Protein Denaturation

AMINO ACIDS AND PROTEINS

Proteins are one of the main constituents of living cells. The

functions of proteins include building of new cells, the
maintenance of cells and the replacement of old cells. All proteins
contain carbon, hydrogen, oxygen and nitrogen. Most proteins
contain sulfur. Elements like phosphorus, iron etc is present in
some special proteins. Some examples of proteins are albumin
(present in eggs), casein (present in milk), collagen (in tissues) and
hemoglobin (present in human blood).

A protein is defined as a polymer made up of several amino

acids. An amino acid is a compound that contains two functional
groups, an amino group (-NH2) and a carboxylic acid group (-
COOH). The side chain (R) distinguishes the amino acids.

side chain
carboxylic acid group
R
H2N C COOH
H
amino group
 Some examples of amino acids are alanine, valine, cysteine,
and tryptophan. Glycine is the simplest amino acid known.

Amino R
acid group
Glycin -H
e
Alanin - CH3
e
Valine CH3
H3C CH

Cystei SH
CH2
ne

Amino acids have both acidic and basic properties. The amino
group (-NH2) lends basic properties whereas the carboxylic acid
group (-COOH) lends acidic properties to the amino acid.

acidic group
R
H2N C COOH
H
basic group
 Several amino acids combine to form peptide bonds. Proteins
contain polypeptide units (several peptide units). When a protein is
hydrolyzed, it breaks down into smaller units (tri and dipeptides),
eventually forming amino acids.

amino acid amino acid amino acid amino acid amino acid

Peptide Peptide Peptide Peptide

bond bond bond bond

 Cross
Structures of proteins can be classified into various levels, primary,
secondary, tertiary and quaternary structure. The sequence of
amino acids present in a protein is known as its primary structure.
The regularly repeating order and spatial arrangements of amino
acids in a protein is known as its secondary structure. The overall
three dimensional shape of the protein is called its tertiary
structure. Quaternary structure is the organization among the

Pr
various polypeptide chains in a protein. The biochemical function
of the protein depends on the three dimensional structure of the
protein.
Denaturation of proteins

Denaturation of proteins involves the disruption and possible

destruction of both the secondary and tertiary structures. Since
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.
Denaturation occurs because the bonding interactions responsible
for the secondary structure (hydrogen bonds to amides) and
tertiary structure are disrupted. In tertiary structure there are four
types of bonding interactions between "side chains" including:
hydrogen bonding, salt bridges, disulfide bonds, and non-polar
hydrophobic interactions. which may be disrupted. Therefore, a
variety of reagents and conditions can cause denaturation. The
most common observation in the denaturation process is the
precipitation or coagulation of the protein.
Effects Of Denaturation on Proteins

The function principle tells us that changing the structure of a

protein will affect its function. Often that means that function is
lost. "Denaturation" of a protein means loss of the protein's
function due to structural change in the protein caused by some
chemical or physical factor such as high temperature or
unfavorable pH. The factor causes the folded protein (the tertiary
structure) to unfold, to unravel.

1\ If the protein functioned as an enzyme, then denaturation

causes it to lose its enzymatic activity.

2\ If the protein was embedded in a cell membrane where it

transported ions or molecules through the membrane, then
denaturation destroys that ability.

3\ If the protein was an antibody, responsible for recognizing an

infectious agent, denaturation will destroy that protective ability.
Consequences Of Denaturation

Loss of Biological Activity

Loss of solubility.

Destruction of toxins.

Improved Digestibility.

Altered Water Binding Capacity.

Factors Of Denaturation

Therefore, a variety of reagents and conditions can cause

denaturation. The most common observation in the denaturation
process is the precipitation or coagulation of the protein.
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.
Alcohol Disrupts Hydrogen Bonding:
Hydrogen bonding occurs between amide groups in the secondary
protein structure. Hydrogen bonding between "side chains" occurs
in tertiary protein structure in a variety of amino acid
combinations. All of these are disrupted by the addition of another
alcohol.
A 70% alcohol solution is used as a disinfectant on the skin. This
concentration of alcohol is able to penetrate the bacterial cell wall
and denature the proteins and enzymes inside of the cell. A 95%
alcohol solution merely coagulates the protein on the outside of
the cell wall and prevents any alcohol from entering the cell.
Alcohol denatures proteins by disrupting the side chain
intramolecular hydrogen bonding. New hydrogen bonds are formed
instead between the new alcohol molecule and the protein side
chains.
In the prion protein, tyr 128 is hydrogen bonded to asp 178, which
cause one part of the chain to be bonding with a part some
distance away. After denaturation, the graphic show substantial
structural changes.

Acids and Bases Disrupt Salt Bridges:

Salt bridges result from the neutralization of an acid and amine on
side chains. The final interaction is ionic between the positive
ammonium group and the negative acid group. Any combination of
the various acidic or amine amino acid side chains will have this
effect.
As might be expected, acids and bases disrupt salt bridges held
together by ionic charges. A type of double replacement reaction
occurs where the positive and negative ions in the salt change
partners with the positive and negative ions in the new acid or
base added. This reaction occurs in the digestive system, when the
acidic gastric juices cause the curdling (coagulating) of milk.
The example below is from the prion protein with the salt bridge of
glutamic acid 200 and lysine 204. In this case a very small loop is
made because there are only three other amino acids are between
them. The salt bridge has the effect of straightening an alpha
helix.
The denaturation reaction on the salt bridge by the addition of an
acid results in a further straightening effect on the protein chain as
shown in the graphic .

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.
The reaction of a heavy metal salt with a protein usually leads to
an insoluble metal protein salt.
This reaction is used for its disinfectant properties in external
applications. For example AgNO3 is used to prevent gonorrhea
infections in the eyes of new born infants. Silver nitrate is also
used in the treatment of nose and throat infections, as well as to
cauterize wounds.
Mercury salts administered as Mercurochrome or Merthiolate have
similar properties in preventing infections in wounds.
This same reaction is used in reverse in cases of acute heavy
metal poisoning. In such a situation, a person may have swallowed
a significant quantity of a heavy metal salt. As an antidote, a
protein such as milk or egg whites may be administered to
precipitate the poisonous salt. Then an emetic is given to induce
vomiting so that the precipitated metal protein is discharged from
the body.
Heavy metals may also disrupt disulfide bonds because of their
high affinity and attraction for sulfur and will also lead to the
denaturation of proteins.
Definitions

coagulation : The collecting into a mass of minute particles of a

solid dispersed throughout a liquid, usually followed by the
precipitation or separation of the solid mass from the liquid. The
casein in milk is coagulated (curdled) by the addition of acetic acid
or citric acid. The albumin in egg white is coagulated by heating.
The clotting of blood is another example of coagulation.
Coagulation usually involves a chemical reaction.

Aggregation: Aggrigation is a general term that encompasses

several types of interactions or characteristics.Protein aggregation
is the aggregation of mis-folded protein and is thought to be
responsible for many degenerative dieses such as Alzheimer's

Gelitation:Gelatine is a substantially pure protein food ingredient,

obtained by the thermal denaturation of collagen , which is the
structural and most common protein in the animal kingdom.
Today gelatine is usually available in granular powder form,
although in Europe, sheet gelatine is still available.

Other Denaturation Factors

Mechanical Treatments:

Whipping ( Egg) .

Ultraviolet.

Though there are exceptions, as a rule denaturation by factors

such as heat, high or low pH, or exposure to organic solvents
(alcohol, e.g.) is irreversible. That is, removal of the "offending"
factor will not cause the protein to fold back into its original shape
and resume its function. Protection of proteins against
denaturation is one result of the buffering of biological solutions
such as blood and the aqueous interior of living cells. If blood pH
changed much from its normal value, proteins in the blood would
begin to pucker, buckle, twist into different shapes, and unravel,
with potential loss of function.

Summary
Disruption of secondary, tertiary and quaternary protein
structure by

heat/organics

Break apart H bonds and disrupt hydrophobic

attractions

acids/ bases

Break H bonds between polar R groups and ionic

bonds

heavy metal ions

React with S-S bonds to form solids

agitation

Stretches chains until bonds break

 University Of Khartoum
Animal Production Faculty
MSC Dairy Production & Technology
(Batch7)

Protein
Denaturation

Prepared By:
Ahmed Munir Yousif Elhakim
Somaya Ahmed Kashif A.Kareem
Safa A.Raheem Abdallah
September 2010