Proteins

Structure, classification and qualitative reactions

Proteins are high-molecular-weight biomolecules that are an
essential component of all living organisms and are widely
distributed in nature.
• Proteins primarily play a structural role, as an integral part of all cells.

• Also, proteins have specific functions:

• Catalytic function - enzymes
• Protective role - antibodies
• Hormonal activity
• Contractile function - actin and myosin
• Transport - hemoglobin transports oxygen and carbon dioxide
• Potential sources of energy
The basic building blocks of
proteins are amino acids.

Although there are hundreds

of amino acids in nature, only
20 amino acids, and
selenocysteine as the 21st
amino acid, are included in
the genetic code and as such
are found in proteins.
• Amino acids consist of:
• -amino group (-NH2), which has a basic character,
• -carboxyl group (-COOH), which has an acidic character, and
• -side chain denoted by -R.

Amino acids are soluble in water, a polar solvent, but do not dissolve in non-
polar solvents such a benzene, ether and hexane.
 The predominant ionic species of an amino acid in solution depends on
its dissociation constant, the pH of the medium and the temperature.

For each amino acid, depending on its dissociation constant and

temperature, there is a specific pH value at which the amino acid behaves as
an electroneutral particle, meaning it exists in the form of a zwitterion. This
pH value is known as the isoelectric point (pI).
• At this point, the amino acid has no net electric charge and is least
soluble, making it prone to precipitation. Additionally, it does not
migrate in an electric field.

• Differences in electric charge and molecular mass have been

utilized in various analytical techniques.
Proteins can be classified into two major groups:
-Simple proteins – which consist solely of amino acid residues.

-Conjugated proteins – apart from amino acid residues, they also

contain other non-protein molecules (prosthetic groups:
carbohydrates, lipids, phosphoric acid, nucleic acids…).
 Physicochemical properties of proteins
determined by their structure, which is based on the qualitative and
quantitative composition of the amino acids that are found in their
molecules

• Denaturation occurs when various physical factors and chemical agents

cause changes in the secondary, tertiary, and quaternary structure of proteins,
while the chemical composition (i.e., the primary structure) remains
unchanged. This process leads to a decrease or complete disappearance of
the biological properties of the proteins.
• If during denaturation the tertiary structure is not disturbed, then the
denaturation can be reversible - renaturation.
• Proteins are characterized by their relatively high reactivity. The
presence of a large number of functional groups in the molecule
allows proteins to react with various compounds.

• The examination of the general properties of proteins mainly

involves two groups of reactions:
• Precipitation reactions that are related to the
physicochemical properties of proteins.
• Color reactions of proteins that are related to their chemical
properties.
 Protein precipitation reactions

Reversible precipitation reactions Irreversible precipitation reactions

1. Precipitation of proteins with 1. Protein precipitation by boiling

ammonium sulfate
2. Precipitation of proteins with 2. Precipitation of proteins by heavy
alcohol or acetone metal salts
3. Precipitation of proteins with
organic acids
Reversible precipitation reactions
 1. Precipitation of proteins with
ammonium sulfate - (NH4)2SO4
Principle
Salts of light metals, such as (NH4)2SO4, can decrease the
solubility of proteins by disrupting the hydration shell around the
protein molecule. This causes protein particles to aggregate and
precipitate. Globulins, which have a higher molecular mass, tend to
precipitate more readily than albumins.
Reagents and equipment
• Aqueous solution of egg white in NaCl
• Saturated solution of ammonium sulfate ((NH4)2SO4)
• Ammonium sulfate crystals
• Test tubes, pipettes, filter funnel, filter paper
Procedure
To 1 mL of egg white solution, add an equal volume of saturated ammonium
sulfate solution, and mix. After a few minutes, observe the formation of a white
precipitate of globulins. Filter the contents and add ammonium sulfate crystals until
saturation, during which a precipitate of albumins is formed. If distilled water is added to
the albumin precipitate, the proteins in the precipitate will dissolve due to the formation
of a hydration shell around them.
 2. Precipitation of proteins with alcohol
(ethanol, methanol) or acetone
Principle
Proteins can be reversibly precipitated with alcohol or acetone.
Ethanol acts on proteins in the same way as the salts of light metals,
removing the hydration shell and causing the proteins to precipitate.
Protein precipitation with alcohol can also be irreversible if water is
not added within the first ten minutes of precipitation.
Reagents and equipment
• Aqueous solution of egg white in NaCl
• Ethanol, 96%
• Test tubes, pipettes.
Procedure
Add 0.25 mL of ethanol to 1 mL of the protein solution. A white protein
precipitate will appear. If water is added, the precipitate will dissolve.
Irreversible precipitation reactions
 1. Protein precipitation by boiling
Principle
Heating proteins in a solution leads to the loss of their
hydration shell, which causes the particles to stick together.
However, they still retain their electric charge, do not settle at the
bottom, and make the solution opaque (opalescent).
Reagents and equipment
• Aqueous solution of protein
• Test tubes, pipettes, alcohol lamp
Procedure
2 mL solution of protein is heated to boiling, and a white turbidity in the solution
is observed.
2. Precipitation of proteins by heavy metal
salts
Principle
Heavy metal salts, such as CuSO4, HgCl2, Pb(CH3COO)2,
and FeCl3, precipitate proteins in the form of insoluble
metalloproteinates. In this reaction, proteins behave as weak acids
and form salts that are insoluble in water with the cations of heavy
metals. Unlike the solutions of light metals, these solutions are
prepared with lower concentrations.
Reagents and equipment
• Aqueous solution of protein
• Copper(II) sulfate, CuSO4 (γ = 50 g/L);
• Test tubes, pipettes.
Procedure
Add 2 mL of protein solution and a few drops of copper (II) sulfate solution to a
test tube. Observe the formation of a white precipitate resulting from denatured proteins.
Note that the precipitate appears blue due to the color of the copper (II) sulfate solution.
 3. Precipitation of proteins with organic
acids
Principle
Organic acids such as sulfosalicylic and trichloroacetic acid
cause proteins to precipitate in the form of white precipitates. This
reaction occurs when proteins in the cationic form (pH lower than
the isoelectric point of proteins) react with the anions of the
mentioned organic acids, resulting in insoluble salts.
Reagents and equipment
• Aqueous solution of protein;
• Trichloroacetic acid, CCl3COOH (γ = 100g/L);
• Test tube, pipettes.
Procedure
• Add 2 mL of protein solution and a few drops of trichloroacetic acid solution to a
test tube. Observe the formation of a white precipitate.
•