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1. By –
Savy Panamkuttiyiel Minal
DENATURATION OF PROTEIN

2. DENATURATION
Denaturation is a process in which a protein loses its native shape due to
the disruption of weak chemical bonds and interactions, thereby becoming
biologically inactive.
When proteins denature, the cells go through a series of changes, first
loosening, then tightening.
In case of proteins :
• A loss of three-dimensional structure, sufficient to cause loss of function
⟰ Loss of secondary, tertiary and quaternary structure of proteins.
⟰ Change in physical, chemical and biological properties of protein
molecules.

3. FOR EXAMPLE:
i. Changing pH denatures proteins because it changes the charges on many of
the side chains. This disrupts electrostatic attractions and hydrogen bonds.
ii. Certain reagents such as urea and guanidine hydrochloride denature
proteins by forming hydrogen bonds to the protein groups that are stronger
than the hydrogen bonds formed between the groups.
iii. Detergents such as sodium dodecyl sulphate denature proteins by
associating with the non-polar groups of protein, thus interfering with the
normal hydrophobic interactions.
iv. Organic solvents such as acetone alcohols denature proteins by disrupting
hydrophobic interactions.
v. Proteins can also be denatured by heat. Heat increase molecular motion
which can disrupt the attractive forces.

4. • None of these agents breaks peptide bonds, so the primary structure of a
protein remains intact when it is denatured.
• When a protein is denatured, it loses its function.
Example:
• A denatured enzyme ceases to function.
• A denatured antibody no longer can bind its antigen.

5. • The denatured state does not necessarily equate with complete unfolding
of the protein and randomization of conformation.
• Under most conditions, denatured proteins exist in a set of partially
folded states that are poorly understood.

6. AGENTS OF DENATURATION
Physical agents :
1. Heat,
2. Violent shaking,
3. X-rays,
4. Hydrostatic Pressure (5,000 – 10,000 atm)
5. UV radiation.

7. HEAT
Most proteins can be denatured by heat, which affects the weak
interactions in a protein (primarily hydrogen bonds) in a complex manner.
If the temperature is increased slowly, a protein’s conformation generally
remains intact until an abrupt loss of structure (and function) occurs over
a narrow temperature range
• In cooking, this stress that causes denaturation is typically heat. As it
heats, its proteins coagulate.
• As high temperatures can denature proteins, and when a cell is exposed to
high temperatures, several types of molecular chaperones swing into
action. For this reason, these chaperones are also called heat-shock
proteins (HSPs).

9. VIOLENT SHAKING
• Agitation also denatures protein.
• We see this clearly in the whipping of egg
whites.
• In nature, you can see the denaturation of
protein in the waves at the beach. The
constant churning creates foam from various
proteins in the sea water.

10. HYDROSTATIC PRESSURE (5,000 – 10,000 atm)
• Pressure destabilization of hydrophobic aggregates by using an
information theory model of hydrophobic interactions.
• Pressure-denatured proteins, unlike heat-denatured proteins,
retain a compact structure with water molecules penetrating
their core.

11. UV RADIATION
• UV radiation supplies kinetic energy to protein molecules,
causing their atoms to vibrate more rapidly and disrupting
relatively weak hydrogen bonding and dispersion forces.
[http://www.ncbi.nlm.nih.gov/pubmed/8933720]

12. CHEMICAL AGENTS :
• Acids and alkalies
• Organic solvents (ether, alcohol),
• Salts of heavy metals (Pb, Hg),
• Chaotropic agents
• Detergents
• Altered pH

13. Acids and alkalies
• Acids and bases disrupt salt bridges
• Acids and bases disrupt salt bridges
held together by ionic charges.
• 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.

14. ACIDIC PROTEIN DENATURANTS INCLUDE:
• Acetic acid
• Trichloroacetic acid 12% in water
• Sulfosalicylic acid
Bases
Bases work similarly to acids in denaturation.
• Sodium bicarbonate

15. ORGANIC SOLVENTS (ETHER, ALCOHOL)
• New hydrogen bonds are formed instead
between the new alcohol molecule and
the protein side chains.
For example :
• 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, there is substantial
structural changes.
Alcohol Disrupts Hydrogen Bonding:

16. Salts of heavy metals (Pb, Hg),
• 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.
“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.”

17. REDUCING AGENTS DISRUPT DISULFIDE BONDS:
• Disulfide bonds are formed by
oxidation of the sulfhydryl groups on
cysteine.
• If oxidizing agents cause the
formation of a disulfide bond, then
reducing agents, of course, act on any
disulfide bonds to split it apart.
Reducing agents add hydrogen atoms
to make the thiol group, -SH.

18. CHAOTROPIC AGENTS
Chaotropic agents include:
1. Urea 6 – 8 mol/l
2. Guanidinium chloride 6 mol/l
3. Lithium perchlorate 4.5 mol/l

19. DETERGENTS
• Detergents are amphiphilic molecules
(both hydrophobic and hydrophilic parts).
Example:
Disrupt hydrophobic interactions:
– hydrophobic parts of the detergent associate with the hydrophobic parts of the protein
(coating with detergent molecules)
– hydrophilic ends of the detergent molecules interact favorably with water (nonpolar
parts of the protein become coated with polar groups that allow their association with
water)
– hydrophobic parts of the protein no longer need to associate with each other
• Dissociation of the non-polar R groups can lead to unfolding of the protein
chain (same effect as in nonpolar solvents).

20. DISULFIDE BOND REDUCERS
Agents that break disulfide bonds by reduction include:
1. 2-Mercaptoethanol
2. Dithiothreitol
3. TCEP (tris(2-carboxyethyl)phosphine)

21. CROSS-LINKING REAGENTS
Cross-linking agents for proteins include:
1. Formaldehyde
2. Glutaraldehyde

22. • According to Wu (1931) denaturation leads mainly to the unfolding of the
peptide chain, thus causing disorganization of the internal structure of protein.
This is evidenced by the fact that the denatured proteins are more easily
hydrolyzed (Mirsky, 1935)
• When the peptide chain or the protein molecules are unrolled, certain bonds
split and new sites of bundles are exposed to the action of certain proteolytic
enzymes causing hydrolysis. Thus, the H-bond linking the 2 peptide chains are
partly freed and the disulphide (– S – S –) bonds also linking the two peptide
chains split open to yield the free sulfhydryl (–SH) groups

23. According to Putnam (1953), the protein , on denaturation, undergo
following charges :
i. Decrease in their solubility,
ii. Cessation of their biochemical activity as enzymes or hormones,
iii. Decrease in size and shape of the molecule,
iv. Increase activity of some radicals present in the molecule such as –
SH group of cysteine –S –S – bond of cystine and phenolic group of
tyrosine.

24. CHARACTERISTICS OF DENATURATION
⧗ The native helical structure of protein is lost
⧗ The primary structure of a protein with peptide linkages remains
intact i.e., peptide bonds are not hydrolyzed.
⧗ The protein loses its biological activity.
⧗ Denatured protein becomes insoluble in the solvent in which it was
originally soluble.
⧗ The viscosity of denatured protein (solution) increases while its
surface tension decreases.
⧗ Denaturation is associated with increase in ionizable and sulfhydryl
groups of protein. This is due to loss of hydrogen and disulfide bonds.

25. ⧗ Denatured protein is more easily digested. This is due to increased
exposure of peptide bonds to enzymes Cooking causes protein
denaturation and, therefore, cooked food (protein) is more easily
digested.
⧗ Denaturation is usually irreversible. For instance, omelet can be
prepared from an egg (protein-albumin) but the reversal is not
possible.
⧗ Careful denaturation is sometimes reversible (known as renaturation).
For example - Hemoglobin undergoes denaturation in the presence of
salicylate. By removal of salicylate, hemoglobin is renatured.

26. HOW DENATURATION OCCURS AT LEVELS OF
PROTEIN STRUCTURE
In quaternary
structure denaturation,
protein sub-units are
dissociated and/or the spatial
arrangement of protein
subunits is disrupted.

27. • Tertiary structure denaturation
involves the disruption of:
– Covalent interactions between
amino acid side-chains (such
as disulfide
bridges between cysteine groups)
– Non-covalent dipole-dipole
interactions between polar amino
acid side-chains (and the
surrounding solvent)
– Van der Waals (induced dipole)
interactions between nonpolar
amino acid side-chains.

28. • In secondary
structure denaturation, proteins
lose all regular repeating
patterns such as alpha-
helices and beta-pleated sheets,
and adopt a random coil
configuration.

29. • Primary structure, such as the sequence of amino acids held
together by covalent peptide bonds, is not disrupted by
denaturation.

30. LOSS OF FUNCTION
• Most biological substrates lose their biological function when
denatured.
• For example, enzymes lose their activity, because the substrates
can no longer bind to the active site,
• The denaturing process and the associated loss of activity can be
measured using techniques such as
• Dual polarization interferometry,
• CD [Circular dichroism], and
• QCMD [Quality Control for Molecular Diagnostics ].

31. CONCLUSION / APPLICATIONS OF DENATURATION
• The study of Denaturation of protein (transporters, enzymes,
hormones) is useful in the field of Proteomics.
• To determines the percentage and conc. of alcohol that could lyse
the bacterial and viral protein and cell wall but not the skin.

32. REFERENCES:
• http://elmhurst.edu/~chm/vchembook/568denaturation.html
• http://en.wikipedia.org/wiki/Denaturation_(biochemistry)#Los
s_of_function
• http://elmhurst.edu/~chm/vchembook/567tertprotein.html

33. THANK YOU