Name:

Course and Year:

Experiment No. 7
PRECIPITATION REACTION OF PROTEINS

OBJECTIVES

1. To be able to know some precipitation reactions of proteins and its application.

RESULT AND DISCUSSION

Protein Precipitation is the process in which protein is separated from any extra contaminants that may
be mixed with it. It is an important part of downstream processing and can be done with a variety of
different techniques.

1. Heat:

Heat coagulation and precipitation occur best near the isoelectric point of the protein.
Albumin coagulates when heated. Aqueous solutions of proteoses, peptones, gelatin and
casein are not coagulated by heat.

a) Heating of 5ml 2% casein

- Upon observation the result of experiment A has an unclear or blurry

appearance.

Casein is heat stable and is not destroyed through cooking. On the other hand, the
proteins beta-lactoglobulin and alpha-lactalbumin are heat sensitive. Therefore
patients who only react to these heat-sensitive proteins may be able to tolerate
products containing milk that has been cooked or baked.

b) The three test tube were prepared and were added with 1ml egg albumin solution:
test tube 1 was added with 0.1 acetic acid, test tube 2 was added with 5N acetic
acid, and test tube 3 was a controlled set up, then it was boiled.
 Upon observation, all three test tube has the same results which is like a milk liquid
or a cloudy appearance with bubbles on top. When the egg albumin is heated, the
heat causes denaturation, hardening the albumen and coloring it opaque white. The
changes in viscosity and color reflect the denaturation of the egg albumin protein. A
simple form of protein, egg albumin protein presents several unique characteristics
that make it extremely useful to pet food manufacturers.

2. Alcohol:

In this procedure, it was observed the result has a white cloudy mixture. The alcohol participates
in a chemical reaction, denaturing or changing the conformation of the protein molecules so they
can form new linkages with each other. As the alcohol diffuses into the egg white, the reaction
proceeds and the egg white turns white.

The solution was then set aside for about 30 minutes and was filtered. After filtration, the filtrate
was subjected to heating in a water bath. It was then observed that the result has a translucent/
clear appearance. An egg white before denaturation of the albumin protein causes the translucent
substance to change in color and viscosity. The heat-caused denaturation in albumin protein in
egg white cause the once translucent, runny substance into one that has white color and has a
firm texture.

3. Neutral Salts:

The third procedure has two test tube with egg albumin solution and here is the result.

Test tube Contents Observation

1 Egg albumin solution with dissolved

sodium chloride

2 Egg albumin solution with Undissolved and cloudy

ammonium sulphate

The addition of NaCl can decrease the amount of hydrosulfonyl exposed on the surface of protein
in the process of heat treatment, increase the surface hydrophobicity, and neutralizes the charge
on the protein surface. When high concentrations of small, highly charged ions such as ammonium
sulfate are added, these groups compete with the proteins to bind to the water molecules. This
 removes the water molecules from the protein and decreases its solubility, resulting in
precipitation. Critical factors that affect the concentration at which a particular protein will
precipitate include: the number and position of polar groups, molecular weight of the protein, pH
of the solution, and temperature at which the precipitation is performed. The concentration at which
antibodies precipitate varies among species; most rabbit antibodies precipitate with a 40%
saturated solution, whereas mouse antibodies require 45-50% saturation.

4. Heavy-Metal Salts

Three test tube were prepared. Each test tubes were added with different types of solution and
here are the result.

Test tube Contents Observation

1 2 ml egg albumin solution with Greenish mixture

1% CuSO4 solution

2 2 ml egg albumin solution with White mixture with dark green

Pb (OAc)2 solution liquid

3 2 ml egg albumin solution with White cloudy mixture

1% AgNO3 solution

When egg albumin is added with copper sulphate, The action of copper sulfate in protein
denaturation derives from its ability to disrupt disulfide Cystine bonds, binding to individual
sulfhydryl groups. When this happens, the sections of the protein which were held in close
proximity by the Cystine bond are free to move apart, which usually allows water molecules to
enter the core of the protein, which leads to further disruptions.

The silver nitrate, on the other hand, it will allow new hydrogen bonds to for. Proteins can be
denatured by a number of different things, including heat, strong acids or bases, and heavy metals.

5. Alkaloidal Reagents:

Three test tubes that contains egg albumin solution were then acidified with one drop of 0.1
normal hydrochloric acid and was then added with 3 different solution.
 Test Contents Observation
tube

1 3 drops of potassium ferrocyanide Reddish liquid with

bubbles on top

2 3 drops of 5% tannic acid Brownish mixture

3 3 drops of saturated picric acid solution Lime yellow mixture

Precipitation of proteins by alkaloid reagents, Alkaloid reagents like tungstic acid, trichloro
acetic acid, sulphosalicylic acid, phosphor tungstic acid, tannic acid & esbach’s reagent
precipitates proteins in acidic medium. This neutralized by negative charge alkaloid
reagents. Alkaloidal reagents precipitates proteins because they combine with positively-
charged amino groups in proteins and disrupt ionic bonds.

CONCLUSIONS

The objective of this experiment is to know the precipitation reaction of proteins and its
application. The method that is most likely the most usable was the denaturing proteins
in the food industry is heat. Heat is the most common way of denaturing proteins when
cooking, such as cooking steaks and eggs because it adds nothing to the food being
cooked unlike other methods. Casein is heat stable and is not destroyed through cooking.
Therefore patients who only react to these heat-sensitive proteins may be able to tolerate
products containing milk that has been cooked or baked. An egg white before
denaturation of the albumin protein causes the translucent substance to change in color
and viscosity. The heat-caused denaturation in albumin protein in egg white cause the
once translucent, runny substance into one that has white color and has a firm texture.
This removes the water molecules from the protein and decreases its solubility, resulting
in precipitation. Critical factors that affect the concentration at which a particular protein
will precipitate include: the number and position of polar groups, molecular weight of the
protein, pH of the solution, and temperature at which the precipitation is performed.
Precipitation of proteins by alkaloid reagents, Alkaloid reagents like tungstic acid, trichloro
acetic acid, sulphosalicylic acid, phosphor tungstic acid, tannic acid & esbach’s reagent
precipitates proteins in acidic medium. This neutralized by negative charge alkaloid
 reagents. Alkaloidal reagents precipitates proteins because they combine with positively-
charged amino groups in proteins and disrupt ionic bonds.

APPLICATION

1. What is the denaturation process of protein?

- Denaturation, in biology, process modifying the molecular structure of a protein.
Denaturation involves the breaking of many of the weak linkages, or bonds,
within a protein molecule that are responsible for the highly ordered structure of
the protein in its natural (native) state. Denatured proteins have a looser, more
random structure; most are insoluble. Denaturation can be brought about in
various ways; by heating, by treatment with alkali, acid, urea, or detergents, and
by vigorous shaking.
2. Cite advantages and disadvantages on the denaturation of protein.

- Protein denaturation becomes a vital process as it way up the digestion process,

it means for the enzymes to work efficiently it require simple form of it,, as in the
stomach there iz oxntic cells that produces HCL and HCL is one of the
denaturation agent thus it makes protein to be denatured into primary structure,,
that facilitate the working ability of pepsin enzymes to convert it into peptides,
thus protein denaturation process seems important in that way. The
disadvantages of this is that Protein denaturation is also a consequence of cell
death. Denatured proteins can exhibit a wide range of characteristics, from
conformational change and loss of solubility to aggregation due to the exposure
of hydrophobic groups. Denatured proteins lose their 3D structure and therefore
cannot function.

3. How are the following denaturation process destroy the protein molecule?

a. Heat- Heat can be used to disrupt hydrogen bonds and non-polar hydrophobic
interactions. This occurs because heat increases the kinetic energy and causes
the molecules to vibrate so rapidly and violently that the bonds are disrupted. The
proteins in eggs denature and coagulate during cooking. Other foods are cooked
to denature the proteins to make it easier for enzymes to digest them. Medical
supplies and instruments are sterilized by heating to denature proteins in bacteria
and thus destroy the bacteria.
 b. Alcohol- Alcohol also denatures proteins. It does this the same way as heat, by breaking
bonds that hold parts of the protein in a folded shape. Sometimes the alcohol
moleculesbond directly to some of the parts of the protein, disrupting the normal way
the proteinwould bond to itself.
c. Neutral salts- Denaturation disrupts the normal alpha-helix and beta sheets in
a protein and uncoils it into a random shape. Denaturation occurs because the bonding
interactions responsible for the secondary structure (hydrogen bonds to amides) and
tertiary structure are disrupted.
d. Alkaloidal reagents- Denaturation reactions are not strong enough to break the peptide
bonds, the primary structure (sequence of amino acids) remains the same after
a denaturation process. Denaturation disrupts the normal alpha-helix and beta sheets in
a protein and uncoils it into a random shape.
e. Heavy-metal salts- Heavy metal salts act to denature proteins in much the same manner
as acids and bases. Heavy metal salts usually contain Hg+2, Pb+2, Ag+1 Tl+1, Cd+2 and
other metals with high atomic weights. Since salts are ionic they disrupt salt bridges
in proteins.
4. Write general equations showing the ionization of a protein in acid medium and in basic
medium. What medium favors acid ionization? Basic ionization?
a. Showing the ionization of a protein in acid medium.

b. In basic medium

Acids are classified as either strong or weak, based on their ionization in water. A strong
acid is an acid which is completely ionized in an aqueous solution. Hydrogen chloride
(HCl) ionizes completely into hydrogen ions and chloride ions in water.

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