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Note 1: Modified from the definition given in ref.[1] Note 2: Denaturation can occur when proteins and nucleic acids are subjected to elevated temperature or to extremes of pH, or to nonphysiological concentrations of salt, organic solvents, urea, or other chemical agents. Note 3: An enzyme loses its catalytic activity when it is denaturized.[2]

Denaturation is a process in which proteins or nucleic acids lose the quaternary structure, tertiary structure and secondary structure which is present in their native state, by application of some external stress or compound such as a strong acid or base, a concentrated inorganic salt, an organic solvent (e.g., alcohol or chloroform), radiation or heat.[3] If proteins in a living cell are denatured, this results in disruption of cell activity and possibly cell death. Protein
Protein
denaturation is also a consequence of cell death.[4][5] Denatured proteins can exhibit a wide range of characteristics, from conformational change and loss of solubility to aggregation due to the exposure of hydrophobic groups. Denatured proteins lose their 3D structure and therefore cannot function. Protein
Protein
folding is key to whether a globular protein or a membrane protein can do its job correctly. It must be folded into the right shape to function. But hydrogen bonds, which play a big part in folding, are rather weak, and it doesn't take much heat, acidity, varying salt concentrations, or other stress to break some and form others, denaturing the protein. This is one reason why tight homeostasis is physiologically necessary in many life forms. This concept is unrelated to denatured alcohol, which is alcohol that has been mixed with additives to make it unsuitable for human consumption.

Contents

1 Common examples 2 Protein
Protein
denaturation

2.1 Background 2.2 How denaturation occurs at levels of protein structure

2.2.1 Loss of function 2.2.2 Reversibility and irreversibility

3 Nucleic acid
Nucleic acid
denaturation

3.1 Biologically-induced denaturation 3.2 Denaturation due to chemical agents

3.2.1 Chemical denaturation as an alternative 3.2.2 Denaturation due to air

3.3 Applications

4 Denaturants

4.1 Protein
Protein
Denaturants

4.1.1 Acids 4.1.2 Bases 4.1.3 Solvents 4.1.4 Cross-linking reagents 4.1.5 Chaotropic agents 4.1.6 Disulfide bond
Disulfide bond
reducers 4.1.7 Other

4.2 Nucleic Acid
Acid
Denaturants

4.2.1 Chemical 4.2.2 Physical

5 See also 6 References 7 External links

Common examples[edit]

(Top) The protein albumin in the egg white undergoes denaturation and loss of solubility when the egg is cooked. (Bottom) Nitinol paperclips provide a visual analogy to help with the conceptualization of the 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 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.[6] 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.[7] Protein
Protein
denaturation[edit] Denatured proteins can exhibit a wide range of characteristics, from loss of solubility to protein aggregation.

Functional proteins have four levels of structural organization: 1) Primary Structure : the linear structure of amino acids in the polypeptide chain 2) Secondary Structure : hydrogen bonds between peptide group chains in an alpha helix or beta sheet 3) Tertiary Structure : three-dimensional structure of alpha helixes and beta helixes folded 4) Quaternary Structure : three-dimensional structure of multiple polypeptides and how they fit together

Process of Denaturation: 1) Functional protein showing a quaternary structure 2) when heat is applied it alters the intramolecular bonds of the protein 3) unfolding of the polypeptides (amino acids)

Background[edit] Proteins or Polypeptides are polymers of amino acids. A protein is created by ribosomes that "read" RNA
RNA
that is encoded by codons in the gene and assemble the requisite amino acid combination from the genetic instruction, in a process known as translation. The newly created protein strand then undergoes posttranslational modification, in which additional atoms or molecules are added, for example copper, zinc, or iron. Once this post-translational modification process has been completed, the protein begins to fold (sometimes spontaneously and sometimes with enzymatic assistance), curling up on itself so that hydrophobic elements of the protein are buried deep inside the structure and hydrophilic elements end up on the outside. The final shape of a protein determines how it interacts with its environment. When a protein is denatured, secondary and tertiary structures are altered but the peptide bonds of the primary structure between the amino acids are left intact. Since all structural levels of the protein determine its function, the protein can no longer perform its function once it has been denatured. This is in contrast to intrinsically unstructured proteins, which are unfolded in their native state, but still functionally active and tend to fold upon binding to their biological target.[8] How denaturation occurs at levels of protein structure[edit] See also: Protein
Protein
structure

In quaternary structure denaturation, protein sub-units are dissociated and/or the spatial arrangement of protein subunits is disrupted. Tertiary structure
Tertiary structure
denaturation involves the disruption of:

Covalent
Covalent
interactions between amino acid side-chains (such as disulfide bridges between cysteine groups) Non-covalent dipole-dipole interactions between polar amino acid side-chains (and the surrounding solvent) Van der Waals (induced dipole) interactions between nonpolar amino acid side-chains.

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.[9]

Loss of function[edit] Most biological substrates lose their biological function when denatured. For example, enzymes lose their activity, because the substrates can no longer bind to the active site, and because amino acid residues involved in stabilizing substrates' transition states are no longer positioned to be able to do so. The denaturing process and the associated loss of activity can be measured using techniques such as dual polarization interferometry, CD, QCM-D and MP-SPR. Reversibility and irreversibility[edit] 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.[10] 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
DNA
that codes for the protein, the so-called "Anfinsen's thermodynamic hypothesis".[11] However, denaturation can also be irreversible. However, this irreversibility is typically a kinetic, not thermodynamic irreversibility, as generally when a protein is folded it has lower free energy. But, through kinetic irreversibility, the fact that the protein is stuck in a local minimum can stop it from ever refolding after it has been irreversibly denatured.[12] Nucleic acid
Nucleic acid
denaturation[edit] Main article: Nucleic acid
Nucleic acid
thermodynamics Nucleic acids (including RNA
RNA
and DNA) are nucleotide polymers synthesized by polymerase enzymes during either transcription or DNA replication. Following 5'-3' synthesis of the backbone, individual nitrogenous bases are capable of interacting with one another via hydrogen bonding, thus allowing for the formation of higher-order structures. Nucleic acid
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
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
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[edit]

DNA
DNA
denaturation occurs when hydrogen bonds between Watson and Crick base pairs are disturbed.

The non-covalent interactions between antiparallel strands in DNA
DNA
can be broken in order to "open" the double helix when biologically important mechanisms such as DNA
DNA
replication, transcription, DNA repair or protein binding are set to occur.[13] The area of partially separated DNA
DNA
is known as the denaturation bubble, which can be more specifically defined as the opening of a DNA
DNA
double helix through the coordinated separation of base pairs.[13] The first model that attempted to describe the thermodynamics of the denaturation bubble was introduced in 1966 and called the Poland-Scheraga Model. This model describes the denaturation of DNA strands as a function of temperature. As the temperature increases, the hydrogen bonds between the Watson and Crick base pairs are increasingly disturbed and "denatured loops" begin to form.[14] However, the Poland-Scheraga Model is now considered elementary because it fails to account for the confounding implications of DNA sequence, chemical composition, stiffness and torsion.[15] Recent thermodynamic studies have inferred that the lifetime of a singular denaturation bubble ranges from 1 microsecond to 1 millisecond.[16] This information is based on established timescales of DNA
DNA
replication and transcription.[16] Currently,[when?] biophysical and biochemical research studies are being performed to more fully elucidate the thermodynamic details of the denaturation bubble.[16] Denaturation due to chemical agents[edit]

Formamide
Formamide
denatures DNA
DNA
by disrupting the hydrogen bonds between Watson and Crick base pairs. Orange, blue, green, and purple lines represent adenine, thymine, guanine, and cytosine respectively. The three short black lines between the bases and the formamide molecules represent newly formed hydrogen bonds.

With polymerase chain reaction (PCR) being among the most popular contexts in which DNA
DNA
denaturation is desired, heating is the most frequent method of denaturation.[17] 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.[18] These chemical denaturing agents lower the melting temperature (Tm) by competing for hydrogen bond donors and acceptors with pre-existing nitrogenous base pairs. Some agents are even able to induce denaturation at room temperature. For example, alkaline agents (e.g. NaOH) have been shown to denature DNA
DNA
by changing pH and removing hydrogen-bond contributing protons.[17] 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.[19] Chemical denaturation as an alternative[edit] 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.[18][20][21] Therefore, depending on the desired effect, chemically denaturing DNA
DNA
can provide a gentler procedure for denaturing nucleic acids than denaturation induced by heat. Studies comparing different denaturation methods such as heating, beads mill of different bead sizes, probe sonification, and chemical denaturation show that chemical denaturation can provide quicker denaturation compared to the other physical denaturation methods described.[17] Particularly in cases where rapid renaturation is desired, chemical denaturation agents can provide an ideal alternative to heating. For example, DNA
DNA
strands denatured with alkaline agents such as NaOH renature as soon as phosphate buffer is added.[17] Denaturation due to air[edit] Small, electronegative molecules such as nitrogen and oxygen, which are the primary gases in air, significantly impact the ability of surrounding molecules to participate in hydrogen bonding.[22] 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.[22] Antiparellel strands in DNA
DNA
double helices are non-covalently bound by hydrogen bonding between Watson and Crick base pairs;[23] nitrogen and oxygen therefore maintain the potential to weaken the integrity of DNA
DNA
when exposed to air.[24] As a result, DNA strands exposed to air require less force to separate and exemplify lower melting temperatures.[24] Applications[edit] 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 DNA
DNA
Sequencing

Denaturants[edit] Protein
Protein
Denaturants[edit] Acids[edit] Acidic protein denaturants include:

Acetic acid[25] Trichloroacetic acid
Trichloroacetic acid
12% in water Sulfosalicylic acid

Bases[edit] Bases work similarly to acids in denaturation. They include:

Sodium bicarbonate

Solvents[edit] Most organic solvents are denaturing, including:[citation needed]

Ethanol alcohol

Cross-linking reagents[edit] Cross-linking agents for proteins include:[citation needed]

Formaldehyde Glutaraldehyde

Chaotropic agents[edit] Chaotropic agents include:[citation needed]

Urea
Urea
6 – 8 mol/l Guanidinium chloride
Guanidinium chloride
6 mol/l Lithium perchlorate
Lithium perchlorate
4.5 mol/l sodium dodecyl sulfate

Disulfide bond
Disulfide bond
reducers[edit] Agents that break disulfide bonds by reduction include:[citation needed]

2-Mercaptoethanol Dithiothreitol TCEP
TCEP
(tris(2-carboxyethyl)phosphine)

Other[edit]

Mechanical agitation Picric acid Radiation Temperature[26]

Nucleic Acid
Acid
Denaturants[edit] Chemical[edit] 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 Urea

Physical[edit]

Thermal denaturation Beads mill Probe sonification Radiation

See also[edit]

Denatured alcohol Fixation (histology) Protein
Protein
folding Random coil

References[edit]

^ Alan D. MacNaught; Andrew R. Wilkinson, eds. (1997). Compendium of Chemical Terminology: IUPAC Recommendations (the "Gold Book"). Blackwell Science. ISBN 0865426848.  ^ Vert, Michel (2012). "Terminology for biorelated polymers and applications (IUPAC Recommendations 2012)" (PDF). Pure and Applied Chemistry. 84 (2): 377–410. doi:10.1351/PAC-REC-10-12-04.  ^ Mosby's Medical Dictionary (8th ed.). Elsevier. 2009. Retrieved September 2013.  Check date values in: access-date= (help) ^ Samson, Andre L.; Ho, Bosco; Au, Amanda E.; Schoenwaelder, Simone M.; Smyth, Mark J.; Bottomley, Stephen P.; Kleifeld, Oded; Medcalf, Robert L. (2016-11-01). "Physicochemical properties that control protein aggregation also determine whether a protein is retained or released from necrotic cells". Open Biology. 6 (11): 160098. doi:10.1098/rsob.160098. ISSN 2046-2441. PMID 27810968.  ^ Samson, Andre L.; Knaupp, Anja S.; Sashindranath, Maithili; Borg, Rachael J.; Au, Amanda E.-L.; Cops, Elisa J.; Saunders, Helen M.; Cody, Stephen H.; McLean, Catriona A. (2012-10-25). "Nucleocytoplasmic coagulation: an injury-induced aggregation event that disulfide crosslinks proteins and facilitates their removal by plasmin". Cell Reports. 2 (4): 889–901. doi:10.1016/j.celrep.2012.08.026. ISSN 2211-1247. PMID 23041318.  ^ Mine, Yoshinori; Noutomi, Tatsushi; Haga, Noriyuki (1990). "Thermally induced changes in egg white proteins". Journal of Agricultural and Food Chemistry. 38 (12): 2122–2125. doi:10.1021/jf00102a004.  ^ "Ceviche: the new sushi," The Times. ^ Dyson, H. Jane; Wright, Peter E. (2005-03-01). "Intrinsically unstructured proteins and their functions" (PDF). Nature Reviews Molecular Cell Biology. 6 (3): 197–208. doi:10.1038/nrm1589. ISSN 1471-0072.  ^ Charles Tanford (1968), " Protein
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denaturation" (PDF), Advances in Protein
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Chemistry, 23: 121–282, doi:10.1016/S0065-3233(08)60401-5, PMID 4882248  ^ Campbell, N. A.; Reece, J.B.; Meyers, N.; Urry, L. A.; Cain, M.L.; Wasserman, S.A.; Minorsky, P.V.; Jackson, R.B. (2009), Biology (8th, Australian version ed.), Sydney: Pearson Education Australia  ^ Anfinsen CB. (1973), "Principles that govern the folding of protein chains", Science, 181 (4096): 223–30, doi:10.1126/science.181.4096.223, PMID 4124164  ^ Wetlaufer, D.B. (1988). "Reversible and irreversible denaturation of proteins in chromatographic systems". Makromolekulare Chemie. Macromolecular Symposia. 17 (1): 17–28. doi:10.1002/masy.19880170104. ISSN 0258-0322.  ^ a b Sicard, François; Destainville, Nicolas; Manghi, Manoel (21 January 2015). " DNA
DNA
denaturation bubbles: Free-energy landscape and nucleation/closure rates". The Journal of Chemical Physics. 142 (3): 034903. arXiv:1405.3867 . doi:10.1063/1.4905668.  ^ Lieu, Simon. "The Poland-Scheraga Model." (2015): 0-5. Massachusetts Institute of Technology, 14 May 2015. Web. 25 Oct. 2016. ^ Richard, C., and A. J. Guttmann. "Poland–Scheraga Models and the DNA
DNA
Denaturation Transition." Journal of Statistical Physics 115.3/4 (2004): 925-47. Web. ^ a b c Altan-Bonnet, Grégoire; Libchaber, Albert; Krichevsky, Oleg (1 April 2003). "Bubble Dynamics in Double-Stranded DNA". Physical Review Letters. 90 (13). Bibcode:2003PhRvL..90m8101A. doi:10.1103/physrevlett.90.138101.  ^ a b c d Wang, X (2014). "Characterization of denaturation and renaturation of DNA
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for DNA
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hybridization". Environmental Health and Toxicology Environ Health Toxicol. 29: e2014007. doi:10.5620/eht.2014.29.e2014007.  ^ a b Marmur, J (1961). "Denaturation of deoxyribonucleic acid by formamide". Biochimica et Biophysica Acta. 51 (1): 91013–7. doi:10.1016/0006-3002(61)91013-7.  ^ "Denaturing Polyacrylamide Gel Electrophoresis of DNA
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& RNA". Electrophoresis. National Diagnostics. Retrieved 13 October 2016.  ^ Tinoco, I; Bustamante, C; Maestre, M (1980). "The Optical Activity of Nucleic Acids and their Aggregates". Annual Review of Biophysics and Bioengineering. 9 (1): 107–141. doi:10.1146/annurev.bb.09.060180.000543.  ^ Fernandes, M (2002). "Calculation of hydrodynamic properties of small nucleic acids from their atomic structure". Nucleic Acids Research. 30 (8): 1782–8. doi:10.1093/nar/30.8.1782.  ^ a b Mathers, T. L.; Schoeffler, G.; McGlynn, S. P. (July 1985). "The effects of selected gases upon ethanol: hydrogen bond breaking by O and N". Canadian Journal of Chemistry. 63 (7): 1864–1869. doi:10.1139/v85-309.  ^ Cox, David L. Nelson, Michael M. (2008). Lehninger principles of biochemistry (5th ed.). New York: W.H. Freeman. ISBN 9780716771081.  ^ a b Mathers, T. L.; Schoeffler, G.; McGlynn, S. P. (1982). "Hydrogen-bond breaking by O/sub 2/ and N/sub 2/. II. Melting curves of DNA". doi:10.2172/5693881.  ^ López-Alonso JP, Bruix M, Font J, Ribó M, Vilanova M, Jiménez MA, Santoro J, González C, Laurents DV (2010), "NMR spectroscopy reveals that RNase A is chiefly denatured in 40% acetic acid: implications for oligomer formation by 3D domain swapping", J. Am. Chem. Soc., 132 (5): 1621–30, doi:10.1021/ja9081638, PMID 20085318  ^ Jaremko, M.; Jaremko Ł; Kim HY; Cho MK; Schwieters CD; Giller K; Becker S; Zweckstetter M. (April 2013). "Cold denaturation of a protein dimer monitored at atomic resolution". Nat. Chem. Biol.
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External links[edit]

McGraw-Hill Online Learning Center — Animation: Protein
Protein

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