Ninhydrin, Xanthoproteic, and Millon’s Test, the nitro derivate show an intensely yellow color
heating. Millon’s test is given by any compound containing
a phenolic hydroxy group Any protein containing
tyrosine will give a positive test of a pink to dark-
red colour. The red colour is probably due
to a mercury salt of nitrated tyrosine
The Hopkins-Cole test determines tryptophan
and an indolenucleus, and is known for creating
the violet ring where the two layers meet. Then
the violet ring appears after the two layers within
an indolenucleus meet, this confirms that
concentrated sulfuric acid was added to a
mixture of some sort that contained glyoxylic acid
and a protein.
IV. DATA AND RESULTS.
Denaturation is a process in which proteins or
(I) Isolation of Proteins nucleic acids lose their tertiary structure and
Weight of Casein  4.25g secondary structure by application of some
external stress or compound, such as a strong
Appearance of Casein  Powdery acid or base, a concentrated inorganic salt, an
(II) Qualitative Tests for Proteins: organic solvent, or heat. In the experiment, two of
the denaturing procedures were used. HCl and
Qualitative Tests Observation heat were added to the skimmed milk to further
Biuret Test Violet denature them until the desired results are
Ninhydrin Test Blue/Violet achieved.
Xanthoproteic Test Yellow
VI. Question and Answer
Millon’s Test Red
Hopkins-Cole Test Violet Ring 1.) What is meant by the isoelectric point of a
Sakaguchi Test Intense Red protein?
Nitroprusside Test Red
 In proteins, the isoelectric point (pI) is defined
Fohl’s Tesgt Black
as the pH at which a protein has no net charge.
When the pH > pI, a protein has a net negative
charge and when the pH < pI, a protein has a net
positive charge. The pI varies for different
V. Discussion proteins.
 The reagents added corresponds to the
ninhydrin test, the xanthropoteic test, the milon9s
test,and the Hopkin’s-Cole test Ninhydrin 2. At what pH is a protein least soluble? Why?
(triketohydrindene hydrate) is an oxidating agent  The solubility of a protein depends on the net
which leads to the oxidative deamination of charge on the surface of the protein molecule.
alpha-amino groups. When reacting with free The net charge depends on the pH of the solvent
amines from the amino acid, blue or purple color and the number and identities of the amino acids
is produced Xanthropoteic test gives a positive that make up the protein. The isoelectric point of
result in those proteins a protein occurs at a specific pH when the
with aminoacids carrying aromatic groups, especi positive and negative charges balance each
ally in the presence of tyrosine
other out and the net charge is zero. At this
isoelectric point a protein is least soluble
3. What type of chemical group is present in all
proteins?
 There is chemical grouping present in all
proteins, as all proteins contain certain elements.
These include hydrogen, oxygen, nitrogen, and
carbon.
4. Give the principle involved for:
 Biuret Test - chemical test for detecting
presence of peptide bonds in the presence of
peptides - Positive visible result: violet
 Ninhydrin Test - shows positive test for alpha
amino acids and proteins that contain free amino
acid groups - Positive visible result: deep blue or
purple color.
 Xanthoproteic Test - yellow acid substance
formed by the reaction of hot HNO3 on
albuminous or protein matter and is changed to a
deep orange-yellow color by the addition of NH3 .
 Millon's Test for Tyrosine - a test developed
by Auguste Millon, a French chemist - This test is
not specific for proteins. It detects phenolic
compounds. A reddish-brown coloration or
precipitate indicates the presence of tyrosine
residue.
 Sakaguchi test for Arginine -positive result:
red
 Hopkins-Cole Test for Tryptophan - positive
visible result: purple ring in the junction where the
two liquids meet
VII. Conclusion
 The purpose of this experiment is to
isolate a protein (casein) from a natural
source (milk) and to demonstrate that the
product obtained is indeed a protein by
performing a number of tests on the
product.
 This experiment also provided a better
understanding in which either or both
proteins and amino acids can be tested
for its presence in a given sample.
Moreover, the experiment gives a closer
look to what is the effect of the different
denaturing agents to a protein.
VIII. Bibliography
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3838 CARUNCHIA WHETSTINE ET AL.
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[7] Legaspi, G.A. 2009. Essentials of
Biochemistry Laboratory
[8] Retrieved from
//http://www.ukessays.com/essays/biochem/isolat
ion-and-characterization-of-proteins.
[9] Retrieved from
/http://fulltimes.wordpress.com/protein/ on July
24, 2014
[10] Retrieved from
/http://www,Elmhurst.edu/~chm/vchembook/568d
enaturation.html/August 1, 2014
IX. Photo Documentary

Reagent Prep

Change in Color
Reagent Prep (2)

X. Certification

Belardo, Eloisa _____________

Berbano, Raziel ____________
Gelisanga, Ronna ____________
Martin, Fe ____________
Rivera, Rycel ___________