Editing Denaturation (biochemistry)

{{Short description|Loss of structure in proteins and nucleic acids due to external stress}}
[[File:Q10 graphs.svg|thumb|400px|The effects of temperature on [[enzyme]] activity.<br> '''Top''': increasing temperature increases the [[rate of reaction]] ([[Q10 (temperature coefficient)|Q10 coefficient]]). <br>'''Middle''': the fraction of folded and functional enzyme decreases above its denaturation temperature.<br> '''Bottom''': consequently, an enzyme's optimal rate of reaction is at an intermediate temperature.]]

{{Quote box
 |width = 30em
 |align = right
 |title = [[International Union of Pure and Applied Chemistry|IUPAC]] definition
 |quote = Process of partial or total alteration of the native secondary, and/or tertiary, and/or quaternary structures of proteins or nucleic acids resulting in a loss of ''bioactivity''.

''Note 1'': Modified from the definition given in ref.<ref>{{cite book|title=Compendium of Chemical Terminology: IUPAC Recommendations (the "Gold Book")|year=1997|publisher=[[Blackwell Science]]|isbn=978-0865426849|editor1=Alan D. MacNaught |editor2=Andrew R. Wilkinson }}</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 ability to alter or speed up a chemical reaction when it is denaturized.<ref>{{cite journal|title=Terminology for biorelated polymers and applications (IUPAC Recommendations 2012)|journal=[[Pure and Applied Chemistry]]|year=2012|volume=84|issue=2|pages=377–410|doi=10.1351/PAC-REC-10-12-04|url=http://pac.iupac.org/publications/pac/pdf/2012/pdf/8402x0377.pdf |archive-url=https://web.archive.org/web/20130927010712/http://pac.iupac.org/publications/pac/pdf/2012/pdf/8402x0377.pdf |archive-date=2013-09-27 |url-status=live | last1 = Vert | first1 = Michel|s2cid=98107080}}</ref>
}}

In [[biochemistry]], '''denaturation''' is a process in which [[protein]]s or [[nucleic acid]]s lose [[Protein structure|folded structure]] present in their [[native state]] due to various factors, including application of some external stress or compound, such as a strong [[acid]] or [[base (chemistry)|base]], a concentrated [[inorganic]] salt, an [[organic compound|organic]] solvent (e.g., [[Alcohol (chemistry)|alcohol]] or [[chloroform]]), agitation, radiation, or [[heat]].<ref>{{cite book|title=Mosby's Medical Dictionary|url=http://medical-dictionary.thefreedictionary.com/Denaturation+%28biochemistry%29|access-date=1 October 2013|edition=8th |year=2009|publisher=[[Elsevier]]}}</ref> If proteins in a living [[Cell (biology)|cell]] are denatured, this results in disruption of cell activity and possibly [[Apoptosis|cell death]]. Protein denaturation is also a consequence of cell death.<ref>{{Cite journal|last1=Samson|first1=Andre L.|last2=Ho|first2=Bosco|last3=Au|first3=Amanda E.|last4=Schoenwaelder|first4=Simone M.|last5=Smyth|first5=Mark J.|last6=Bottomley|first6=Stephen P.|last7=Kleifeld|first7=Oded|last8=Medcalf|first8=Robert L.|date=2016-11-01|title=Physicochemical properties that control protein aggregation also determine whether a protein is retained or released from necrotic cells|journal=Open Biology|volume=6|issue=11|doi=10.1098/rsob.160098|url=https://zenodo.org/record/1065526|issn=2046-2441|pmid=27810968|page=160098|pmc=5133435}}</ref><ref>{{Cite journal|last1=Samson|first1=Andre L.|last2=Knaupp|first2=Anja S.|last3=Sashindranath|first3=Maithili|last4=Borg|first4=Rachael J.|last5=Au|first5=Amanda E.-L.|last6=Cops|first6=Elisa J.|last7=Saunders|first7=Helen M.|last8=Cody|first8=Stephen H.|last9=McLean|first9=Catriona A.|date=2012-10-25|title=Nucleocytoplasmic coagulation: an injury-induced aggregation event that disulfide crosslinks proteins and facilitates their removal by plasmin|journal=Cell Reports|volume=2|issue=4|pages=889–901|doi=10.1016/j.celrep.2012.08.026|issn=2211-1247|pmid=23041318|doi-access=free}}</ref> Denatured proteins can exhibit a wide range of characteristics, from [[conformational change]] and loss of [[solubility]] or dissociation of [[Cofactor (biochemistry)|cofactors]] to [[Protein aggregation|aggregation]] due to the exposure of [[hydrophobic]] groups. The loss of solubility as a result of denaturation is called [[Precipitation (chemistry)|''coagulation'']].<ref>{{Cite web |date=2019-07-15 |title=2.5: Denaturation of proteins |url=https://chem.libretexts.org/Courses/University_of_Arkansas_Little_Rock/CHEM_4320_5320%3A_Biochemistry_1/02%3A__Protein_Structure/2.5%3A_Denaturation_of_proteins |access-date=2022-04-25 |website=Chemistry LibreTexts |language=en}}</ref> Denatured proteins, e.g., [[metalloenzyme]]s, lose their [[Protein structure|3D structure]] or metal cofactor, and therefore, cannot function.

Proper [[protein folding]] is key to whether a [[globular protein|globular]] or [[membrane protein]] can do its job correctly; it must be folded into the native shape to function. However, [[hydrogen bond]]s and cofactor-protein binding, which play a crucial role in folding, are rather weak, and thus, easily affected by heat, acidity, varying salt concentrations, [[Chelation|chelating agents]], and other stressors which can denature the protein. This is one reason why cellular [[homeostasis]] is [[physiology|physiologically]] necessary in most [[life|life forms]].

== <span id="Cooking"></span> Common examples ==
[[File:Protein Denaturation.png|thumb|(Top) The protein ''[[albumin]]'' in the egg white undergoes denaturation and loss of solubility when the egg is cooked. (Bottom) Paperclips provide a visual analogy to help with the conceptualization of the denaturation process.]]

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 [[ovalbumin|egg albumins]] in water. Fresh from the eggs, egg whites are transparent and liquid. Cooking the [[Thermostability|thermally unstable]] whites turns them opaque, forming an interconnected solid mass.<ref>{{Cite journal|last1=Mine|first1=Yoshinori|last2=Noutomi|first2=Tatsushi|last3=Haga|first3=Noriyuki|title=Thermally induced changes in egg white proteins|journal=Journal of Agricultural and Food Chemistry|language=en|volume=38|issue=12|pages=2122–2125|doi=10.1021/jf00102a004|year=1990|bibcode=1990JAFC...38.2122M }}</ref> 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.<ref>[https://archive.today/20081012034157/http://www.timesonline.co.uk/tol/life_and_style/food_and_drink/article4220254.ece "Ceviche: the new sushi,"] The Times.</ref>

== Protein denaturation ==
{{also|Equilibrium unfolding}}

Denatured proteins can exhibit a wide range of characteristics, from loss of [[solubility]] to [[protein aggregation]].

[[File:Levels of structural organization of a protein.svg|thumb| Functional proteins have four levels of structural organization:{{ordered list
 | list_style=margin-left:0; list-style-position:inside;
 | Primary structure: the linear structure of amino acids in the polypeptide chain
 | Secondary structure: hydrogen bonds between peptide group chains in an alpha helix or beta sheet
 | Tertiary structure: three-dimensional structure of alpha helixes and beta helixes folded
 | Quaternary structure: three-dimensional structure of multiple polypeptides and how they fit together
}}]]

[[File:Process of Denaturation.svg|thumb|Process of denaturation:{{ordered list
 | list_style=margin-left:0; list-style-position:inside;
 | Functional protein showing a quaternary structure
 | When heat is applied it alters the intramolecular bonds of the protein
 | Unfolding of the polypeptides (amino acids)
}}]]

=== Background ===
[[Protein]]s or [[polypeptide]]s are polymers of [[amino acid]]s. A protein is created by [[ribosome]]s that "read" RNA that is encoded by [[codon]]s in the gene and assemble the requisite amino acid combination from the [[DNA|genetic]] instruction, in a process known as [[translation (genetics)|translation]]. The newly created protein strand then undergoes [[posttranslational modification]], in which additional [[atom]]s or [[molecule]]s 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.

Protein folding consists of a balance between a substantial amount of weak intra-molecular interactions within a protein (Hydrophobic, [[Electrostatics|electrostatic]], and Van Der Waals Interactions) and protein-solvent interactions.<ref name=":01">{{Cite journal|last=Bondos|first=Sarah|date=2014|title=Protein folding |journal=Access Science|language=en|doi=10.1036/1097-8542.801070}}</ref> As a result, this process is heavily reliant on environmental state that the protein resides in.<ref name=":01" /> These environmental conditions include, and are not limited to, [[temperature]], [[salinity]], [[pressure]], and the solvents that happen to be involved.<ref name=":01" /> 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.<ref name="test">{{Cite journal|date=2006-04-03|title= Denaturation|url=http://link.galegroup.com/apps/doc/CV2431500175/SCIC?sid=SCIC&xid=fc5b75c9|journal=Science in Context|language=en}}</ref>

When a protein is denatured, secondary and tertiary structures are altered but the [[peptide bond]]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]], which are unfolded in their [[native state]], but still functionally active and tend to fold upon binding to their biological target.<ref>{{Cite journal|author-link=Jane Dyson|last1=Dyson|first1=H. Jane|last2=Wright|first2=Peter E.|date=2005-03-01|title=Intrinsically unstructured proteins and their functions|journal=Nature Reviews Molecular Cell Biology|language=en|volume=6|issue=3|pages=197–208|doi=10.1038/nrm1589|pmid=15738986|s2cid=18068406|issn=1471-0072}}</ref>

=== How denaturation occurs at levels of protein structure ===
{{See also|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]] interactions between amino acid [[Side chain|side-chains]] (such as [[disulfide bridge]]s between [[cysteine]] groups)
** Non-covalent [[dipole]]-dipole interactions between polar amino acid side-chains (and the surrounding [[solvent]])
** [[van der Waals force|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 helix|alpha-helices]] and [[beta sheet|beta-pleated sheets]], and adopt a [[random coil]] configuration.
* '''[[Protein primary structure|Primary structure]]''', such as the sequence of amino acids held together by covalent peptide bonds, is not disrupted by denaturation.<ref>{{citation |author=Charles Tanford |year=1968 |title=Protein denaturation |journal=Advances in Protein Chemistry |volume=23 |pages=121–282 |doi=10.1016/S0065-3233(08)60401-5 |pmid=4882248 |url=http://garfield.library.upenn.edu/classics1980/A1980JC93500001.pdf |archive-url=https://web.archive.org/web/20051110145538/http://garfield.library.upenn.edu/classics1980/A1980JC93500001.pdf |archive-date=2005-11-10 |url-status=live|isbn=9780120342235 }}</ref>

==== Loss of function ====
Most biological substrates lose their biological function when denatured. For example, [[enzyme]]s lose their [[catalysis|activity]], because the substrates can no longer bind to the [[active site]],<ref>{{citation |author=Biology Online Dictionary |title=Denaturation Definition and Examples |date=2 December 2020 |url=https://www.biology-online.org/dictionary/Denaturation }}</ref> and because amino acid residues involved in stabilizing substrates' [[transition state]]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]], [[Circular dichroism|CD]], [[Quartz crystal microbalance with dissipation monitoring|QCM-D]] and [[multi-parametric surface plasmon resonance|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.<ref name=":22">{{cite journal|last1=Tamás|first1=Markus J.|last2=Sharma|first2=Sandeep K.|last3=Ibstedt|first3=Sebastian|last4=Jacobson|first4=Therese|last5=Christen|first5=Philipp|title=Heavy Metals and Metalloids As a Cause for Protein Misfolding and Aggregation|journal=Biomolecules|date=2014-03-04|volume=4|issue=1|pages=252–267|doi=10.3390/biom4010252|pmid=24970215|pmc=4030994|doi-access=free}}</ref> Heavy metals fall into categories consisting of transition metals as well as a select amount of [[metalloid]].<ref name=":22" /> These metals, when interacting with native, folded proteins, tend to play a role in obstructing their biological activity.<ref name=":22" /> 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.<ref name=":22" /> Heavy metals also play a role in oxidizing amino acid side chains present in protein.<ref name=":22" />  Along with this, when interacting with metalloproteins, heavy metals can dislocate and replace key metal ions.<ref name=":22" /> 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'''.<ref>{{citation |author1=Campbell, N. A. |author2=Reece, J.B. |author3=Meyers, N. |author4=Urry, L. A. |author5=Cain, M.L. |author6=Wasserman, S.A. |author7=Minorsky, P.V. |author8=Jackson, R.B. |year=2009 |title=Biology |edition=8th, Australian version |place=Sydney |publisher=Pearson Education Australia}}</ref> 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 "[[Christian B. Anfinsen|Anfinsen's]] [[Anfinsen's dogma|thermodynamic hypothesis]]".<ref>{{citation |author=Anfinsen CB. |s2cid=10151090 |year=1973 |title=Principles that govern the folding of protein chains |journal=Science |volume=181 |issue=4096 |pages=223–30 |doi=10.1126/science.181.4096.223 |pmid=4124164|bibcode=1973Sci...181..223A }}</ref>

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.<ref name="Wetlaufer1988">{{cite journal|last1=Wetlaufer|first1=D.B.|title=Reversible and irreversible denaturation of proteins in chromatographic systems|journal=Makromolekulare Chemie. Macromolecular Symposia|volume=17|issue=1|year=1988|pages=17–28|issn=0258-0322|doi=10.1002/masy.19880170104}}</ref>

==== 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.<ref name=":0">{{Cite book|last=Konermann|first=Lars|title=Encyclopedia of Life Sciences |date=2012-05-15|chapter=Protein Unfolding and Denaturants|journal=eLS|language=en|location=Chichester, UK|publisher=John Wiley & Sons, Ltd|doi=10.1002/9780470015902.a0003004.pub2|isbn=978-0470016176}}</ref> Acid-induced unfolding often occurs between pH 2 and 5, base-induced unfolding usually requires pH 10 or higher.<ref name=":0" />

== Nucleic acid denaturation ==
{{main|Nucleic acid thermodynamics}}

[[Nucleic acid]]s (including [[RNA]] and [[DNA]]) are [[nucleotide]] polymers synthesized by [[Polymerase|polymerase enzymes]] during either [[transcription (genetics)|transcription]] or [[DNA replication]]. Following 5'-3' synthesis of the backbone, individual [[Nucleobase|nitrogenous bases]] are capable of interacting with one another via [[hydrogen bond]]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 [[Annealing (biology)|annealed]] strands. For example, denaturation of DNA due to high temperatures results in the disruption of  [[base pair]]s and the separation of the double stranded helix into two single strands. Nucleic acid strands are capable of re-annealling when "[[Polymerase chain reaction|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 ===
[[Image:DNA Denaturation.png|thumb|DNA denaturation occurs when hydrogen bonds between base pairs are disturbed.]]
The [[non-covalent interactions]] between [[Antiparallel (biochemistry)|antiparallel strands]] in DNA can be broken in order to "open" the [[Nucleic acid double helix|double helix]] when biologically important mechanisms such as DNA replication, transcription, [[DNA repair]] or protein binding are set to occur.<ref name="1st">{{cite journal|last2=Destainville|first2=Nicolas|last3=Manghi|first3=Manoel|date=21 January 2015|title=DNA denaturation bubbles: Free-energy landscape and nucleation/closure rates|journal=The Journal of Chemical Physics|volume=142|issue=3|pages=034903|doi=10.1063/1.4905668|pmid=25612729|last1=Sicard|first1=François|arxiv=1405.3867|bibcode=2015JChPh.142c4903S|s2cid=13967558}}</ref> 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.<ref name="1st" />

The first model that attempted to describe the [[Nucleic acid thermodynamics|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 base pairs are increasingly disturbed and "denatured loops" begin to form.<ref>Lieu, Simon. "The Poland-Scheraga Model." (2015): 0-5. Massachusetts Institute of Technology, 14 May 2015. Web. 25 Oct. 2016.</ref> However, the Poland-Scheraga Model is now considered elementary because it fails to account for the confounding implications of [[Nucleic acid sequence|DNA sequence]], chemical composition, [[stiffness]] and [[torsion (mechanics)|torsion]].<ref>Richard, C., and A. J. Guttmann. "Poland–Scheraga Models and the DNA Denaturation Transition." ''Journal of Statistical Physics'' 115.3/4 (2004): 925-47. Web.</ref>

Recent thermodynamic studies have inferred that the lifetime of a singular denaturation bubble ranges from 1 microsecond to 1 millisecond.<ref name="2nd">{{cite journal|last2=Libchaber|first2=Albert|last3=Krichevsky|first3=Oleg|date=1 April 2003|title=Bubble Dynamics in Double-Stranded DNA|journal=Physical Review Letters|volume=90|issue=13|pages=138101|doi=10.1103/physrevlett.90.138101|pmid=12689326|last1=Altan-Bonnet|first1=Grégoire|s2cid=1427570|bibcode=2003PhRvL..90m8101A}}</ref> This information is based on established timescales of DNA replication and transcription.<ref name="2nd" /> Currently,{{when|date=December 2017}} biophysical and biochemical research studies are being performed to more fully elucidate the thermodynamic details of the denaturation bubble.<ref name="2nd" />

=== Denaturation due to chemical agents ===
[[File:DNA Denaturation by Formamide.png|thumb|Formamide denatures DNA by disrupting the hydrogen bonds between 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.<ref name=":02">{{cite journal|date=2014|title=Characterization of denaturation and renaturation of DNA for DNA hybridization|journal=Environmental Health and Toxicology|volume=29|doi=10.5620/eht.2014.29.e2014007|pmid=25234413|pmc=4168728|last1=Wang|first1=X|page=e2014007}}</ref> 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]].<ref name="ReferenceA">{{cite journal|date=1961|title=Denaturation of deoxyribonucleic acid by formamide|volume=51|issue=1|pages=91013–7|last1=Marmur|first1=J|journal=Biochimica et Biophysica Acta|doi=10.1016/0006-3002(61)91013-7|pmid=13767022}}</ref> These chemical denaturing agents lower the melting temperature (T<sub>m</sub>) 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, [[Alkalinity|alkaline]] agents (e.g. NaOH) have been shown to denature DNA by changing [[pH]] and removing hydrogen-bond contributing protons.<ref name=":02"/> These denaturants have been employed to make [[Temperature gradient gel electrophoresis|Denaturing Gradient Gel Electrophoresis gel]] (DGGE), which promotes denaturation of nucleic acids in order to eliminate the influence of nucleic acid shape on their [[Gel electrophoresis of nucleic acids|electrophoretic]] mobility.<ref>{{cite web|url=https://www.nationaldiagnostics.com/electrophoresis/article/denaturing-polyacrylamide-gel-electrophoresis-dna-rna|title=Denaturing Polyacrylamide Gel Electrophoresis of DNA & RNA|website=Electrophoresis|date=15 August 2011 |publisher=National Diagnostics|access-date=13 October 2016}}</ref>

==== Chemical denaturation as an alternative ====
The [[Optical rotation|optical activity]] (absorption and scattering of light) and hydrodynamic properties ([[Rotational diffusion|translational diffusion]], [[sedimentation coefficient]]s, and [[rotational correlation time]]s) of [[formamide]] denatured nucleic acids are similar to those of heat-denatured nucleic acids.<ref name="ReferenceA"/><ref>{{cite journal|last2=Bustamante|first2=C|last3=Maestre|first3=M|date=1980|title=The Optical Activity of Nucleic Acids and their Aggregates|journal=Annual Review of Biophysics and Bioengineering|volume=9|issue=1|pages=107–141|doi=10.1146/annurev.bb.09.060180.000543|pmid=6156638|last1=Tinoco|first1=I}}</ref><ref>{{cite journal|date=2002|title=Calculation of hydrodynamic properties of small nucleic acids from their atomic structure|journal=Nucleic Acids Research|volume=30|issue=8|pages=1782–8|doi=10.1093/nar/30.8.1782|pmid=11937632|pmc=113193|last1=Fernandes|first1=M}}</ref> 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.<ref name=":02"/> Particularly in cases where rapid renaturation is desired, chemical denaturation agents can provide an ideal alternative to heating. For example, DNA strands denatured with [[Alkalinity|alkaline agents]] such as [[Sodium hydroxide|NaOH]] renature as soon as [[Phosphate-buffered saline|phosphate buffer]] is added.<ref name=":02" />

==== Denaturation due to air ====
Small, [[Electronegativity|electronegative]] molecules such as [[nitrogen]] and [[oxygen]], which are the primary gases in [[Atmosphere of Earth|air]], significantly impact the ability of surrounding molecules to participate in [[hydrogen bond]]ing.<ref name=":1">{{cite journal|last2=Schoeffler|first2=G.|last3=McGlynn|first3=S. P.|date=July 1985|title=The effects of selected gases upon ethanol: hydrogen bond breaking by O and N|journal=Canadian Journal of Chemistry|volume=63|issue=7|pages=1864–1869|doi=10.1139/v85-309|last1=Mathers|first1=T. L.|doi-access=free}}</ref> 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.<ref name=":1" /> [[Antiparallel (biochemistry)|Antiparellel strands]] in DNA double helices are non-covalently bound by hydrogen bonding between base pairs;<ref>{{cite book|title=Lehninger principles of biochemistry|date=2008|publisher=W.H. Freeman|isbn=9780716771081|edition=5th|location=New York|last1=Cox|first1=David L. Nelson, Michael M.|url-access=registration|url=https://archive.org/details/lehningerprincip00lehn_1}}</ref> nitrogen and oxygen therefore maintain the potential to weaken the integrity of DNA when exposed to air.<ref name="DNA Air">{{cite journal|last2=Schoeffler|first2=G.|last3=McGlynn|first3=S. P.|date=1982|title=Hydrogen-bond breaking by O/sub 2/ and N/sub 2/. II. Melting curves of DNA|doi=10.2172/5693881|last1=Mathers|first1=T. L.|osti=5693881|url=https://digital.library.unt.edu/ark:/67531/metadc1089485/m2/1/high_res_d/5693881.pdf |archive-url=https://web.archive.org/web/20180724122925/https://digital.library.unt.edu/ark:/67531/metadc1089485/m2/1/high_res_d/5693881.pdf |archive-date=2018-07-24 |url-status=live}}</ref> As a result, DNA strands exposed to air require less force to separate and exemplify lower [[Nucleic acid thermodynamics|melting temperatures]].<ref name="DNA Air" />

=== 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:
* [[Polymerase chain reaction|PCR]]
* [[Southern blot]]
* [[Northern blot]]
* [[DNA sequencing]]

== Denaturants ==

=== Protein denaturants ===

==== Acids ====
[[Acid]]ic protein denaturants include:
* [[Acetic acid]]<ref>{{citation|title=NMR spectroscopy reveals that RNase A is chiefly denatured in 40% acetic acid: implications for oligomer formation by 3D domain swapping|year=2010|journal=J. Am. Chem. Soc.|volume=132|issue=5|pages=1621–30|doi=10.1021/ja9081638|pmid=20085318|vauthors=López-Alonso JP, Bruix M, Font J, Ribó M, Vilanova M, Jiménez MA, Santoro J, González C, Laurents DV|url=https://figshare.com/articles/NMR_Spectroscopy_Reveals_that_RNase_A_is_Chiefly_Denatured_in_40_Acetic_Acid_Implications_for_Oligomer_Formation_by_3D_Domain_Swapping/2792884}}</ref>
* [[Trichloroacetic acid]] 12% in water
* [[Sulfosalicylic acid]]

==== Bases ====
[[Base (chemistry)|Bases]] work similarly to acids in denaturation.  They include:
* [[Sodium bicarbonate]]

==== Solvents ====
Most organic [[solvent]]s are denaturing, including:{{citation needed|date=May 2013}}
* [[Ethanol]]

==== Cross-linking reagents ====
[[Cross-link]]ing agents for proteins include:{{citation needed|date=May 2013}}
* [[Formaldehyde]]
* [[Glutaraldehyde]]

==== Chaotropic agents ====
[[Chaotropic agent]]s include:{{citation needed|date=May 2013}}
* [[Urea]] 6–8&nbsp;[[molarity|mol/L]]
* [[Guanidinium chloride]] 6&nbsp;mol/L
* [[Lithium perchlorate]] 4.5&nbsp;mol/L
* [[Sodium dodecyl sulfate]]

==== Disulfide bond reducers ====
Agents that break [[disulfide bond]]s by reduction include:{{citation needed|date=May 2013}}
* [[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<ref>{{cite journal|last=Jaremko|first=M.|date=April 2013|title=Cold denaturation of a protein dimer monitored at atomic resolution|journal=[[Nat. Chem. Biol.]]|volume=9|issue=4|pages=264–70|doi=10.1038/nchembio.1181|pmid=23396077|author2=Jaremko Ł|author3=Kim HY|author4=Cho MK|author5=Schwieters CD|author6=Giller K|author7=Becker S|author8=Zweckstetter M.|pmc=5521822}}</ref>
* [[Joule heating]]

=== Nucleic acid denaturants ===

==== Chemical ====
[[Acid]]ic nucleic acid denaturants include:
* [[Acetic acid]]
* HCl
* Nitric acid

[[Acid|Basic]] nucleic acid denaturants include:
* NaOH

Other nucleic acid denaturants include:
* [[Dimethyl sulfoxide|DMSO]]
* [[Formamide]]
* [[Guanidine]]
* [[Sodium salicylate]]
* [[Propylene glycol]]
* [[Urea]]

==== Physical ====
* Thermal denaturation
* Beads mill
* Probe [[sonication]]
* [[Radiation]]

== See also ==
* [[Denatured alcohol]]
* [[Equilibrium unfolding]]
* [[Fixation (histology)]]
* [[Molten globule]]
* [[Protein folding]]
* [[Random coil]]

== References ==
{{reflist|30em}}

== External links ==
* [http://highered.mcgraw-hill.com/sites/0072943696/student_view0/chapter2/animation__protein_denaturation.html McGraw-Hill Online Learning Center&nbsp;— Animation: Protein Denaturation]
{{Biochemistry topics|state=collapsed}}{{DEFAULTSORT:Denaturation (Biochemistry)}}

[[Category:Biochemical reactions]]
[[Category:Nucleic acids]]
[[Category:Protein structure]]