Denaturation (biochemistry)
Denaturation refers to a structural modification of biomolecules such as for example, proteins ( proteins) or deoxyribonucleic acid ( DNA), which is connected, in most cases, a loss of the biological function of these molecules. Denaturation may be due to either physical or chemical influences. Denaturation at the primary structure remains unchanged.
- 3.1 Acid and alkali denaturation
- 3.2 denaturation by salts
- 3.3 denaturation by ethanol
- 3.4 denaturation by pure water
- 3.5 denaturation by modification and networking
Principle of denaturation
Occurs caused by external influences change in the protein structure, particularly secondary and tertiary structure of a protein (and thus possibly also of its quaternary structure ), without changing the sequence of amino acids ( primary structure). The protein loses its original folding shape, which is also referred to as a native configuration or conformation. The process can be reversible ( reversible ) or irreversible ( irreversible ). The reversal of the denaturation is also called renaturation. Reversible changes in molecular structure are, for example, in the thermal denaturation of DNA, if it is heated during a polymerase chain reaction (PCR) and cooled. For the irreversible change in molecular structure of the original three-dimensional structure of the molecule can not be restored. This happens for example in cooking the Frühstückeis, which can not be cooked again soft when it is first of all become a " hard-boiled egg ." The denaturation of proteins usually leads to the fact that the molecule is inactivated, that is, that the molecule its biological function can not run to a lesser extent or more only.
Denaturierungsvorgängen all have in common that covalent bonds (except for the disulfide bonds in proteins ) not cleaved. The chain structure, and thus the sequence of blocks ( primary structure) is thus obtained. By supplying energy but are the individual building blocks, namely nucleotides and amino acids, or even the whole molecular chain, so much brought into vibration that bonds and acting forces (ionic, polar and van der Waals interactions, hydrogen bonds, hydrophobic effects ) can be split or suspended between different areas of the molecular chain. Disulfide bridges are commonly cleaved by reduction with sulfhydryls.
Denaturation by physical influences
The most common denaturation under physical influence of the heat denaturation and the Strahlungsdenaturierung. Physically, denaturation next to be caused by high pressure, strong stirring, shaking, through the action of ultrasound and by interfacial absorption.
Heat denaturation
The heating or thermal denaturation is a denaturation of the type in which a change in the molecular structure is brought about by an increase in temperature. In this case, no covalent chemical bonds by the action of heat usually broken or formed, ie the primary structure remains unchanged. Instead, hydrogen bonds are broken or formed, which are usually bonds between chain segments, which often occurs a change in the tertiary structure in enzymes and other proteins. This usually has a loss of biological activity and a decrease in solubility result. The latter becomes then as " flocculation " or " coagulation " noticeable. Since the protein folding and hydrophobic effects play a role, the denaturation is also generated by the removal of the hydrophobic effect of increasing temperature. A heat denaturation (like other denaturing ) be reversible when the structural changes are not too profound, but often it is irreversible ( irreversible ). The temperature at which denaturation of proteins occurs is quite different depending on the structure and the body. The enzymes hyper- thermophilic archaea have to withstand temperatures well above 80 ° C.
Autoclaving pathogens are inactivated on objects by means of denaturing of vital biopolymers. When autoclaving temperature must be maintained well above 100 ° C at elevated pressure for a predetermined time to safely sterilize.
Denature nucleic acids within a fairly narrow temperature interval, also called " melting point", which is usually above 80 ° C. The denaturation is reversible. By cooling the nucleic acids, the single strands lie down together again. This operation makes it in molecular biology in performing PCR in order to reproduce specific genes from an organism in vitro: DNA extracted at high temperatures in a reaction vessel, melting (denaturation ). Then the temperature is lowered again to a predetermined temperature. This annealing temperature is dependent on the primers and typically is 2-3 ° C below its melting point ( 50-65 ° C). The primer templates contained in the solution are deposited ( called annealing or primer hybridization ) to the DNA single strands at. Then, with the help of a Taq polymerase, the strands again completed ( elongation). The cycle of denaturation, annealing and elongation starts again. It will be carried out for about 25 to 50 cycles. So you go to the reversible denaturation of DNA up to 50 times advantage of in order to reproduce a sought gene of an organism.
Denaturation by pressure
Since the reaction volumes are very small in protein folding, you have to spend cash to unfold proteins in the control pressure of several 1000. Nevertheless, in practice, the high-pressure treatment of foods is becoming increasingly important. For this purpose, the food, usually packed in foil, in a printing medium, such as Of water was added, and the pressure is applied to this medium. In this " non-thermal process ," one high pressure pasteurization, undesirable microorganisms and enzymes are inactivated and made the food preserved. Loss of quality, as the application of heat are avoided.
In general, the tertiary and quaternary structure of proteins is influenced by the pressure, while the secondary structure may be hardly changed.
Denaturation by high-energy radiation
As the transfer of energy by means of infrared light, any other energy transfer, for example, act by means of UV light, microwaves, or other radiation, denaturing. However, high-energy radiation such as UV light, gamma and X-rays in particular especially interact with covalent bonds of nucleic acids and lead to continued fractions ( depolymerization ). Conversely, high-energy radiation also cause additional covalent bonds (eg dimerization in nucleic acids).
Denaturation by chemical influences
Cause of protein denaturation may be, for example, chemical substances such as acids, bases, salts, detergents (for example, one percent solution of sodium lauryl sulfate solution), urea or guanidinium salts. Protein structures can also be affected by heavy metals, as ion complex structures form with the amino acid residues and thus changing the structure of the biologically active protein.
Acid and alkali denaturation
The acid denaturation leads to charge transfers between the molecules and in the end the same result as the heat denaturation, a refolding of the protein in the under the respective conditions energetically favorable state. The acid are protons (H ), thus causing the change in charge in the protein structure, so that the hydrogen bonds are partially destroyed and the like charges repel each other. Additionally gives the acid protons (H ) from the carboxylate group (COO - ), so that a carboxyl group -COOH is formed and the previous negative charge disappears. This means that no ionic interaction between the carboxy group and the positive charges in the protein are possible.
The same can cause alkaline solutions, they also change the composition of the ions on the pH, but amino groups of lysine or arginine are deprotonated, resulting in less positive charges occur in the protein that could interact with negatively charged groups. In addition, the carboxylic acid groups are deprotonated to carboxylate, which hydrogen bonds can be destroyed and occur more negative charges in the protein, which repel each other.
Wherein the acid or can simultaneously Laugendenaturierung hydrolysis of the protein occurs.
Denaturation by salts
Salts have an influence on the hydrophobic effect, and therefore can cause denaturation, which can also go in the direction of restoration depending on the salt, the influence. One speaks then, with respect to the precipitation, by " salting " and " salting out ". The relative influence of the salts forming anions and cations is described by the " Hofmeister series ".
Denaturation by ethanol
According to the acid denaturation of ethanol or other water- soluble organic solvent may interfere with the necessary in biopolymers maintain the structure of hydrogen bonds and hydrophobic interactions by interfering as polar organic solvent. 50 - to 70 -percent ethanol denatured most proteins and nucleic acids. Since by the release of the membrane lipids and the destruction of the spatial structure, the membrane proteins lose their function and burst the cells in question seifenblasen like, can so with higher percent alcohol disinfected ( eg, ethanol, isopropanol): Bacterial and fungal cells are on the denaturation of their membrane proteins irreversibly inactivated enveloped viruses, their lipid envelope are accordingly deprived in the sit Andockproteine .
Denaturation by pure water
Proteins are present in their natural environment in the presence of other proteins, dissolved salts, cofactors or metabolites before that stabilize the native protein structure in a more or less complex manner. If you remove salts and other small molecules by dialysis of a protein solution against doubly distilled water - preferably in the cold - one can often achieve selective ( and reversible) denaturation mainly of large proteins that are precipitated under these conditions ( precipitate ).
Denaturation by modification and networking
By the use of molecular markers Fixierungsslösungen, covalent crosslinking (for example, formaldehyde, paraformaldehyde or glutaraldehyde ), and solutions forming by stable complexes of heavy metal ions the catalytic center or binding site of a protein is changed such that some functions are not satisfied occasionally. In this case, the protein is not (as in the chaotropic or pH-dependent denaturation) unfolds it can, however, be altered or fixed in a non - native conformation and lose functionality. Remain unaffected necessary functions of the protein from the fixation, as well as other properties such as the biological half-life can also be changed, refer networking. In the course of antigen retrieval is trying to make the effects of fixation reversed.
Renaturation
After denaturation of a protein (e.g. an enzyme ) during the purification of a protein mixture, a return of the protein in the native form is necessary for the measurement of biological activity. However, this is only for proteins whose native conformation is also the lowest energy state under isotonic conditions, but not in metastable proteins. Renaturation can be achieved by slow dilution of the denaturant, accompanied by a recovery of the cofactors and of the isotonic environment. A reconstitution can occur following.
Demarcation from other changes
Are referred to as non- denaturing the proteins mediated by structural changes:
At very high temperature, it may also lead to the cleavage of covalent bonds and thus continued fractions ( depolymerization ). Such changes in the primary structure are not counted among the denaturation. Similarly acids as alkaline solutions can result in high concentrations and reaction temperatures for the cleavage of covalent bonds. Hydrolysis then changes the primary structure. Such changes of the primary structure are ordinary chemical reactions and are not expected to denaturation.
A borderline case is the cleavage of disulfide bridges between two protein strands. Here, although a covalent chemical bond is broken, the amino acid sequence in each strand is retained, however, so one such reductive cleavage of disulfide bonds, which is in principle reversible, to the denaturation.