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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 an ...
, denaturation is a process in which proteins or
nucleic acid Nucleic acids are biopolymers, macromolecules, essential to all known forms of life. They are composed of nucleotides, which are the monomers made of three components: a 5-carbon sugar, a phosphate group and a nitrogenous base. The two main cl ...
s lose the quaternary structure,
tertiary structure Protein tertiary structure is the three dimensional shape of a protein. The tertiary structure will have a single polypeptide chain "backbone" with one or more protein secondary structures, the protein domains. Amino acid side chains may inte ...
, and
secondary structure Protein secondary structure is the three dimensional form of ''local segments'' of proteins. The two most common secondary structural elements are alpha helices and beta sheets, though beta turns and omega loops occur as well. Secondary structur ...
which is present in their
native state In biochemistry, the native state of a protein or nucleic acid is its properly folded and/or assembled form, which is operative and functional. The native state of a biomolecule may possess all four levels of biomolecular structure, with the ...
, by application of some external stress or compound such as a strong
acid In computer science, ACID ( atomicity, consistency, isolation, durability) is a set of properties of database transactions intended to guarantee data validity despite errors, power failures, and other mishaps. In the context of databases, a s ...
or base, a concentrated
inorganic In chemistry, an inorganic compound is typically a chemical compound that lacks carbon–hydrogen bonds, that is, a compound that is not an organic compound. The study of inorganic compounds is a subfield of chemistry known as ''inorganic chemistr ...
salt, an organic solvent (e.g.,
alcohol Alcohol most commonly refers to: * Alcohol (chemistry), an organic compound in which a hydroxyl group is bound to a carbon atom * Alcohol (drug), an intoxicant found in alcoholic drinks Alcohol may also refer to: Chemicals * Ethanol, one of se ...
or
chloroform Chloroform, or trichloromethane, is an organic compound with formula C H Cl3 and a common organic solvent. It is a colorless, strong-smelling, dense liquid produced on a large scale as a precursor to PTFE. It is also a precursor to various ref ...
), 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 al ...
. 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 In biochemistry, a conformational change is a change in the shape of a macromolecule, often induced by environmental factors. A macromolecule is usually flexible and dynamic. Its shape can change in response to changes in its environment or othe ...
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, t ...
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 or
membrane protein Membrane proteins are common proteins that are part of, or interact with, biological membranes. Membrane proteins fall into several broad categories depending on their location. Integral membrane proteins are a permanent part of a cell membrane ...
can do its job correctly; it must be folded into the right shape to function. However,
hydrogen bond In chemistry, a hydrogen bond (or H-bond) is a primarily electrostatic force of attraction between a hydrogen (H) atom which is covalently bound to a more electronegative "donor" atom or group (Dn), and another electronegative atom bearing ...
s, 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 i ...
is physiologically necessary in many life forms. This concept is unrelated to
denatured alcohol Denatured alcohol (also called methylated spirits in Australia, Canada, Ireland, New Zealand, South Africa, and the United Kingdom; wood spirit; and denatured rectified spirit) is ethanol that has additives to make it poisonous, bad-tasting, foul ...
, 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 miscible ...
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 Ceviche () is a Peruvian dish typically made from fresh raw fish cured in fresh citrus juices, most commonly lime or lemon. It is also spiced with '' ají'', chili peppers or other seasonings, and julienned red onions, salt, and cilantro are ...
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 solubil ...
to
protein aggregation In molecular biology, protein aggregation is a phenomenon in which intrinsically-disordered or mis-folded proteins aggregate (i.e., accumulate and clump together) either intra- or extracellularly. Protein aggregates have been implicated in a wi ...
.


Background

Proteins or
polypeptide Peptides (, ) are short chains of amino acids linked by peptide bonds. Long chains of amino acids are called proteins. Chains of fewer than twenty amino acids are called oligopeptides, and include dipeptides, tripeptides, and tetrapeptides. A ...
s 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 a ...
s. A protein is created by
ribosome Ribosomes ( ) are macromolecular machines, found within all cells, that perform biological protein synthesis (mRNA translation). Ribosomes link amino acids together in the order specified by the codons of messenger RNA (mRNA) molecules to fo ...
s that "read" RNA that is encoded by
codon The genetic code is the set of rules used by living cells to translate information encoded within genetic material ( DNA or RNA sequences of nucleotide triplets, or codons) into proteins. Translation is accomplished by the ribosome, which links p ...
s 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 ''transl ...
. 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, ...
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 bioch ...
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 pinkish- ...
,
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 periodic t ...
, 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 fro ...
. Once this post-translational modification process has been completed, the protein begins to fold (sometimes spontaneously and sometimes with
enzymatic 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. ...
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, t ...
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 no ...
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 Salinity () is the saltiness or amount of salt dissolved in a body of water, called saline water (see also soil salinity). It is usually measured in g/L or g/kg (grams of salt per liter/kilogram of water; the latter is dimensionless and equal t ...
,
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 and ...
, 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 bond In organic chemistry, a peptide bond is an amide type of covalent chemical bond linking two consecutive alpha-amino acids from C1 (carbon number one) of one alpha-amino acid and N2 (nitrogen number two) of another, along a peptide or protein ch ...
s 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 In molecular biology, an intrinsically disordered protein (IDP) is a protein that lacks a fixed or ordered three-dimensional structure, typically in the absence of its macromolecular interaction partners, such as other proteins or RNA. IDPs ra ...
, which are unfolded in their
native state In biochemistry, the native state of a protein or nucleic acid is its properly folded and/or assembled form, which is operative and functional. The native state of a biomolecule may possess all four levels of biomolecular structure, with the ...
, 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 Cysteine (symbol Cys or C; ) is a semiessential proteinogenic amino acid with the formula . The thiol side chain in cysteine often participates in enzymatic reactions as a nucleophile. When present as a deprotonated catalytic residue, sometim ...
groups) ** Non-covalent
dipole In physics, a dipole () is an electromagnetic phenomenon which occurs in two ways: *An electric dipole deals with the separation of the positive and negative electric charges found in any electromagnetic system. A simple example of this system ...
-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 In polymer chemistry, a random coil is a conformation of polymers where the monomer subunits are oriented randomly while still being bonded to adjacent units. It is not one specific shape, but a statistical distribution of shapes for all the ch ...
configuration. *
Primary structure Protein primary structure is the linear sequence of amino acids in a peptide or protein. By convention, the primary structure of a protein is reported starting from the amino-terminal (N) end to the carboxyl-terminal (C) end. Protein biosynthes ...
, 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 In biology and biochemistry, the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. The active site consists of amino acid residues that form temporary bonds with the substrate (binding 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 wi ...
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 Dual-polarization interferometry (DPI) is an analytical technique that probes molecular layers adsorbed to the surface of a waveguide using the evanescent wave of a laser beam. It is used to measure the conformational change in proteins, or oth ...
, 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 acid Nucleic acids are biopolymers, macromolecules, essential to all known forms of life. They are composed of nucleotides, which are the monomers made of three components: a 5-carbon sugar, a phosphate group and a nitrogenous base. The two main cl ...
s (including
RNA Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydra ...
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 biomolecule ...
polymers synthesized by polymerase enzymes during either
transcription Transcription refers to the process of converting sounds (voice, music etc.) into letters or musical notes, or producing a copy of something in another medium, including: Genetics * Transcription (biology), the copying of DNA into RNA, the fir ...
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 inheritance ...
. Following 5'-3' synthesis of the backbone, individual nitrogenous bases are capable of interacting with one another via
hydrogen bond In chemistry, a hydrogen bond (or H-bond) is a primarily electrostatic force of attraction between a hydrogen (H) atom which is covalently bound to a more electronegative "donor" atom or group (Dn), and another electronegative atom bearing ...
ing, 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 In chemistry, a non-covalent interaction differs from a covalent bond in that it does not involve the sharing of electrons, but rather involves more dispersed variations of electromagnetic interactions between molecules or within a molecule. The ...
between antiparallel strands in DNA can be broken in order to "open" the
double helix A double is a look-alike or doppelgänger; one person or being that resembles another. Double, The Double or Dubble may also refer to: Film and television * Double (filmmaking), someone who substitutes for the credited actor of a character * ...
when biologically important mechanisms such as DNA replication, transcription,
DNA repair DNA repair is a collection of processes by which a cell (biology), cell identifies and corrects damage to the DNA molecules that encode its genome. In human cells, both normal metabolism, metabolic activities and environmental factors such as r ...
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 o ...
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 DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine. The ...
, 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 bo ...
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 Formamide is an amide derived from formic acid. It is a colorless liquid which is miscible with water and has an ammonia-like odor. It is chemical feedstock for the manufacture of sulfa drugs and other pharmaceuticals, herbicides and pesticides ...
,
guanidine Guanidine is the compound with the formula HNC(NH2)2. It is a colourless solid that dissolves in polar solvents. It is a strong base that is used in the production of plastics and explosives. It is found in urine predominantly in patients experie ...
,
sodium salicylate Sodium salicylate is a sodium salt of salicylic acid. It can be prepared from sodium phenolate and carbon dioxide under higher temperature and pressure. Historically, it has been synthesized by refluxing methyl salicylate ( wintergreen oil) wit ...
,
dimethyl sulfoxide Dimethyl sulfoxide (DMSO) is an organosulfur compound with the formula ( CH3)2. This colorless liquid is the sulfoxide most widely used commercially. It is an important polar aprotic solvent that dissolves both polar and nonpolar compounds ...
(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 is ...
, and
urea Urea, also known as carbamide, is an organic compound with chemical formula . This amide has two amino groups (–) joined by a carbonyl functional group (–C(=O)–). It is thus the simplest amide of carbamic acid. Urea serves an important ...
. These chemical denaturing agents lower the melting temperature (Tm) by competing for hydrogen bond donors and acceptors with pre-existing
nitrogenous base Nucleobases, also known as ''nitrogenous bases'' or often simply ''bases'', are nitrogen-containing biological compounds that form nucleosides, which, in turn, are components of nucleotides, with all of these monomers constituting the basic b ...
pairs. Some agents are even able to induce denaturation at room temperature. For example,
alkaline In chemistry, an alkali (; from ar, القلوي, al-qaly, lit=ashes of the saltwort) is a basic, ionic salt of an alkali metal or an alkaline earth metal. An alkali can also be defined as a base that dissolves in water. A solution of a s ...
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 Electrophoresis, from Ancient Greek ἤλεκτρον (ḗlektron, "amber") and φόρησις (phórēsis, "the act of bearing"), is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric fie ...
mobility.


Chemical denaturation as an alternative

The
optical activity Optical rotation, also known as polarization rotation or circular birefringence, is the rotation of the orientation of the plane of polarization about the optical axis of linearly polarized light as it travels through certain materials. Circula ...
(absorption and scattering of light) and hydrodynamic properties ( translational diffusion,
sedimentation coefficient The sedimentation coefficient () of a particle characterizes its sedimentation during centrifugation. It is defined as the ratio of a particle's sedimentation velocity to the applied acceleration causing the sedimentation. : s = \frac The sedim ...
s, and rotational correlation times) of
formamide Formamide is an amide derived from formic acid. It is a colorless liquid which is miscible with water and has an ammonia-like odor. It is chemical feedstock for the manufacture of sulfa drugs and other pharmaceuticals, herbicides and pesticides ...
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 A sonicator at the Weizmann Institute of Science during sonicationSonication is the act of applying sound energy to agitate particles in a sample, for various purposes such as the extraction of multiple compounds from plants, microalgae and seawe ...
, 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 Sodium hydroxide, also known as lye and caustic soda, is an inorganic compound with the formula NaOH. It is a white solid ionic compound consisting of sodium cations and hydroxide anions . Sodium hydroxide is a highly caustic base and alkali t ...
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 d ...
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 sevent ...
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 as ...
, which are the primary gases in air, significantly impact the ability of surrounding molecules to participate in
hydrogen bond In chemistry, a hydrogen bond (or H-bond) is a primarily electrostatic force of attraction between a hydrogen (H) atom which is covalently bound to a more electronegative "donor" atom or group (Dn), and another electronegative atom bearing ...
ing. 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 A Southern blot is a method used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detecti ...
*
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 sample.Ke ...
*
DNA Sequencing DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine. T ...


Denaturants


Protein denaturants


Acids

Acid In computer science, ACID ( atomicity, consistency, isolation, durability) is a set of properties of database transactions intended to guarantee data validity despite errors, power failures, and other mishaps. In the context of databases, a s ...
ic protein denaturants include: *
Acetic acid Acetic acid , systematically named ethanoic acid , is an acidic, colourless liquid and organic compound with the chemical formula (also written as , , or ). Vinegar is at least 4% acetic acid by volume, making acetic acid the main compon ...
* Trichloroacetic acid 12% in water *
Sulfosalicylic acid Sulfosalicylic acid is used in urine tests to determine urine protein content. The chemical causes the precipitation of dissolved proteins, which is measured from the degree of turbidity.Bases work similarly to acids in denaturation. They include: *
Sodium bicarbonate Sodium bicarbonate ( IUPAC name: sodium hydrogencarbonate), commonly known as baking soda or bicarbonate of soda, is a chemical compound with the formula NaHCO3. It is a salt composed of a sodium cation ( Na+) and a bicarbonate anion ( HCO3− ...


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-link In chemistry and biology a cross-link is a bond or a short sequence of bonds that links one polymer chain to another. These links may take the form of covalent bonds or ionic bonds and the polymers can be either synthetic polymers or natural ...
ing 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 Glutaraldehyde is an organic compound with the formula . The molecule consists of a five carbon chain doubly terminated with formyl (CHO) groups. It is usually used as a solution in water, and such solutions exists as a collection of hydrates, c ...


Chaotropic agents

Chaotropic agent A chaotropic agent is a molecule in water solution that can disrupt the hydrogen bonding network between water molecules (i.e. exerts chaotropic activity). This has an effect on the stability of the native state of other molecules in the solution, ...
s include: *
Urea Urea, also known as carbamide, is an organic compound with chemical formula . This amide has two amino groups (–) joined by a carbonyl functional group (–C(=O)–). It is thus the simplest amide of carbamic acid. Urea serves an important ...
6–8  mol/L *
Guanidinium chloride Guanidinium chloride or guanidine hydrochloride, usually abbreviated GdmCl and sometimes GdnHCl or GuHCl, is the hydrochloride salt of guanidine. Structure Guanidinium chloride crystallizes in orthorhombic space group ''Pbca''. The crystal stru ...
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 bond In biochemistry, a disulfide (or disulphide in British English) refers to a functional group with the structure . The linkage is also called an SS-bond or sometimes a disulfide bridge and is usually derived by the coupling of two thiol groups. In ...
s by reduction include: * 2-Mercaptoethanol *
Dithiothreitol Dithiothreitol (DTT) is the common name for a small-molecule redox reagent also known as Cleland's reagent, after W. Wallace Cleland. DTT's formula is C4H10O2S2 and the chemical structure of one of its enantiomers in its reduced form is shown on ...
* 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 Picric acid is an organic compound with the formula (O2N)3C6H2OH. Its IUPAC name is 2,4,6-trinitrophenol (TNP). The name "picric" comes from el, πικρός (''pikros''), meaning "bitter", due to its bitter taste. It is one of the most acidic ...
* Radiation * Temperature


Nucleic acid denaturants


Chemical

Acid In computer science, ACID ( atomicity, consistency, isolation, durability) is a set of properties of database transactions intended to guarantee data validity despite errors, power failures, and other mishaps. In the context of databases, a s ...
ic nucleic acid denaturants include: *
Acetic acid Acetic acid , systematically named ethanoic acid , is an acidic, colourless liquid and organic compound with the chemical formula (also written as , , or ). Vinegar is at least 4% acetic acid by volume, making acetic acid the main compon ...
* HCl * Nitric acid
Basic BASIC (Beginners' All-purpose Symbolic Instruction Code) is a family of general-purpose, high-level programming languages designed for ease of use. The original version was created by John G. Kemeny and Thomas E. Kurtz at Dartmouth College ...
nucleic acid denaturants include: * NaOH Other nucleic acid denaturants include: *
DMSO Dimethyl sulfoxide (DMSO) is an organosulfur compound with the formula ( CH3)2. This colorless liquid is the sulfoxide most widely used commercially. It is an important polar aprotic solvent that dissolves both polar and nonpolar compounds an ...
*
Formamide Formamide is an amide derived from formic acid. It is a colorless liquid which is miscible with water and has an ammonia-like odor. It is chemical feedstock for the manufacture of sulfa drugs and other pharmaceuticals, herbicides and pesticides ...
*
Guanidine Guanidine is the compound with the formula HNC(NH2)2. It is a colourless solid that dissolves in polar solvents. It is a strong base that is used in the production of plastics and explosives. It is found in urine predominantly in patients experie ...
*
Sodium salicylate Sodium salicylate is a sodium salt of salicylic acid. It can be prepared from sodium phenolate and carbon dioxide under higher temperature and pressure. Historically, it has been synthesized by refluxing methyl salicylate ( wintergreen oil) wit ...
*
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 is ...
*
Urea Urea, also known as carbamide, is an organic compound with chemical formula . This amide has two amino groups (–) joined by a carbonyl functional group (–C(=O)–). It is thus the simplest amide of carbamic acid. Urea serves an important ...


Physical

* Thermal denaturation * Beads mill * Probe
sonication A sonicator at the Weizmann Institute of Science during sonicationSonication is the act of applying sound energy to agitate particles in a sample, for various purposes such as the extraction of multiple compounds from plants, microalgae and seawe ...
*
Radiation In physics, radiation is the emission or transmission of energy in the form of waves or particles through space or through a material medium. This includes: * ''electromagnetic radiation'', such as radio waves, microwaves, infrared, vi ...


See also

*
Denatured alcohol Denatured alcohol (also called methylated spirits in Australia, Canada, Ireland, New Zealand, South Africa, and the United Kingdom; wood spirit; and denatured rectified spirit) is ethanol that has additives to make it poisonous, bad-tasting, foul ...
* Equilibrium unfolding *
Fixation (histology) In the fields of histology, pathology, and cell biology, fixation is the preservation of biological tissues from decay due to autolysis or putrefaction. It terminates any ongoing biochemical reactions and may also increase the treated tissues' me ...
*
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 In polymer chemistry, a random coil is a conformation of polymers where the monomer subunits are oriented randomly while still being bonded to adjacent units. It is not one specific shape, but a statistical distribution of shapes for all the ch ...


References


External links


McGraw-Hill Online Learning Center — Animation: Protein Denaturation
{{DEFAULTSORT:Denaturation (Biochemistry) Protein structure Nucleic acids