Laboratory Report

Experiment No. 2

Title: Isolation and Qualitative Tests

of Proteins

Biochemistry Lab, BSN 1B Date Submitted: September 13, 2019

Group No. 3 Date Performed: September 6, 2019

Class Schedule: Friday, 7AM-1PM

Group Members: BUGHAO, Maria Angelika A.

CAGUITLA, France Jerald L.

CASIM, Ryzza Mae A.

CASTILLO, Axle Anne B.

I. Objectives

1. To isolate the protein, casein, in a skimmed milk through isoelectric precipitation.

2. To test for the specific chemical groupings on the protein structure of a casein from skimmed
milk.

II. Introduction

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
residues.

In order to perform in vitro analysis, a protein must be purified away from other
molecular components. A mixture can be purified using ultracentrifugation, precipitation (salting
out), chromatography, electrophoresis, spectroscopy, or by enzyme assays.

A number of qualitative color reactions have been devised which are useful for detection
of proteins. These tests are to be used with the knowledge that they test for the specific chemical
groupings on the protein structure.
III. Materials

A. Equipment

(2) 250-mL Beaker Litmus paper

pH meter or pH indicator Filter paper

(2) 5-mL pipet (5)-10-mL Test tubes

Hot Plate

B. Reagents

Skimmed milk 0.1 M HCl

Hopkins-Cole Reagent 10% NaOH

Millon’s Reagent (freshly prepared) 0.02% Naphthol solution

0.1 % Ninhydrin Solution 2% NaOBr (freshly prepared)

0.01 M CuSO4 Conc. H2SO4

2.5 M NaOH Conc. HNO3

IV. Procedure (Schematic Diagram)
V. Results

I. Isolation of Protein

Weight of Casein: 11.2 g

Physical Appearance: Slightly wet and the casein itself sticks on the filter paper. (It stays white)

II. A. Qualitative Test for Proteins

Qualitative Test Observation

Biuret Test It has purple precipitate

Ninhydrin Test It forms blue precipitate underneath

Xanthoproteic Test It forms yellow precipitate

Millon’s Test It forms red precipitate

Hopkins-Cole Test It forms two layers brownish and yellowish

Sakaguchi Test Forms two layers

Nitroprusside Test Yellowish precipitate

Fohl’s Test Brown precipitate

B. Quantitative Test for Isoelectric Precipitation of Casein

Formula: % Casein = Wt. of Casein x 100

Wt. of Skimmed
Milk

Wt. of Beaker 114. 6 g

Wt. of Beaker + Skimmed Milk 134.8 g
Wt. of Skimmed Milk 20.2 g
Wt. of Filter Paper 1.8 g
Wt. of Filter Paper + 13.0 g
Casein
Wt. of Casein 11. 2 g
% of Casein 55.45 %
VI. Discussion

A. Qualitative Test for Proteins

The observation is every test had changes when we put the test tubes in a boiling
water, we observe what will be the changes since we put it in. The Biuret Test Had a
purple precipitate, the Ninhydrin Test forms to blue precipitate, the Xanthoproteic Test
forms to yellow precipitate, the Millon’s Test forms to red precipitate, the Hopkins-Cole
Test forms to two layers brownish and yellowish, the Sakaguchi Test forms to two layers,
Nitroprusside Test has yellowish precipitate, and the last one is the Fohl’s Test has brown
precipitate.
Ninhydrin detects the presence of amines and indicates the presence of amino
acids and/or proteins, the Biuret test detects the presence of peptide bonds, and specific
analytical tests determine the presence of individual amino acids. Biuret solution is used
to identify the presence of protein. Biuret reagent is a blue solution that, when it reacts
with protein, will change color to pink-purple. The xanthoproteic reaction is a method
that can be used to determine the amount of protein soluble in a solution, using
concentrated nitric acid. Millon's reagent. Millon's reagent is an analytical reagent used
to detect the presence of soluble proteins. A few drops of the reagent are added to the test
solution, which is then heated gently. A reddish-brown coloration or precipitate indicates
the presence of tyrosine residue which occur in nearly all proteins. The Hopkins-Cole
reaction, also known as the glyoxylic acid reaction, is a chemical test used for detecting
the presence of tryptophan in proteins. The Sakaguchi test is a chemical test used for
detecting the presence of arginine in proteins. It is named after the Japanese Food
Scientist and Organic Chemist, Shoyo Sakaguchi (1900–1995) who described the test in
1925. Sakaguchi reagent consists of 1-Naphthol and a drop of sodium hypobromite. The
nitroprusside test is specific for cysteine, the only amino acid containing a sulfhydryl
group (—SH). The group reacts with nitroprusside in alkaline solution to yield a red
complex. The nitroprusside test is specific for cysteine, the only amino acid containing a
sulfhydryl group (—SH). The group reacts with nitroprusside in alkaline solution to yield
a red complex. Fohl’s Test or the Pauly reaction is a chemical test used for detecting the
presence of tyrosine or histidine in proteins.

B. Isoelectric Precipitation of Casein

Quantitative test of Isoelectric Precipitation of Casein. The weight of beaker is
144.6 grams, beaker with skimmed milk is 134.8 grams, skimmed milk is 20.2 grams,
filter paper is 1.8 grams, filter paper with casein is 13.0 grams, casein is 11.2 grams. The
percentage of the casein is 55.45%.
Casein is a heterogeneous mixture of phosphorous containing proteins in milk. ...
That pH value is known as the isoelectric point (IEP) of the protein and is generally the
pH at which the protein is least soluble. For casein, the IEP is approximately 4.6 and it is
 the pH value at which acid casein is precipitated. The isoelectric point (pI) is the pH of a
solution at which the net primary charge of a protein becomes zero. At a solution pH that
is above the pI the surface of the protein is predominantly negatively charged and
therefore like-charged molecules will exhibit repulsive forces. The isoelectric point of
casein is 4.6. Since milk's pH is 6.6, casein has a negative charge in milk. The purified
protein is water-insoluble

VII. Conclusion

As for the conclusion, there is changes in color appearance, the size of it and the the
shape is also changed. Casein pronounced "kay-seen" in British English, is a family of related.
Phosphoproteins. These proteins are commonly found in mammalian milk, comprising c. 80% of
the proteins in cow's milk and between 20% and 45% of the proteins in human milk. It is a slow-
digesting dairy protein that people often take as a supplement. It releases amino acids slowly, so
people often take it before bed to help with recovery and reduce muscle breakdown while they
sleep. Several studies have shown it helps boost muscle growth, along with a ton of other
benefits. Because casein is a protein, it's found mostly in dairy products with moderate-to-high
protein content such as low-fat milk, yogurt, kefir, cheese, ice cream.

Skimmed milk, or skim milk, is made when all the milk fat is removed from whole milk.
It tends to contain around 0.1% fat. It's lower in fat and calories than whole milk, and marginally
higher in calcium, but some experts suggest that the saturated fat in dairy may not be a problem
in terms of heart health. In fact, by drinking skimmed we may be missing out on fat-soluble
nutrients like vitamins A and E. The composition of non‐fat solids of skim milk is: 52.15%
lactose, 38.71% protein (31.18% casein, 7.53% whey protein), 1.08% fat, and 8.06% ash. The
main function of the casein micelle is to provide fluidity to casein molecules and solubilize
phosphate and calcium.

In the experiment, casein was successfully isolated from the Milk Magic non-fat
milkthrough isoelectric precipitation. The isolated casein was subjected to acid and
alkalinehydrolysis which yielded visible changes comparing the appearance of the isolate before
andafter autoclaving it.The protein suspension gave positive results for Biuret test and Hopkins-
Cole test,from these results, it is determined that the protein suspension components are a peptide
bond nitrogen and tryptophan. Alternatively, the basic hydrolyzate yielded a positive result only
for Xanthoproteic test, Ninhydrin test and Hopkins Cole test, is made up of an aminoacid, free
amino group and tryptophan. In the protein assay via Bradford method, the calculated
concentration of the unknown protein solution.
VIII. Assessment

1. What is the meant by the isoelectric point of a protein?

The isoelectric point (pI) is the pH value at which the molecule carries no
electrical charge. The concept is particularly important for zwitterionic molecules such as
amino acids, peptides, and proteins. For an amino acid, the isoelectric point is the average
of pKa values for the amine and the carboxyl group. For complex molecules such as
proteins, the isoelectric point is useful in the description of acidic or basic character,
where individual pKa values are not relevant.

2. At what pH is a protein least soluble? Why?

For most proteins this occurs in the pH range of 5.5 to 8. A protein gets more
soluble if there is a net charge at the protein surface, since it prefers to interact with
water, rather than with other protein molecules. Without a charge at the surface proteins
are likely precipitate and aggregate.

3. What type of chemical grouping is present in all proteins?

Proteins are polymers of amino acids. Each amino acid contains a central carbon,
a hydrogen, a carboxyl group, an amino group, and a variable R group. The R group
specifies which class of amino acids it belongs to: electrically charged hydrophilic side
chains, polar but uncharged side chains, nonpolar hydrophobic side chains, and special
cases.

4. Give the principle involved and the chemical structure responsible for the positive Biuret
test, Ninhydrin test, Xanthoproteic test, Millon’s test, Hopkins-Cole test, and Sakaguchi
test.
 Biuret Test
The Biuret Test is often used to determine the presence of peptide bonds in
protein. Biuret test is based on the ability of Cu (II) ions to form a violet-coloured
chelate complex with peptide bonds (-CONH- groups) in alkaline conditions.
 Ninhydrin Test
This test is a general test and thus given by all amino acids. This test is due to a
reaction between a amino group of free amino acid and ninhydrin. Ninhydrin is a
powerful oxidizing agent and its presence, amino acid undergo oxidative deamination
liberating ammonia, CO2, a corresponding aldehyde and reduced form of ninhydrin (
hydrindantin). The NH3 formed from a amino group reacts with another molecule of
ninhydrin and is reduced product ( hydrindatin) to give a blue substance diketohydrin
( Ruhemanns complex).

 Xanthoproteic test
Xanthoproteic test is used to detect amino acids containing an aromatic nucleus
(tyrosine, tryptophan and phenylalanine) in a protein solution which gives yellow
color nitro derivatives on heating with conc. HNO3. The aromatic benzene ring
undergoes nitration to give yellow colored product.

 Millon’s test
Compounds containing hydroxybenzene radical react with Millon’s reagent to form
red complexes. The only amino acid having hydroxybenzene ring is tyrosine. 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.
 Hopkins-Cole Test
To detect amino acid tryptophan present in protein. The indole group of tryptophan
reacts with glyoxylic acid in the presence of conc. H2SO4 to give a purple colored
complex.

 Sakaguchi Test
The Sakaguchi test is a chemical test used for detecting the presence of arginine in
proteins. The arginine reacts with α – napththol and an oxidizing agent such as
bromine water or sodium hypochlorite/sodium hypobromite to give a red colored
product. The other guanidinium containing compounds other than amino acid also
give this reaction.
IX. References

(n.d.). Isoelectric Point. Retrieved from

https://www.sciencedirect.com/topics/chemistry/isoelectric-point

Huns, V. et al. (2013, April 17). Answer to Question #28649 in Inorganic Chemistry for
Christian. Retrieved from https://www.assignmentexpert.com/homework-
answers/chemistry/inorganic-chemistry/question-28649

Learning, L. (n.d.). Biology for Majors I. Retrieved from

https://courses.lumenlearning.com/suny-wmopen-biology1/chapter/proteins/

(n.d.). Retrieved from

https://www.google.com/search?ei=B0t6XbXkM9OlwAOO47qYDQ&q=Isoelectric+Precipitatio
n+of+Casein&oq=Isoelectric+Precipitation+of+Casein&gs_l=psy-
ab.3..0j0i22i30l2.610017.610017..610478...0.5..0.190.190.0j1......0....2j1..gws-wiz.......0i71.-
mbSloK6KK0&ved=0ahUKEwj1g4LbtMvkAhXTEnAKHY6xDtMQ4dUDCAs&uact=5

Ninhydrin Test: Principle, Requirements, Procedure and Result. (2018, December 18).
Retrieved from https://www.onlinebiologynotes.com/ninhydrin-test-principle-requirements-
procedure-and-result/

Xanthoproteic test: Objective, Principle, Reagents, Procedure and Result. (2018,

December 18). Retrieved from https://www.onlinebiologynotes.com/xanthoproteic-test-
objective-principle-reagents-procedure-and-result/

X. Conforme

Prepared by: CASIM, Ryzza Mae A. Signature:

Performed by:

BUGHAO, Maria Angelika A. Signature:

CAGUITLA, France Jerald L. Signature:

CASTILLO, Axle Anne B. Signature: