In
biochemistry
Biochemistry or biological chemistry is the study of chemical processes within and relating to living organisms. A sub-discipline of both chemistry and biology, biochemistry may be divided into three fields: structural biology, enzymology and ...
, denaturation is a process in which
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, res ...
s 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
Organic may refer to:
* Organic, of or relating to an organism, a living entity
* Organic, of or relating to an anatomical organ
Chemistry
* Organic matter, matter that has come from a once-living organism, is capable of decay or is the product ...
solvent (e.g.,
alcohol or
chloroform), agitation and radiation or
heat
In thermodynamics, heat is defined as the form of energy crossing the boundary of a thermodynamic system by virtue of a temperature difference across the boundary. A thermodynamic system does not ''contain'' heat. Nevertheless, the term is ...
. 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
In chemistry, hydrophobicity is the physical property of a molecule that is seemingly repelled from a mass of water (known as a hydrophobe). In contrast, hydrophiles are attracted to water.
Hydrophobic molecules tend to be nonpolar and, ...
groups. The loss of solubility as a result of denaturation is called
''coagulation''. Denatured proteins lose their 3D structure and therefore cannot function.
Protein folding
Protein folding is the physical process by which a protein chain is translated to its native three-dimensional structure, typically a "folded" conformation by which the protein becomes biologically functional. Via an expeditious and reproduc ...
is key to whether a
globular
A globular cluster is a spheroidal conglomeration of stars. Globular clusters are bound together by gravity, with a higher concentration of stars towards their centers. They can contain anywhere from tens of thousands to many millions of membe ...
or
membrane protein can do its job correctly; it must be folded into the right shape to function. However,
hydrogen bonds, which play a big part in folding, are rather weak and thus easily affected by heat, acidity, varying salt concentrations, and other stressors which can denature the protein. This is one reason why
homeostasis
In biology, homeostasis ( British also homoeostasis) (/hɒmɪə(ʊ)ˈsteɪsɪs/) is the state of steady internal, physical, and chemical conditions maintained by living systems. This is the condition of optimal functioning for the organism and ...
is
physiologically
Physiology (; ) is the scientific study of functions and mechanisms in a living system. As a sub-discipline of biology, physiology focuses on how organisms, organ systems, individual organs, cells, and biomolecules carry out the chemical a ...
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 consumption.
Common examples
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
Acetone (2-propanone or dimethyl ketone), is an organic compound with the formula . It is the simplest and smallest ketone (). It is a colorless, highly volatile and flammable liquid with a characteristic pungent odour.
Acetone is miscibl ...
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.
Protein denaturation
Denatured proteins can exhibit a wide range of characteristics, from loss of
solubility
In chemistry, solubility is the ability of a substance, the solute, to form a solution with another substance, the solvent. Insolubility is the opposite property, the inability of the solute to form such a solution.
The extent of the solubi ...
to
protein aggregation.
Background
Protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, res ...
s or
polypeptides are polymers of
amino acid
Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha ...
s. 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
Translation is the communication of the meaning of a source-language text by means of an equivalent target-language text. The English language draws a terminological distinction (which does not exist in every language) between ''transla ...
. The newly created protein strand then undergoes
posttranslational modification
Post-translational modification (PTM) is the covalent and generally enzymatic modification of proteins following protein biosynthesis. This process occurs in the endoplasmic reticulum and the golgi apparatus. Proteins are synthesized by ribos ...
, in which additional
atom
Every atom is composed of a nucleus and one or more electrons bound to the nucleus. The nucleus is made of one or more protons and a number of neutrons. Only the most common variety of hydrogen has no neutrons.
Every solid, liquid, gas, a ...
s or
molecule
A molecule is a group of two or more atoms held together by attractive forces known as chemical bonds; depending on context, the term may or may not include ions which satisfy this criterion. In quantum physics, organic chemistry, and b ...
s are added, for example
copper
Copper is a chemical element with the symbol Cu (from la, cuprum) and atomic number 29. It is a soft, malleable, and ductile metal with very high thermal and electrical conductivity. A freshly exposed surface of pure copper has a pink ...
,
zinc
Zinc is a chemical element with the symbol Zn and atomic number 30. Zinc is a slightly brittle metal at room temperature and has a shiny-greyish appearance when oxidation is removed. It is the first element in group 12 (IIB) of the periodi ...
, or
iron
Iron () is a chemical element with symbol Fe (from la, ferrum) and atomic number 26. It is a metal that belongs to the first transition series and group 8 of the periodic table. It is, by mass, the most common element on Earth, right in ...
. 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
In chemistry, hydrophobicity is the physical property of a molecule that is seemingly repelled from a mass of water (known as a hydrophobe). In contrast, hydrophiles are attracted to water.
Hydrophobic molecules tend to be nonpolar and, ...
elements of the protein are buried deep inside the structure and
hydrophilic
A hydrophile is a molecule or other molecular entity that is attracted to water molecules and tends to be dissolved by water.Liddell, H.G. & Scott, R. (1940). ''A Greek-English Lexicon'' Oxford: Clarendon Press.
In contrast, hydrophobes are n ...
elements end up on the outside. The final shape of a protein determines how it interacts with its environment.
Protein folding consists of a balance between a substantial amount of weak intra-molecular interactions within a protein (Hydrophobic, electrostatic, and Van Der Waals Interactions) and protein-solvent interactions.
As a result, this process is heavily reliant on environmental state that the protein resides in.
These environmental conditions include, and are not limited to,
temperature
Temperature is a physical quantity that expresses quantitatively the perceptions of hotness and coldness. Temperature is measured with a thermometer.
Thermometers are calibrated in various temperature scales that historically have relied o ...
,
salinity,
pressure
Pressure (symbol: ''p'' or ''P'') is the force applied perpendicular to the surface of an object per unit area over which that force is distributed. Gauge pressure (also spelled ''gage'' pressure)The preferred spelling varies by country a ...
, and the solvents that happen to be involved.
Consequently, any exposure to extreme stresses (e.g. heat or radiation, high inorganic salt concentrations, strong acids and bases) can disrupt a protein's interaction and inevitably lead to denaturation.
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 disrupted.
* Tertiary structure denaturation involves the disruption of:
**
Covalent
A covalent bond is a chemical bond that involves the sharing of electrons to form electron pairs between atoms. These electron pairs are known as shared pairs or bonding pairs. The stable balance of attractive and repulsive forces between atom ...
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
A solvent (s) (from the Latin '' solvō'', "loosen, untie, solve") is a substance that dissolves a solute, resulting in a solution. A solvent is usually a liquid but can also be a solid, a gas, or a supercritical fluid. Water is a solvent for ...
)
**
Van der Waals (induced dipole) interactions between nonpolar amino acid side-chains.
* 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,
enzyme
Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products ...
s lose their
activity, because the substrates can no longer bind to the
active site, and because amino acid residues involved in stabilizing substrates'
transition state
In chemistry, the transition state of a chemical reaction is a particular configuration along the reaction coordinate. It is defined as the state corresponding to the highest potential energy along this reaction coordinate. It is often marked ...
s 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.
Loss of activity due to heavy metals and metalloids
By targeting proteins, heavy metals have been known to disrupt the function and activity carried out by proteins.
It is important to note that heavy metals fall into categories consisting of transition metals as well as a select amount of metalloid.
These metals, when interacting with native, folded proteins, tend to play a role in obstructing their biological activity.
This interference can be carried out in a different number of ways. These heavy metals can form a complex with the functional side chain groups present in a protein or form bonds to free thiols.
Heavy metals also play a role in oxidizing amino acid side chains present in protein.
Along with this, when interacting with metalloproteins, heavy metals can dislocate and replace key metal ions.
As a result, heavy metals can interfere with folded proteins, which can strongly deter protein stability and activity.
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".
Denaturation can also be irreversible. This irreversibility is typically a kinetic, not thermodynamic irreversibility, as a folded protein generally has lower free energy than when it is unfolded. 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.
Protein denaturation due to pH
Denaturation can also be caused by changes in the pH which can affect the chemistry of the amino acids and their residues. The ionizable groups in amino acids are able to become ionized when changes in pH occur. A pH change to more acidic or more basic conditions can induce unfolding.
Acid-induced unfolding often occurs between pH 2 and 5, base-induced unfolding usually requires pH 10 or higher.
Nucleic acid denaturation
Nucleic acids (including
RNA and
DNA) are
nucleotide
Nucleotides are organic molecules consisting of a nucleoside and a phosphate. They serve as monomeric units of the nucleic acid polymers – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both of which are essential biomolecu ...
polymers synthesized by
polymerase enzymes during either
transcription or
DNA replication
In molecular biology, DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule. DNA replication occurs in all living organisms acting as the most essential part for biological inheritan ...
. 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 structures. 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 strands. 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.
Biologically-induced denaturation
The
non-covalent interactions between
antiparallel strands in DNA can be broken in order to "open" the
double helix when biologically important mechanisms such as DNA replication, transcription,
DNA repair
DNA repair is a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. In human cells, both normal metabolic activities and environmental factors such as radiation can cause DNA d ...
or protein binding are set to occur.
The area of partially separated 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
Thermodynamics is a branch of physics that deals with heat, work, and temperature, and their relation to energy, entropy, and the physical properties of matter and radiation. The behavior of these quantities is governed by the four laws ...
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
Temperature is a physical quantity that expresses quantitatively the perceptions of hotness and coldness. Temperature is measured with a thermometer.
Thermometers are calibrated in various temperature scales that historically have relied o ...
. 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
Stiffness is the extent to which an object resists deformation in response to an applied force.
The complementary concept is flexibility or pliability: the more flexible an object is, the less stiff it is.
Calculations
The stiffness, k, of a ...
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 of DNA replication and transcription.
Currently, biophysical and biochemical research studies are being performed to more fully elucidate the thermodynamic details of the denaturation bubble.
Denaturation due to chemical agents
With
polymerase chain reaction
The polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete or partial) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of it) ...
(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
Propylene glycol ( IUPAC name: propane-1,2-diol) is a viscous, colorless liquid, which is nearly odorless but possesses a faintly sweet taste. Its chemical formula is CH3CH(OH)CH2OH.
Containing two alcohol groups, it is classed as a diol. It ...
, and
urea.
These chemical denaturing agents lower the melting temperature (T
m) 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, chemically denaturing 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
sonication, 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 example, DNA strands denatured with
alkaline agents such as
NaOH renature as soon as
phosphate buffer is added.
Denaturation due to air
Small,
electronegative
Electronegativity, symbolized as , is the tendency for an atom of a given chemical element to attract shared electrons (or electron density) when forming a chemical bond. An atom's electronegativity is affected by both its atomic number and the ...
molecules such as
nitrogen
Nitrogen is the chemical element with the symbol N and atomic number 7. Nitrogen is a nonmetal and the lightest member of group 15 of the periodic table, often called the pnictogens. It is a common element in the universe, estimated at se ...
and
oxygen
Oxygen is the chemical element with the symbol O and atomic number 8. It is a member of the chalcogen group in the periodic table, a highly reactive nonmetal, and an oxidizing agent that readily forms oxides with most elements ...
, 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.
Applications
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:
*
PCR
*
Southern blot
*
Northern blot
The northern blot, or RNA blot,Gilbert, S. F. (2000) Developmental Biology, 6th Ed. Sunderland MA, Sinauer Associates. is a technique used in molecular biology research to study gene expression by detection of RNA (or isolated mRNA) in a sampl ...
*
DNA Sequencing
Denaturants
Protein denaturants
Acids
Acidic protein denaturants include:
*
Acetic acid
*
Trichloroacetic acid 12% in water
*
Sulfosalicylic acid
Bases
Bases work similarly to acids in denaturation. They include:
*
Sodium bicarbonate
Solvents
Most organic
solvent
A solvent (s) (from the Latin '' solvō'', "loosen, untie, solve") is a substance that dissolves a solute, resulting in a solution. A solvent is usually a liquid but can also be a solid, a gas, or a supercritical fluid. Water is a solvent for ...
s are denaturing, including:
*
Ethanol
Ethanol (abbr. EtOH; also called ethyl alcohol, grain alcohol, drinking alcohol, or simply alcohol) is an organic compound. It is an alcohol with the chemical formula . Its formula can be also written as or (an ethyl group linked to a ...
Cross-linking reagents
Cross-linking agents for proteins include:
*
Formaldehyde
Formaldehyde ( , ) ( systematic name methanal) is a naturally occurring organic compound with the formula and structure . The pure compound is a pungent, colourless gas that polymerises spontaneously into paraformaldehyde (refer to section ...
*
Glutaraldehyde
Chaotropic agents
Chaotropic agents include:
*
Urea 6–8
mol/L
*
Guanidinium chloride 6 mol/L
*
Lithium perchlorate
Lithium perchlorate is the inorganic compound with the formula LiClO4. This white or colourless crystalline salt is noteworthy for its high solubility in many solvents. It exists both in anhydrous form and as a trihydrate.
Applications Inorgan ...
4.5 mol/L
*
Sodium dodecyl sulfate
Disulfide bond reducers
Agents that break
disulfide bonds by reduction include:
*
2-Mercaptoethanol
*
Dithiothreitol
*
TCEP (tris(2-carboxyethyl)phosphine)
Chemically reactive agents
Agents such as hydrogen peroxide, elemental chlorine, hypochlorous acid (chlorine water), bromine, bromine water, iodine, nitric and oxidising acids, and ozone react with sensitive moieties such as sulfide/thiol, activated aromatic rings (phenylalanine) in effect damage the protein and render it useless.
Other
* Mechanical agitation
*
Picric acid
* Radiation
* Temperature
Nucleic acid denaturants
Chemical
Acidic nucleic acid denaturants include:
*
Acetic acid
* HCl
* Nitric acid
Basic nucleic acid denaturants include:
* NaOH
Other nucleic acid denaturants include:
*
DMSO
*
Formamide
*
Guanidine
*
Sodium salicylate
*
Propylene glycol
Propylene glycol ( IUPAC name: propane-1,2-diol) is a viscous, colorless liquid, which is nearly odorless but possesses a faintly sweet taste. Its chemical formula is CH3CH(OH)CH2OH.
Containing two alcohol groups, it is classed as a diol. It ...
*
Urea
Physical
* Thermal denaturation
* Beads mill
* Probe
sonication
*
Radiation
See also
*
Denatured alcohol
*
Equilibrium unfolding
In biochemistry, equilibrium unfolding is the process of unfolding a protein or RNA molecule by gradually changing its environment, such as by changing the temperature or pressure, pH, adding chemical denaturants, or applying force as with an ...
*
Fixation (histology)
*
Protein folding
Protein folding is the physical process by which a protein chain is translated to its native three-dimensional structure, typically a "folded" conformation by which the protein becomes biologically functional. Via an expeditious and reproduc ...
*
Random coil
References
External links
McGraw-Hill Online Learning Center — Animation: Protein Denaturation
{{DEFAULTSORT:Denaturation (Biochemistry)
Protein structure
Nucleic acids