Denaturation (biochemistry)

Denaturation (biochemistry)

Overview
Denaturation is a process in which proteins or nucleic acids lose their tertiary structure
Tertiary structure
In biochemistry and molecular biology, the tertiary structure of a protein or any other macromolecule is its three-dimensional structure, as defined by the atomic coordinates.-Relationship to primary structure:...

 and secondary structure
Secondary structure
In biochemistry and structural biology, secondary structure is the general three-dimensional form of local segments of biopolymers such as proteins and nucleic acids...

 by application of some external stress or compound, such as a strong acid
Acid
An acid is a substance which reacts with a base. Commonly, acids can be identified as tasting sour, reacting with metals such as calcium, and bases like sodium carbonate. Aqueous acids have a pH of less than 7, where an acid of lower pH is typically stronger, and turn blue litmus paper red...

 or base
Base (chemistry)
For the term in genetics, see base A base in chemistry is a substance that can accept hydrogen ions or more generally, donate electron pairs. A soluble base is referred to as an alkali if it contains and releases hydroxide ions quantitatively...

, a concentrated inorganic salt, an organic
Organic compound
An organic compound is any member of a large class of gaseous, liquid, or solid chemical compounds whose molecules contain carbon. For historical reasons discussed below, a few types of carbon-containing compounds such as carbides, carbonates, simple oxides of carbon, and cyanides, as well as the...

 solvent (e.g., alcohol
Alcohol
In chemistry, an alcohol is an organic compound in which the hydroxy functional group is bound to a carbon atom. In particular, this carbon center should be saturated, having single bonds to three other atoms....

 or chloroform
Chloroform
Chloroform is an organic compound with formula CHCl3. It is one of the four chloromethanes. The colorless, sweet-smelling, dense liquid is a trihalomethane, and is considered somewhat hazardous...

), or heat
Heat
In physics and thermodynamics, heat is energy transferred from one body, region, or thermodynamic system to another due to thermal contact or thermal radiation when the systems are at different temperatures. It is often described as one of the fundamental processes of energy transfer between...

. If proteins in a living cell are denatured, this results in disruption of cell activity and possibly cell death. Denatured proteins can exhibit a wide range of characteristics, from loss of solubility to communal aggregation
Communal aggregation
Communal aggregation is the phenomenon of aggregation of the hydrophobic proteins to come closer and form the bonding between them, so as to reduce the total area exposed to water. It is a very common problem with the hydrophobic protein to make aggregates. Such aggregates hamper the filtration...

.

This concept is unrelated to denatured alcohol, which is alcohol that has been mixed with additives to make it unsuitable for human consumption.

When food is cooked, some of its proteins become denatured.
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Encyclopedia
Denaturation is a process in which proteins or nucleic acids lose their tertiary structure
Tertiary structure
In biochemistry and molecular biology, the tertiary structure of a protein or any other macromolecule is its three-dimensional structure, as defined by the atomic coordinates.-Relationship to primary structure:...

 and secondary structure
Secondary structure
In biochemistry and structural biology, secondary structure is the general three-dimensional form of local segments of biopolymers such as proteins and nucleic acids...

 by application of some external stress or compound, such as a strong acid
Acid
An acid is a substance which reacts with a base. Commonly, acids can be identified as tasting sour, reacting with metals such as calcium, and bases like sodium carbonate. Aqueous acids have a pH of less than 7, where an acid of lower pH is typically stronger, and turn blue litmus paper red...

 or base
Base (chemistry)
For the term in genetics, see base A base in chemistry is a substance that can accept hydrogen ions or more generally, donate electron pairs. A soluble base is referred to as an alkali if it contains and releases hydroxide ions quantitatively...

, a concentrated inorganic salt, an organic
Organic compound
An organic compound is any member of a large class of gaseous, liquid, or solid chemical compounds whose molecules contain carbon. For historical reasons discussed below, a few types of carbon-containing compounds such as carbides, carbonates, simple oxides of carbon, and cyanides, as well as the...

 solvent (e.g., alcohol
Alcohol
In chemistry, an alcohol is an organic compound in which the hydroxy functional group is bound to a carbon atom. In particular, this carbon center should be saturated, having single bonds to three other atoms....

 or chloroform
Chloroform
Chloroform is an organic compound with formula CHCl3. It is one of the four chloromethanes. The colorless, sweet-smelling, dense liquid is a trihalomethane, and is considered somewhat hazardous...

), or heat
Heat
In physics and thermodynamics, heat is energy transferred from one body, region, or thermodynamic system to another due to thermal contact or thermal radiation when the systems are at different temperatures. It is often described as one of the fundamental processes of energy transfer between...

. If proteins in a living cell are denatured, this results in disruption of cell activity and possibly cell death. Denatured proteins can exhibit a wide range of characteristics, from loss of solubility to communal aggregation
Communal aggregation
Communal aggregation is the phenomenon of aggregation of the hydrophobic proteins to come closer and form the bonding between them, so as to reduce the total area exposed to water. It is a very common problem with the hydrophobic protein to make aggregates. Such aggregates hamper the filtration...

.

This concept is unrelated to denatured alcohol, which is alcohol that has been mixed with additives to make it unsuitable for human consumption.

Common examples


When food is cooked, some of its proteins become denatured. This is why boiled eggs become hard and cooked meat becomes firm.

A classic example of denaturing in proteins comes from egg white
Egg white
Egg white is the common name for the clear liquid contained within an egg. In chickens it is formed from the layers of secretions of the anterior section of the hen's oviduct during the passage of the egg. It forms around either fertilized or unfertilized egg yolks...

s, which are largely egg albumin
Ovalbumin
Ovalbumin is the main protein found in egg white, making up 60-65% of the total protein. Ovalbumin displays sequence and three-dimensional homology to the serpin superfamily, but unlike most serpins it is not a serine protease inhibitor...

s in water. Fresh from the eggs, egg whites are transparent and liquid. Cooking the thermally unstable
Thermostability
Thermostability is the quality of a substance to resist irreversible change in its chemical or physical structure at a high relative temperature.Thermostable materials may be used industrially as fire retardants...

 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
Acetone is the organic compound with the formula 2CO, a colorless, mobile, flammable liquid, the simplest example of the ketones.Acetone is miscible with water and serves as an important solvent in its own right, typically as the solvent of choice for cleaning purposes in the laboratory...

 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
Ceviche is a seafood dish popular in the coastal regions of the Americas, especially Central and South America. The dish is typically made from fresh raw fish marinated in citrus juices such as lemon or lime and spiced with chilli peppers. Additional seasonings such as onion, salt,...

 is prepared by chemically "cooking" raw fish and shellfish in an acidic citrus marinade, without heat.

Although denaturing egg whites is irreversible, in many other cases denaturing is reversible.

Protein denaturation


Denatured proteins can exhibit a wide range of characteristics, from loss of solubility
Solubility
Solubility is the property of a solid, liquid, or gaseous chemical substance called solute to dissolve in a solid, liquid, or gaseous solvent to form a homogeneous solution of the solute in the solvent. The solubility of a substance fundamentally depends on the used solvent as well as on...

 to communal aggregation
Communal aggregation
Communal aggregation is the phenomenon of aggregation of the hydrophobic proteins to come closer and form the bonding between them, so as to reduce the total area exposed to water. It is a very common problem with the hydrophobic protein to make aggregates. Such aggregates hamper the filtration...

. Communal aggregation is the phenomenon of aggregation of the hydrophobic proteins to come closer and form the bonding between them, so as to reduce the total area exposed to water.

Background


Protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

s are amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...

 polymers. A protein is created by ribosome
Ribosome
A ribosome is a component of cells that assembles the twenty specific amino acid molecules to form the particular protein molecule determined by the nucleotide sequence of an RNA molecule....

s that "read" RNA that is encoded by codons in the gene and assemble the requisite amino acid combination from the genetic
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...

 instruction, in a process known as translation
Translation (genetics)
In molecular biology and genetics, translation is the third stage of protein biosynthesis . In translation, messenger RNA produced by transcription is decoded by the ribosome to produce a specific amino acid chain, or polypeptide, that will later fold into an active protein...

. The newly created protein strand then undergoes posttranslational modification
Posttranslational modification
Posttranslational modification is the chemical modification of a protein after its translation. It is one of the later steps in protein biosynthesis, and thus gene expression, for many proteins....

, in which additional atom
Atom
The atom is a basic unit of matter that consists of a dense central nucleus surrounded by a cloud of negatively charged electrons. The atomic nucleus contains a mix of positively charged protons and electrically neutral neutrons...

s or molecule
Molecule
A molecule is an electrically neutral group of at least two atoms held together by covalent chemical bonds. Molecules are distinguished from ions by their electrical charge...

s are added, for example copper
Copper
Copper is a chemical element with the symbol Cu and atomic number 29. It is a ductile metal with very high thermal and electrical conductivity. Pure copper is soft and malleable; an exposed surface has a reddish-orange tarnish...

, zinc
Zinc
Zinc , or spelter , is a metallic chemical element; it has the symbol Zn and atomic number 30. It is the first element in group 12 of the periodic table. Zinc is, in some respects, chemically similar to magnesium, because its ion is of similar size and its only common oxidation state is +2...

, or iron
Iron
Iron is a chemical element with the symbol Fe and atomic number 26. It is a metal in the first transition series. It is the most common element forming the planet Earth as a whole, forming much of Earth's outer and inner core. It is the fourth most common element in the Earth's crust...

. 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, the secondary
Secondary structure
In biochemistry and structural biology, secondary structure is the general three-dimensional form of local segments of biopolymers such as proteins and nucleic acids...

 and tertiary structure
Tertiary structure
In biochemistry and molecular biology, the tertiary structure of a protein or any other macromolecule is its three-dimensional structure, as defined by the atomic coordinates.-Relationship to primary structure:...

s are altered but the peptide bond
Peptide bond
This article is about the peptide link found within biological molecules, such as proteins. A similar article for synthetic molecules is being created...

s of the primary structure between the amino acids are left intact. Since all structural levels of the protein determines its function, the protein can no longer perform its function once it has been denatured. This is in contrast to intrinsically unstructured proteins
Intrinsically unstructured proteins
Intrinsically unstructured proteins, often referred to as naturally unfolded proteins or disordered proteins, are proteins characterized by lack of stable tertiary structure when the protein exists as an isolated polypeptide chain under physiological conditions in vitro...

, which are unfolded in their native state
Native state
In biochemistry, the native state of a protein is its operative or functional form. While all protein molecules begin as simple unbranched chains of amino acids, once completed they assume highly specific three-dimensional shapes; that ultimate shape, known as tertiary structure, is the folded...

, but still functionally active.

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 interactions between amino acid side-chains (such as disulfide bridges between cysteine
    Cysteine
    Cysteine is an α-amino acid with the chemical formula HO2CCHCH2SH. It is a non-essential amino acid, which means that it is biosynthesized in humans. Its codons are UGU and UGC. The side chain on cysteine is thiol, which is polar and thus cysteine is usually classified as a hydrophilic amino acid...

     groups)
  • Noncovalent dipole
    Dipole
    In physics, there are several kinds of dipoles:*An electric dipole is a separation of positive and negative charges. The simplest example of this is a pair of electric charges of equal magnitude but opposite sign, separated by some distance. A permanent electric dipole is called an electret.*A...

    -dipole interactions between polar amino acid side-chains (and the surrounding solvent
    Solvent
    A solvent is a liquid, solid, or gas that dissolves another solid, liquid, or gaseous solute, resulting in a solution that is soluble in a certain volume of solvent at a specified temperature...

    )
  • Van der Waals (induced dipole) interactions
    Van der Waals force
    In physical chemistry, the van der Waals force , named after Dutch scientist Johannes Diderik van der Waals, is the sum of the attractive or repulsive forces between molecules other than those due to covalent bonds or to the electrostatic interaction of ions with one another or with neutral...

     between nonpolar amino acid side-chains.
  • In secondary structure denaturation, proteins lose all regular repeating patterns such as alpha-helices
    Alpha helix
    A common motif in the secondary structure of proteins, the alpha helix is a right-handed coiled or spiral conformation, in which every backbone N-H group donates a hydrogen bond to the backbone C=O group of the amino acid four residues earlier...

     and beta-pleated sheets
    Beta sheet
    The β sheet is the second form of regular secondary structure in proteins, only somewhat less common than the alpha helix. Beta sheets consist of beta strands connected laterally by at least two or three backbone hydrogen bonds, forming a generally twisted, pleated sheet...

    , and adopt a random coil
    Random coil
    A random coil is a polymer conformation 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 chains in a population of macromolecules...

     configuration.
  • Primary structure, such as the sequence of amino acids held together by covalent peptide bond
    Peptide bond
    This article is about the peptide link found within biological molecules, such as proteins. A similar article for synthetic molecules is being created...

    s, is not disrupted by denaturation.

Loss of function


Most biological substrates lose their biological function when denatured. For example, enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...

s lose their activity
Catalysis
Catalysis is the change in rate of a chemical reaction due to the participation of a substance called a catalyst. Unlike other reagents that participate in the chemical reaction, a catalyst is not consumed by the reaction itself. A catalyst may participate in multiple chemical transformations....

, because the substrates can no longer bind to the active site
Active site
In biology the active site is part of an enzyme where substrates bind and undergo a chemical reaction. The majority of enzymes are proteins but RNA enzymes called ribozymes also exist. The active site of an enzyme is usually found in a cleft or pocket that is lined by amino acid residues that...

, and because amino acid residues involved in stabilizing substrates' transition state
Transition state
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 energy along this reaction coordinate. At this point, assuming a perfectly irreversible reaction, colliding reactant molecules will always...

s are no longer positioned to be able to do so. The denaturing process and the associated loss of activity can be measured using techniques such as dual polarization interferometry, CD
Circular dichroism
Circular dichroism refers to the differential absorption of left and right circularly polarized light. This phenomenon was discovered by Jean-Baptiste Biot, Augustin Fresnel, and Aimé Cotton in the first half of the 19th century. It is exhibited in the absorption bands of optically active chiral...

, and QCMD.

Reversibility and irreversibility


In many proteins (unlike egg whites), 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
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...

 that codes for the protein, the so called "Anfinsen's
Christian B. Anfinsen
Christian Boehmer Anfinsen, Jr. was an American biochemist. He shared the 1972 Nobel Prize in Chemistry with Stanford Moore and William Howard Stein for work on ribonuclease, especially concerning the connection between the amino acid sequence and the biologically active conformation...

 thermodynamic hypothesis
Anfinsen's dogma
Anfinsen's dogma is a postulate in molecular biology championed by the Nobel Prize Laureate Christian B. Anfinsen...

".

Nucleic acid denaturation


The denaturation of nucleic acid
Nucleic acid
Nucleic acids are biological molecules essential for life, and include DNA and RNA . Together with proteins, nucleic acids make up the most important macromolecules; each is found in abundance in all living things, where they function in encoding, transmitting and expressing genetic information...

s such as DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...

 due to high temperatures is the separation of a double strand into two single strands, which occurs when the hydrogen bond
Hydrogen bond
A hydrogen bond is the attractive interaction of a hydrogen atom with an electronegative atom, such as nitrogen, oxygen or fluorine, that comes from another molecule or chemical group. The hydrogen must be covalently bonded to another electronegative atom to create the bond...

s between the strands are broken. This may occur during polymerase chain reaction
Polymerase chain reaction
The polymerase chain reaction is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence....

. Nucleic acid strands realign when "normal" conditions are restored during annealing. If the conditions are restored too quickly, the nucleic acid strands may realign imperfectly.

Acids


Acid
Acid
An acid is a substance which reacts with a base. Commonly, acids can be identified as tasting sour, reacting with metals such as calcium, and bases like sodium carbonate. Aqueous acids have a pH of less than 7, where an acid of lower pH is typically stronger, and turn blue litmus paper red...

ic protein denaturants include:
  • Acetic acid
    Acetic acid
    Acetic acid is an organic compound with the chemical formula CH3CO2H . It is a colourless liquid that when undiluted is also called glacial acetic acid. Acetic acid is the main component of vinegar , and has a distinctive sour taste and pungent smell...

  • Trichloroacetic acid
    Trichloroacetic acid
    Trichloroacetic acid is an analogue of acetic acid in which the three hydrogen atoms of the methyl group have all been replaced by chlorine atoms....

     12% in water
  • Sulfosalicylic acid

Solvents


Most organic solvent
Solvent
A solvent is a liquid, solid, or gas that dissolves another solid, liquid, or gaseous solute, resulting in a solution that is soluble in a certain volume of solvent at a specified temperature...

s are denaturing, including:
  • Ethanol
    Ethanol
    Ethanol, also called ethyl alcohol, pure alcohol, grain alcohol, or drinking alcohol, is a volatile, flammable, colorless liquid. It is a psychoactive drug and one of the oldest recreational drugs. Best known as the type of alcohol found in alcoholic beverages, it is also used in thermometers, as a...

  • Methanol
    Methanol
    Methanol, also known as methyl alcohol, wood alcohol, wood naphtha or wood spirits, is a chemical with the formula CH3OH . It is the simplest alcohol, and is a light, volatile, colorless, flammable liquid with a distinctive odor very similar to, but slightly sweeter than, ethanol...


Cross-linking reagents


Cross-link
Cross-link
Cross-links are bonds that link one polymer chain to another. They can be covalent bonds or ionic bonds. "Polymer chains" can refer to synthetic polymers or natural polymers . When the term "cross-linking" is used in the synthetic polymer science field, it usually refers to the use of...

ing agents for proteins include:
  • Formaldehyde
    Formaldehyde
    Formaldehyde is an organic compound with the formula CH2O. It is the simplest aldehyde, hence its systematic name methanal.Formaldehyde is a colorless gas with a characteristic pungent odor. It is an important precursor to many other chemical compounds, especially for polymers...

  • Glutaraldehyde
    Glutaraldehyde
    Glutaraldehyde is an organic compound with the formula CH22. A pungent colorless oily liquid, glutaraldehyde is used to disinfect medical and dental equipment...


Chaotropic agents


Chaotropic agent
Chaotropic agent
A denaturating agent is a substance which disrupts the three dimensional structure in macromolecules such as proteins, DNA, or RNA and denatures them. A denaturating agent is a chaotropic agent, but chaotropic agents aren't necessarily denaturating agents...

s include:
  • Urea
    Urea
    Urea or carbamide is an organic compound with the chemical formula CO2. The molecule has two —NH2 groups joined by a carbonyl functional group....

     6 - 8 mol/l
  • Guanidinium chloride
    Guanidinium chloride
    Guanidinium chloride or guanidine hydrochloride, usually abbreviated GdmCl and sometimes GndCl or GuHCl, is the hydrochloride salt of guanidine.-Use in protein denaturation:...

     6 mol/l
  • Lithium perchlorate
    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.-Uses:...

     4.5 mol/l

Disulfide bond reducers


Agents that break disulfide bond
Disulfide bond
In chemistry, a disulfide bond is a covalent bond, usually derived by the coupling of two thiol groups. The linkage is also called an SS-bond or disulfide bridge. The overall connectivity is therefore R-S-S-R. The terminology is widely used in biochemistry...

s by reduction include:
  • 2-Mercaptoethanol
    2-Mercaptoethanol
    2-Mercaptoethanol is the chemical compound with the formula HOCH2CH2SH. It is a hybrid of ethylene glycol, HOCH2CH2OH, and 1,2-ethanedithiol, HSCH2CH2SH...

  • Dithiothreitol
    Dithiothreitol
    Dithiothreitol is the common name for a small-molecule redox reagent known as Cleland's reagent. DTT's formula is C4H10O2S2 and the molecular structure of its reduced form is shown at the right; its oxidized form is a disulfide-bonded 6-membered ring . Its name derives from the four-carbon...

  • TCEP
    TCEP
    TCEP is a reducing agent frequently used in biochemistry and molecular biology applications. It is often prepared and used as a hydrochloride salt with a molecular weight of 286.65 gram/mol...

     (tris(2-carboxyethyl)phosphine)

See also

  • Protein folding
    Protein folding
    Protein folding is the process by which a protein structure assumes its functional shape or conformation. It is the physical process by which a polypeptide folds into its characteristic and functional three-dimensional structure from random coil....

  • Random coil
    Random coil
    A random coil is a polymer conformation 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 chains in a population of macromolecules...

  • Fixation (histology)
    Fixation (histology)
    In the fields of histology, pathology, and cell biology, fixation is a chemical process by which biological tissues are preserved from decay, thereby preventing autolysis or putrefaction...

  • Denatured alcohol

External links