MAKERERE UNIVERSITY

COLLEGE OF NATURAL SCIENCES

SCHOOL OF BIOSCIENCES
DEPARTMENT OF BIOLOGICAL SCIENCES
BACHELOR OF SCIENCE IN BIOTECHNOLOGY
COURSE UNIT: BASIC PLANT PHYSIOLOGY
COURSE CODE: BBT 1204
REPORT

NAME: AMUTUHAIRE ROBERT
REG NO: 12/U/2640
STUDENT NO: 212002056


LECTURER: PROF. ORYEM ORIGA

Objectives of the experiment:
Demonstrate the presence of albumin in wheat flour.
To confirm the presence of proteins in the albumen
For quantitative determination and detection of amino acids present
Obtain knowledge about protein and amino acid structure
Practical application of knowledge through performing protein and amino acid color and
precipitation reactions
To identify different amino acids present in a protein using different reagents

INTRODUCTION:
The protein tests are used to determine the presence and concentration of proteins present in different
food sources. Nitrogenous reserve materials occurring in plants are various kinds of protein and amino
acids and amines.

Materials and requirements used
1g of dry peas, beakers, Grinding mill, Distilled water, maize flour, Concentrated nitric acid, alcohol
solution, Ammonia solution, 5% copper ii sulphate solution, Millons reagent, 5% lead and HaCl
2
,
Reduced oxalic acid, barium chloride solution, Conc. Sulphuric acid, 3% tannic acid, 40% sodium
hydroxide solution, 0.5% ninhydrin solution, 1% copper sulphate solution, glutamic acid, 5% lead
acetate, 5% Ferric Chloride solution, Wheat flour, Heat source.
ABSTRACT:
No class of compounds is of more fundamental significance than the proteins. The matrix of protoplasm
largely consists of proteins in the colloidal state, and, without doubt, they occur to some extent in the
same condition in the cell-sap. They are also found in the cell in the solid state, in the form of either
amorphous granules, termed aleurone, or crystalline or semi-crystalline bodies, termed crystalloids.
Both solid forms constitute "reserve material" and are often found in seeds, tubers, bulbs, buds and
roots.
Although they are present in every cell in all parts of plants, little, however, is known of plant proteins,
except of those in seeds, because of the difficulties of obtaining them in sufficiently large quantities, and
of separating them from each other.
Proteins are in the colloidal state when in so-called solution, and are unable to diffuse through
parchment membranes. The proteoses and peptones, however, which have simpler molecules, can
diffuse through such membranes.
The vegetable proteins are soluble in various solvents according to the nature of the protein; some are
soluble in water, others in dilute salt solutions, others, again, in dilute alkalis, and a few in dilute
alcohol. Vegetable albumins are coagulated from solution on boiling, but most of the globulins, unlike
the corresponding animal products, are only imperfectly coagulated on heating and some not at all. The
precipitate formed when coagulation is complete will not go into solution again either in water, acid,
alkali or salts. Alcohol precipitates the proteins; in the case of animal proteins, the precipitate becomes
coagulated and insoluble if allowed to remain in contact with the alcohol but this does not appear to be
so with plant proteins.
SUMMARY OF THE OBSERVATIONS:
The observations were obtained from reactions that are summarized as general reactions, color
reactions and precipitation reaction.
I. General reactions and their observations.s
The ninhydrin reaction gives a purple colored complex after heating.
The biuret reaction gives a violet complex due to the reaction with the peptide bonds in the proteins.
II. Color reactions.
The reaction gives a dark brown complex of lead sulphide after heating with lead acetate in alkaline
medium.
The Xanthroproteic reaction gives a yellow color that is formed due to nitration in proteins containing
tyrosine and an orange color in proteins containing tryptophan.
The Millons reagent gives a red precipitate with proteins that contain the phenolic amino acid tyrosine.
III. Precipitation reactions
Heavy metals like copper, lead and mercury have cations with positive charges that counteract with
negative charge of the carbohydrate group in proteins to give precipitates. The precipitates have varying
turbidities.
A. PROTEINS
Weigh about 1 g of dry peas (pisum sp), grind them to powder in a grinding mill and then add 100ml of
distilled water to it. Stir thoroughly and allow the mixture to stand for one hour. Filter and make the
following tests with the filtrate. (A solution of pure/chemical protein may be used instead)

TEST OBSERVATIONS DEDUCTIONS
1. Xanthroproteic reaction
To 2ml of the protein solution in a test-
tube 1ml of concentrated nitric acid.
Observe and record. Heat the mixture to
boiling and observe any change taking
place. Cool the solution under ramming
tap water and strong ammonia solution
until the reaction becomes alkaline.
Observe and record

A white precipitate is formed
on addition of conc. Nitric
acid. The precipitate turns
yellow on boiling

Aromatic amino acids
present
2. Millons reaction
To 2ml of protein solution in a test-tube,
add 1ml of Millons reagent, observe and
record. Heat the mixture, observe and
record.

A white precipitate is formed
on addition of Millons
reagent. The precipitate
turns brick red on heating

Phenolic amino acids
present
3. The glyoxylic reaction
To 2ml of the protein solution add an
equal volume of reduced oxalic acid and
shake to mix the solutions. Then add equal
volume of concentrated sulphuric acid by
pouring it down the side of the tube.
Observe and record. Shake the tube
gently, observe and record your
observation.

A purple ring forms at the
junction of the two liquids.
The purple color spreads
through the solution on
shaking

Aromatic amino acids
present
4. The Buiret reaction
To 2ml of solution add 1ml of 40% sodium
hydroxide solution and one drop of 1% of
copper sulphate solution. Observe and
record.

A clear solution is formed on
addition of sodium hydroxide
A violet solution is formed on
addition of copper sulphate



A protein with a long
polypeptide chain was
present

5. The Sulphur reaction
To 2ml of the protein solution in a test-
tube, add equal volume of 40% sodium
hydroxide solution and heat to boiling for
2 minutes. Then add 1-2 drops of 5% lead

The solution turns brownish.
A dark brown precipitate is
formed on addition of lead

Sulphur containing
amino acids present
acetate solution. Observe and record. acetate
6. Test for leucosin in wheat flour
Weigh 10g of wheat flour and add 100ml
of distilled water and stir thoroughly.
Allow the mixture to stand while stirring
periodically for 2-6 hours. Filter and slow
heat the filtrate on boiling. Observe and
record.

Precipitate of coagulated
protein is formed.

Leucosin is present in
wheat flour
7. Extraction of zein and prolamine
of maize.
Weigh 100g of maize flour and add 250ml
of hot 70% ethanol. Stir the mixture
thoroughly and filter. Concentrate the
filtrate in a hot water bath. Add a few
drops of the concentrated filtrate to 1ml
of absolute alcohol and distilled water
respectively. Observe and record.


Turbid solution turned
colorless on warming in the
hot bath. Fine colloidal
suspension is formed on
addition of ethanol and
distilled water.

Ethanol added
dehydrated the zein and
prolamine proteins in
the turbid solution. This
dehydration effect lead
to unfolding of these
proteins forming
colloidal suspensions.



EXPLANATION FOR THE RESULTS:
1. Xanthroproteic reaction.
The Xanthroproteic test is a method that can be used to determine the amount of protein soluble in a
solution, using concentrated nitric acid. The test gives a positive result in those proteins with amino
acids carrying aromatic groups, especially in the presence of tyrosine. If the test is positive the proof is
neutralized with an alkali, turning dark yellow.
A white precipitate is formed (except in the case of proteoses, peptones, etc.). On boiling, the
precipitate turns yellow, and partly dissolves to give a yellow solution. The yellow color becomes orange.
The precipitate is due to the fact that metaprotein is formed by the action of acid on albumins or
globulins, and this metaprotein is insoluble in strong acids. The yellow color is the result of the
formation of a nitro-compound of some aromatic component of the protein, such as tyrosine,
tryptophan and phenylalanine.
2. Millons reaction
Millon's reagent is an analytical reagent used to detect the presence of soluble proteins. For the above
test, the white precipitate is due to the action of the mercuric nitrate on the proteins. The reaction is
characteristic of all aromatic substances which contain a hydroxyl group attached to the benzene ring.
The aromatic complex in the protein to which the reaction is due is tyrosine.
3. The glyoxylic reaction
The reaction is for detection of proteins originally described by Adamkiewicz consisted in the addition of
strong sulphuric acid to the acetic acid solution of the substance under investigation. In the presence of
proteins a reddish violet ring is produced at the junction of the fluids which spreads throughout the
solution on standing or on gentle shaking. This test was subsequently investigated by Hopkins and Cole,
who found that proteins did not uniformly yield a positive reaction and that the differences were
attributable to the acetic acid employed in the test.
A purple ring forms at the junction of the two liquids. If the liquids are mixed by shaking the tube gently,
the purple color will spread throughout the solution. The substance in the protein molecule to which the
reaction is due is tryptophan. If carbohydrates are present in the liquid to be tested, the color is not
good, owing to blackening produced by the charring with the strong sulphuric acid.
4. The Buiret reaction
The biuret test for proteins identifies the presence of proteins in solution with a deep violet color.
Buiret, H2NCONHCONH2, reacts with copper (ii) ions in a basic solution to form a deep violet complex.
The peptide linkages resemble those in the biuret and also form deep violet complexes with basic
copper (ii) ions in solution. The figure below shows the protein-copper(ii) ion complex, also called the
buiret complex
For the test above, the reaction is given by nearly all substances containing two CONH groups attached
to one another, to the same nitrogen atom, or to the same carbon atom. The cause of the reaction with
proteins is the presence of one or more groupings formed by the condensation of the carboxylic group
of an amino- acid with the amino group of another amino-acid.
5. The Sulphur reaction
This reaction is due to the formation of sodium sulphide by the action of the strong alkali on the sulphur
of the protein. On addition of the lead salt, either a black precipitate, or dark color, due to lead sulphide
is formed. The sulphur in the protein molecule is mainly present as cystine. Sulphur containing amino
acids, such as cysteine and cystine upon boiling with sodium hydroxide (hot alkali) yield sodium
sulphide. This reaction is due to partial conversion of the organic sulphur to inorganic sulphide, which
can detected by precipitating it to lead sulphide, using lead acetate solution.
6. Test for leucosin in wheat flour
The grain of Wheat contains some proteose and a small percentage of an albumin, leucosin. The
albumen in wheat is sensitive to heat and was denatured on heating the solution to boiling. The
denatured protein caused a white coagulant. Hydrogen bonds were denatured causing the protein to
the denaturation coagulation
7. Extraction of zein and prolamine of maize
Zein is a protein that is only found in corn; however, there are proteins which share similar prolamine
characteristics to that of the zein found in corn. The extracted prolamine proteins from these cereals
have each been shown to have industrial importance, but zein is favored because of higher yields and
the large volume of corn products available for extraction. Ethanol added dehydrated the zein and
prolamine proteins in the turbid solution. This dehydration effect lead to unfolding of these proteins
forming colloidal suspensions


For the following reactions, a purified protein solution e.g. egg albumen should be used.
TEST OBSERVATIONS DEDUCTION
1. Precipitation by alcohol
To 2ml of the protein solution,
add a few drops of absolute
alcohol and observe and record.
A white precipitate Proteins present
2. Reaction with 5%
CuSO
4
, 5% Lead and
HgCl
2

To 2ml of protein solution in
three test-tubes add
respectively a little of:
5% copper sulphate solution and
record your observation





A blue precipitate





Protein precipitated
5% lead acetate solution and
record your observation.
A white coagulant was formed Protein precipitated
5% mercuric chloride Solution
and record your observation.
A white coloration Protein precipitated
3. Precipitation by alkaline
earth metals
To 2ml of protein solution in a
test-tube, add 5ml of barium
chloride solution and record
your observation.
A precipitate is formed on
standing
Proteins present
4. Precipitation by tannic
acid
To 2ml of protein solution add a
little 3% of tannic solution and
record your observation.
A precipitate is formed Proteins present


A. AMINO ACIDS
There is every reason to believe, since they always arise in hydrolysis of proteins, that amino-acids are
universally distributed in the plant. It is, however difficult to isolate and detect them, except in certain
special cases, as, for instance, in germinating seeds when a large store of protein is being rapidly
hydrolyzed and translocated.
TEST OBSERVATIONS DEDUCTIONS
1. Ninhydrin reaction
Heat to boiling 2ml of 0.5% glutamic
acid with 1ml of 0.25% Ninhydrin
solution in a test-tube. Observe and
record the color changes.

A purple solution is formed

Glutamic acid is an alpha
amino acid
2. Copper Sulphate test.
Add a few drops of 5% copper
sulphate solution to a 2ml glutamic
acid solution in a test-tube and
observe and record.

A blue solution is formed

Glutamic acid is an aliphatic
amino acid
3. Ferric chloride test.
Add a few drops of 5% ferric
chloride solution to 2ml of 5%
glutamic acid solution and record
your observation.

A pale brown solution is
formed

Glutamic acid is an aliphatic
amino acid

DISCUSSION AND EXPLANATION OF THE RESULTS
Precipitation by alcohol
Alcohol disrupts the hydrogen bonding between water and the surface polar residues favoring the
exposure of more hydrophobic molecules buried inside. This denaturation brings about the white
precipitate. This therefore confirms presence of proteins in the albumen
Ninhydrin test.
Amino acids contain a free amino group and a free carboxylic group that reacts 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 groups 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.
A color reaction given by amino acids and peptides on heating with the chemical ninhydrin. Ninhydrin is
a powerful oxidizing agent which reacts with all amino acids between pH 4-8 to produce a purple
colored-compound.
The reaction is given by primary amines and ammonia but without the liberation of carbon dioxide. The
amino acid proline reacts but produces a yellow color.
-amino acid + 2 ninhydrin---CO
2
+aldehyde + final complex (purple) +3H
2
O.
In summary, ninhydrin, which is originally yellow, reacts with amino acids and turns to deep purple
which is the color detected by this method.
Precipitation by tannic acid
Tannic acid disrupts the salt bridges held by ionic charge in the proton. A replacement reaction occurs
where the positive ions in the salt change patterns with the positive ions in the tannic acid. This changes
the structure of proteins hence forming a white precipitate
Precipitation by alkali earth metals.
Barium chloride disrupts the ionic bonding in the protein by changing its secondary and tertiary
structures. As a result of this, a white coagulant is formed.
Ferric chloride test
The ferric chloride solution reacts with the amino acids in the glutamic acidreleasing Fe
3+
ions that
turned the solution brown in color.




References
Mueiel wheldale Onslow (1923) PRACTICAL PLANT BIOCHEMISTRY, (second edition), Cambridge
University Press, London. Pages 133-145