Determination of Proteins and Amino Acids

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BIOC 201 2010-2011 www.yahoo.com, www. Wikipeadia.com
Determination of proteins and amino acids
Proteins are sequences of amino acids, its contain C, H, O, and Nitrogen,

Common structure: Central C, with a H, amino group (NH2), and an acid group
(COOH), and a side group, proteins made up of about 20 different amino acids;
Unique Side Groups, Differ in size, shape, electrical charge.
Proteins are probably the most important class of biochemical molecules,

although of course lipids and carbohydrates are also essential for life. Proteins
are the basis for the major structural components of animal and human tissue. It
can be hydrolyzed by acids, bases or specific enzymes.
Classification of Proteins
Conjugated
Simple Proteins Derived Proteins
Proteins
contains non- Formed by the

Contain only impact of some of
amino acids. amino acid
component in the vital factors
addition to of natural or
amino acids. This chemical change
Example: non-amino acid of proteins and
Egg albumin component is natural partially
called the installed so that
prosthetic group . they retain their
properties
Example: General
Phosphoprotein
( Casin ) Example:
Peptone, Gelatin
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1- Solubility of proteins
Identify the solubility of different types of proteins in different solvents.
Principle:
Simple proteins dissolve easily in water while soluble protein complex by low
solubility in distilled water. The proteins are derived insoluble in water but
soluble in alkaline solutions.
How to perform the test:
1. Test the solubility of each (albumin, casein, gelatin, peptone) in cold water
and then in hot water and then in sodium hydroxide solution.
2. Record solubility of all proteins in cold water and hot water and sodium
hydroxide solution in the following table of results.
2-Precipitation of proteins
(A) Precipitation of proteins by heavy metals:
Principle:
Proteins are precipitated in alkaline medium with heavy metals due to the
direct union of cation (Cu++, Ag+, Ba++, Pb++) with anionic groups of proteins,
which are formed in basic medium.
At alkaline pH 7 and above, proteins are usually negatively charged so the
addition of positively charged ions will neutralize this charge and the proteins
come out of solution (i.e. heavy metals combine with proteins forming
insoluble metalloproteine).
1- Put 2ml from protein solution (albumin, casein, gelatin, and peptone) in
4 test tubes.
2- Add 3 drops from silver nitrate (AgNO3). Or other heavy metals are
found in lab.
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3- Add 10% NaOH to the solution.

4- Precipitate will be formed is a white colour.
5- Record your result in the table.
(B) Precipitation of proteins by concentrated acid:

Principle:
Proteins are precipitated in acidic medium with some reagents such as
TCA, picric acid and tannic acid due to the direct union of the anionic group
with the cationic groups of the proteins, which are formed in acidic medium.
These compounds carry large negative charges which neutralize the
positively charged protein to form insoluble salt complex with protein.
The acidic reagents are therefore most effective at acidic medium where
proteins are positively charged.
1-Put 2ml from protein solution (albumin, casein, gelatin, and peptone) in 4
test tubes.
2- Add 3 drops from few drops of concentrated acid (H2SO4 or HNO3).
3- Add slowly from 10% NaOH to the solution.
4- Precipitate will be formed is a white colour.
3-Color Tests
(A) Biuret Test:
Principle:
It is the general test for all proteins. Biuret reagent is dilute CuSO4 in strong
alkaline medium. Alkaline CuSO4 reacts with all compounds containing 2 or
more peptide bonds to give a blue-violet color.
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test tubes.
2- Add 1 ml of biuret reagent ( 2 ml of NaOH 10% + 1 drop of CuSO4
1% ).
3- Mix well
4- Formed blue-violet color.
)B) Ninhydrin Test

Principle:
Amino acids contain a free amino group and a free carboxylic acid group
that react together with ninhydrin to produce a colored product. When an
amino group is attached to the first, or alpha, carbon on the amino acids carbon
chain, the amino group’s nitrogen atom is part of a blue-purple product, as
shown in Equation 2. Proteins also contain free amino groups on the alpha-
carbon and can react with ninhydrin to produce a blue-purple product.
test tubes.
2-Add 1 ml of 0.5% ninhydrin – ethanol reagent solution .
3-Mix well
4-Place the test tubes into the boiling-water bath for 5 min , remove from
Bwb,
5- Formed a blue-purple.
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4- Coagulation (clotting) Test:

Principle:
In this test we see the denaturating proteins at high temperature due to
change shape, chemical and physical properties of proteins
test tubes.
2-Heat the flame directly on the tube and slightly inclined.
3- Notice formation of coagulation of the solution (give with albumin only).

5- FreezingTest:
Principle:
In this test we see the change shape at low temperatrue due to change shape,
chemical and physical properties of proteins.
test tubes.
2- Put the tube in the Cup by the snow and leave for five minutes.
3- Notice formation of freezing of the solution (give with gelatin only).
6- Sulfur test:
Principle:
The basic idea of this test is to detect the presence of amino acids that contain
sulfur such as cystein.
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test tubes.
2 - Add 1 mL of alkaline solution of 40% NaOH.
3 - Heat in a boiling- water bath for 10 minutes.
4-Cool solution and add 1 ml 1% lead acetate.
7- The black precipitate or brown (gives with albumin).
7- Phosphorus Test
Principle:
test tubes.
2 - Add 3 mL of alkaline solution of 20% NaOH.
3 - Heat in a boiling- water bath for 10 minutes.
4-Add in the order as follows: (1 ml of Molbidic acid and + 1 ml sodium
sulphate + 1 ml of 0.5% hydroquinone ) respectively.
5- Formed blue colour (gives with casein only).
6-Record your result in the table.
8-Millon's test:
Principle:
The basic idea of this test is to detect the presence of amino acid by the
hydroxyl group in proteins such as phenyl acid tyrosine.
test tubes.
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2- Add 5 drops out of a solution of Mellon.

3-Shake well, then Heat in a water bath for 10 minutes.
4-Cool under tap water and add 5 drops of a solution of sodium nitrite 1%.
5-Notice formation of red precipitate (give with albumin and casein).
9- Hopkin-cole Test
Principle:
indol group such as tryptophan.
test tubes.
2 - Add 3 mL of Hopkin-cole reagent.
3 – Mix well.
4-Add carfuley conc H2SO4 down side of tube so to form two layers.
5- Formed purple ring colour.
10- Sakaguchi Test

Principle:
guanidine group such asarginine.
test tubes.
2 - Add 2 mL NaOH.
3 – Add 1 ml ethanolic 0.02% α-naphthol.
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4-Mix well, cool in ice.

5- Add 1 ml alkaline hypochlorite solution.
6- Formed red colour.
11- Xanthoproteic Test

Principle:
benzene ring such phenylalanine, tryptophan and tyrosin.
test tubes.
2 - Add 1 mL HNO3.
3 – Add 1 ml ethanolic 0.02% α-naphthol.
4-Heat the mixture in water bath for 30 second.
5- Cool and add drop-wise 40% NaOH to render the solution alkaline.
6- Formed yellow to orange colour.
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When you are finished collecting data, determine the identity of each
unknown. Record chemical properties of your test, observation and results in
the tables below.
Unknown (1)
Test Observation Results

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Unknown (2)

11
Unknown (3)

12
Unknown (4)

Determination of Proteins and Amino Acids

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BIOC 201 2010-2011 www.yahoo.com, www. Wikipeadia.com

Determination of proteins and amino acids

Proteins are sequences of amino acids, its contain C, H, O, and Nitrogen,

Proteins are probably the most important class of biochemical molecules,

contains non- Formed by the

3- Add 10% NaOH to the solution.

(B) Precipitation of proteins by concentrated acid:

How to perform the test:

)B) Ninhydrin Test

4- Coagulation (clotting) Test:

3- Notice formation of coagulation of the solution (give with albumin only).

How to perform the test:

2- Add 5 drops out of a solution of Mellon.

10- Sakaguchi Test

4-Mix well, cool in ice.

11- Xanthoproteic Test

Test Observation Results

Test Observation Results

Test Observation Results

Test Observation Results

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