Note 1: Modified from the definition given in ref.
Note 2: Denaturation can occur when proteins and nucleic acids are
subjected to elevated temperature or to extremes of pH, or to
nonphysiological concentrations of salt, organic solvents, urea, or
other chemical agents.
Note 3: An enzyme loses its catalytic activity when it is
Denaturation is a process in which proteins or nucleic acids lose the
quaternary structure, tertiary structure and secondary structure which
is present in their native state, by application of some external
stress or compound such as a strong acid or base, a concentrated
inorganic salt, an organic solvent (e.g., alcohol or chloroform),
radiation or heat. If proteins in a living cell are denatured, this
results in disruption of cell activity and possibly cell death.
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.
Protein folding is key to whether a globular protein or a membrane
protein can do its job correctly. It must be folded into the right
shape to function. But hydrogen bonds, which play a big part in
folding, are rather weak, and it doesn't take much heat, acidity,
varying salt concentrations, or other stress to break some and form
others, denaturing the protein. This is one reason why tight
homeostasis is physiologically necessary in many life forms.
This concept is unrelated to denatured alcohol, which is alcohol that
has been mixed with additives to make it unsuitable for human
1 Common examples
2.2 How denaturation occurs at levels of protein structure
2.2.1 Loss of function
2.2.2 Reversibility and irreversibility
Nucleic acid denaturation
3.1 Biologically-induced denaturation
3.2 Denaturation due to chemical agents
3.2.1 Chemical denaturation as an alternative
3.2.2 Denaturation due to air
4.1.4 Cross-linking reagents
4.1.5 Chaotropic agents
Disulfide bond reducers
5 See also
7 External links
(Top) The protein albumin in the egg white undergoes denaturation and
loss of solubility when the egg is cooked. (Bottom) Nitinol paperclips
provide a visual analogy to help with the conceptualization of the
When food is cooked, some of its proteins become denatured. This is
why boiled eggs become hard and cooked meat becomes firm.
A classic example of denaturing in proteins comes from egg whites,
which are typically largely egg albumins in water. Fresh from the
eggs, egg whites are transparent and liquid. Cooking the thermally
unstable whites turns them opaque, forming an interconnected solid
mass. The same transformation can be effected with a denaturing
chemical. Pouring egg whites into a beaker of acetone will also turn
egg whites translucent and solid. The skin that forms on curdled milk
is another common example of denatured protein. The cold appetizer
known as ceviche is prepared by chemically "cooking" raw fish and
shellfish in an acidic citrus marinade, without heat.
Denatured proteins can exhibit a wide range of characteristics, from
loss of solubility to protein aggregation.
Functional proteins have four levels of structural organization:
1) Primary Structure : the linear structure of amino acids in the
2) Secondary Structure : hydrogen bonds between peptide group
chains in an alpha helix or beta sheet
3) Tertiary Structure : three-dimensional structure of alpha
helixes and beta helixes folded
4) Quaternary Structure : three-dimensional structure of multiple
polypeptides and how they fit together
Process of Denaturation: 1) Functional protein showing a quaternary
structure 2) when heat is applied it alters the intramolecular bonds
of the protein 3) unfolding of the polypeptides (amino acids)
Proteins or Polypeptides are polymers of amino acids. A protein is
created by ribosomes that "read"
RNA that is encoded by codons in the
gene and assemble the requisite amino acid combination from the
genetic instruction, in a process known as translation. The newly
created protein strand then undergoes posttranslational modification,
in which additional atoms or molecules are added, for example copper,
zinc, or iron. Once this post-translational modification process has
been completed, the protein begins to fold (sometimes spontaneously
and sometimes with enzymatic assistance), curling up on itself so that
hydrophobic elements of the protein are buried deep inside the
structure and hydrophilic elements end up on the outside. The final
shape of a protein determines how it interacts with its environment.
When a protein is denatured, secondary and tertiary structures are
altered but the peptide bonds of the primary structure between the
amino acids are left intact. Since all structural levels of the
protein determine its function, the protein can no longer perform its
function once it has been denatured. This is in contrast to
intrinsically unstructured proteins, which are unfolded in their
native state, but still functionally active and tend to fold upon
binding to their biological target.
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
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
In secondary structure denaturation, proteins lose all regular
repeating patterns such as alpha-helices and beta-pleated sheets, and
adopt a random coil configuration.
Primary structure, such as the sequence of amino acids held together
by covalent peptide bonds, is not disrupted by denaturation.
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, and because amino
acid residues involved in stabilizing substrates' transition states
are no longer positioned to be able to do so. The denaturing process
and the associated loss of activity can be measured using techniques
such as dual polarization interferometry, CD, QCM-D and MP-SPR.
Reversibility and irreversibility
In many cases, denaturation is reversible (the proteins can regain
their native state when the denaturing influence is removed). This
process can be called renaturation. This understanding has led to
the notion that all the information needed for proteins to assume
their native state was encoded in the primary structure of the
protein, and hence in the
DNA that codes for the protein, the
so-called "Anfinsen's thermodynamic hypothesis".
However, denaturation can also be irreversible. However, this
irreversibility is typically a kinetic, not thermodynamic
irreversibility, as generally when a protein is folded it has lower
free energy. But, through kinetic irreversibility, the fact that the
protein is stuck in a local minimum can stop it from ever refolding
after it has been irreversibly denatured.
Nucleic acid denaturation
Nucleic acid thermodynamics
Nucleic acids (including
RNA and DNA) are nucleotide polymers
synthesized by polymerase enzymes during either transcription or DNA
replication. Following 5'-3' synthesis of the backbone, individual
nitrogenous bases are capable of interacting with one another via
hydrogen bonding, thus allowing for the formation of higher-order
Nucleic acid denaturation occurs when hydrogen bonding
between nucleotides is disrupted, and results in the separation of
previously annealed strands. For example, denaturation of
DNA due to
high temperatures results in the disruption of Watson and Crick base
pairs and the separation of the double stranded helix into two single
Nucleic acid strands are capable of re-annealling when
"normal" conditions are restored, but if restoration occurs too
quickly, the nucleic acid strands may re-anneal imperfectly resulting
in the improper pairing of bases.
DNA denaturation occurs when hydrogen bonds between Watson and Crick
base pairs are disturbed.
The non-covalent interactions between antiparallel strands in
be broken in order to "open" the double helix when biologically
important mechanisms such as
DNA replication, transcription, DNA
repair or protein binding are set to occur. The area of partially
DNA is known as the denaturation bubble, which can be more
specifically defined as the opening of a
DNA double helix through the
coordinated separation of base pairs.
The first model that attempted to describe the thermodynamics of the
denaturation bubble was introduced in 1966 and called the
Poland-Scheraga Model. This model describes the denaturation of DNA
strands as a function of temperature. As the temperature increases,
the hydrogen bonds between the Watson and Crick base pairs are
increasingly disturbed and "denatured loops" begin to form.
However, the Poland-Scheraga Model is now considered elementary
because it fails to account for the confounding implications of DNA
sequence, chemical composition, stiffness and torsion.
Recent thermodynamic studies have inferred that the lifetime of a
singular denaturation bubble ranges from 1 microsecond to 1
millisecond. This information is based on established timescales
DNA replication and transcription. Currently,[when?]
biophysical and biochemical research studies are being performed to
more fully elucidate the thermodynamic details of the denaturation
Denaturation due to chemical agents
DNA by disrupting the hydrogen bonds between
Watson and Crick base pairs. Orange, blue, green, and purple lines
represent adenine, thymine, guanine, and cytosine respectively. The
three short black lines between the bases and the formamide molecules
represent newly formed hydrogen bonds.
With polymerase chain reaction (PCR) being among the most popular
contexts in which
DNA denaturation is desired, heating is the most
frequent method of denaturation. Other than denaturation by heat,
nucleic acids can undergo the denaturation process through various
chemical agents such as formamide, guanidine, sodium salicylate,
dimethyl sulfoxide (DMSO), propylene glycol, and urea. These
chemical denaturing agents lower the melting temperature (Tm) by
competing for hydrogen bond donors and acceptors with pre-existing
nitrogenous base pairs. Some agents are even able to induce
denaturation at room temperature. For example, alkaline agents (e.g.
NaOH) have been shown to denature
DNA by changing pH and removing
hydrogen-bond contributing protons. These denaturants have been
employed to make Denaturing Gradient Gel Electrophoresis gel (DGGE),
which promotes denaturation of nucleic acids in order to eliminate the
influence of nucleic acid shape on their electrophoretic mobility.
Chemical denaturation as an alternative
The optical activity (absorption and scattering of light) and
hydrodynamic properties (translational diffusion, sedimentation
coefficients, and rotational correlation times) of formamide denatured
nucleic acids are similar to those of heat-denatured nucleic
acids. Therefore, depending on the desired effect,
DNA can provide a gentler procedure for
denaturing nucleic acids than denaturation induced by heat. Studies
comparing different denaturation methods such as heating, beads mill
of different bead sizes, probe sonification, and chemical denaturation
show that chemical denaturation can provide quicker denaturation
compared to the other physical denaturation methods described.
Particularly in cases where rapid renaturation is desired, chemical
denaturation agents can provide an ideal alternative to heating. For
DNA strands denatured with alkaline agents such as NaOH
renature as soon as phosphate buffer is added.
Denaturation due to air
Small, electronegative molecules such as nitrogen and oxygen, which
are the primary gases in air, significantly impact the ability of
surrounding molecules to participate in hydrogen bonding. These
molecules compete with surrounding hydrogen bond acceptors for
hydrogen bond donors, therefore acting as "hydrogen bond breakers" and
weakening interactions between surrounding molecules in the
environment. Antiparellel strands in
DNA double helices are
non-covalently bound by hydrogen bonding between Watson and Crick base
pairs; nitrogen and oxygen therefore maintain the potential to
weaken the integrity of
DNA when exposed to air. As a result, DNA
strands exposed to air require less force to separate and exemplify
lower melting temperatures.
Many laboratory techniques rely on the ability of nucleic acid strands
to separate. By understanding the properties of nucleic acid
denaturation, the following methods were created:
Acidic protein denaturants include:
Trichloroacetic acid 12% in water
Bases work similarly to acids in denaturation. They include:
Most organic solvents are denaturing, including:
Cross-linking agents for proteins include:
Chaotropic agents include:
Urea 6 – 8 mol/l
Guanidinium chloride 6 mol/l
Lithium perchlorate 4.5 mol/l
sodium dodecyl sulfate
Disulfide bond reducers
Agents that break disulfide bonds by reduction include:[citation
Acidic nucleic acid denaturants include:
Basic nucleic acid denaturants include:
Other nucleic acid denaturants include:
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