Denaturation of proteins.
The structure/function principle we covered in class tells us that changing the structure of a protein will affect its function. Often that means that function is lost. "Denaturation" of a protein means loss of the protein's function due to structural change in the protein caused by some chemical or physical factor such as high temperature or unfavorable pH. The factor causes the folded protein (the tertiary structure) to unfold, to unravel. If the protein functioned as an enzyme, then denaturation causes it to lose its enzymatic activity. If the protein was embedded in a cell membrane where it transported ions or molecules through the membrane, then denaturation destroys that ability. If the protein was an antibody, responsible for recognizing an infectious agent, denaturation will destroy that protective ability. Though there are exceptions, as a rule denaturation by factors such as heat, high or low pH, or exposure to organic solvents (alcohol, e.g.) is irreversible. That is, removal of the "offending" factor will not cause the protein to fold back into its original shape and resume its function. Protection of proteins against denaturation is one result of the buffering of biological solutions such as blood and the aqueous interior of living cells. If blood pH changed much from its normal value, proteins in the blood would begin to pucker, buckle, twist into different shapes, and unravel, with potential loss of function.
To understand how factors such as high temperature and high or low pH cause denaturation, you need to consider the forces that hold a protein in its folded 3-D shape (secondary structure, tertiary structure and, if applicable, quaternary structure) and how those forces would be affected by the changes in temperature or pH. For example, H bonds, such as in C=O∙∙∙∙H-N, are important in 2°, 3°, and 4° structure. As the temperature of a solution containing the protein is raised, the extra heat causes twisting, rotating, and bending of bonds and functional groups within the molecule; the higher the temperature, the more of this there is. Since individual H bonds are weak, they are easily broken. When one is ruptured, then others nearby are more susceptible to rupture.
As another example, ionic interactions occur between charged amino and carboxyl groups in the side chains of some amino acids in the folded protein. If the pH of the solution is changed, some of those functional groups will gain or lose a proton (recall reversible dissociation) and, therefore, will lose their charge or become charged, depending on which way the pH is changed and by how much. That will eliminate some, perhaps many, of the ionic interactions that were necessary for maintenance of the folded shape of the protein.
Finally, exposure of a protein to an organic solvent, such as an alcohol, may also cause denaturation. Recall the hydrophobic effect in tertiary structure. The surface of the protein, in contact with water, will bristle with lots of hydrophilic (polar) amino acid side chains, while the hydrophobic (nonpolar) amino acid side chains tend to be tucked away toward the protein's interior. That interaction with the surrounding solvent (water) is an important factor in determining the ultimate stability of the folded protein. When an organic solvent such as an alcohol is mixed with the aqueous solution, the amino acid side chains on the surface don't dissolve in the water-alcohol mixture as well. The protein molecule twists and flexes (through free bond rotations within) as the hydrophilic side chains shun the alcohol. At the same time some interior hydrophobic side chains twist to the surface where they interact favorably with the organic solvent. The net effect is as though the protein molecule were trying to turn itself inside out in response to the change in the surrounding solvent. In the process H bonds and ionic interactions will be broken. The result is a structurally altered, perhaps unraveled, protein molecule.
As for why denaturation tends to be irreversible as a rule, consider a familiar case. Think for a moment about frying an egg. The "white" before cooking isn't white; it's transparent and viscous. It's an aqueous solution of egg protein (ovalbumin) and a bit of salt. When you apply heat to the skillet, the ovalbumin is denatured, and the molecules stick to each other, forming a dense network. To the eye, you see that the transparent solution becomes white, opaque, and rubbery. The yolk, which also contains protein, undergoes a similar transition from fluid to solid, for the same reason. Now, can you reverse this denaturation by letting the egg cool? A denatured protein represents a great many disrupted bonds and interactions. It is unlikely that all of those would reform in the same way to regenerate the original folded structure of the molecule. Read what your textbook says about chaperonins. When cells make proteins, the folding process is not random.
| Introduction: Denaturation of proteins involves the disruption and possible destruction of both the secondary and tertiary structures. Since denaturation reactions are not strong enough to break the peptide bonds, the primary structure (sequence of amino acids) remains the same after a denaturation process. Denaturation disrupts the normal alpha-helix and beta sheets in a protein and uncoils it into a random shape. Denaturation occurs because the bonding interactions responsible for the secondary structure (hydrogen bonds to amides) and tertiary structure are disrupted. In tertiary structure there are four types of bonding interactions between "side chains" including: hydrogen bonding, salt bridges, disulfide bonds, and non-polar hydrophobic interactions. which may be disrupted. Therefore, a variety of reagents and conditions can cause denaturation. The most common observation in the denaturation process is the precipitation or coagulation of the protein. Heat: Heat can be used to disrupt hydrogen bonds and non-polar hydrophobic interactions. This occurs because heat increases the kinetic energy and causes the molecules to vibrate so rapidly and violently that the bonds are disrupted. The proteins in eggs denature and coagulate during cooking. Other foods are cooked to denature the proteins to make it easier for enzymes to digest them. Medical supplies and instruments are sterilized by heating to denature proteins in bacteria and thus destroy the bacteria. |
Alcohol Disrupts Hydrogen Bonding:
Hydrogen bonding occurs between amide groups in the secondary protein structure. Hydrogen bonding between "side chains" occurs in tertiary protein structure in a variety of amino acid combinations. All of these are disrupted by the addition of another alcohol.
A 70% alcohol solution is used as a disinfectant on the skin. This concentration of alcohol is able to penetrate the bacterial cell wall and denature the proteins and enzymes inside of the cell. A 95% alcohol solution merely coagulates the protein on the outside of the cell wall and prevents any alcohol from entering the cell. Alcohol denatures proteins by disrupting the side chain intramolecular hydrogen bonding. New hydrogen bonds are formed instead between the new alcohol molecule and the protein side chains.
In the prion protein, tyr 128 is hydrogen bonded to asp 178, which cause one part of the chain to be bonding with a part some distance away. After denaturation, the graphic show substantial structural changes.
Acids and Bases Disrupt Salt Bridges:
Salt bridges result from the neutralization of an acid and amine on side chains. The final interaction is ionic between the positive ammonium group and the negative acid group. Any combination of the various acidic or amine amino acid side chains will have this effect.
As might be expected, acids and bases disrupt salt bridges held together by ionic charges. A type of double replacement reaction occurs where the positive and negative ions in the salt change partners with the positive and negative ions in the new acid or base added. This reaction occurs in the digestive system, when the acidic gastric juices cause the curdling (coagulating) of milk.
The example on the left is from the prion protein with the salt bridge of glutamic acid 200 and lysine 204. In this case a very small loop is made because there are only three other amino acids are between them. The salt bridge has the effect of straightening an alpha helix.
The denaturation reaction on the salt bridge by the addition of an acid results in a further straightening effect on the protein chain as shown in the graphic on the left.
Prion Protein
Heavy Metal Salts:
Heavy metal salts act to denature proteins in much the same manner as acids and bases. Heavy metal saltsusually contain Hg+2, Pb+2, Ag+1 Tl+1, Cd+2 and other metals with high atomic weights. Since salts are ionic they disrupt salt bridges in proteins. The reaction of a heavy metal salt with a protein usually leads to an insoluble metal protein salt.
This reaction is used for its disinfectant properties in external applications. For example AgNO3 is used to prevent gonorrhea infections in the eyes of new born infants. Silver nitrate is also used in the treatment of nose and throat infections, as well as to cauterize wounds.
Mercury salts administered as Mercurochrome or Merthiolate have similar properties in preventing infections in wounds.
This same reaction is used in reverse in cases of acute heavy metal poisoning. In such a situation, a person may have swallowed a significant quantity of a heavy metal salt. As an antidote, a protein such as milk or egg whites may be administered to precipitate the poisonous salt. Then an emetic is given to induce vomiting so that the precipitated metal protein is discharged from the body.
Heavy Metal Salts Disrupt Disulfide Bonds:
Heavy metals may also disrupt disulfide bonds because of their
high affinity and attraction for sulfur and will also lead to the
denaturation of proteins.
Reducing Agents Disrupt Disulfide Bonds:
Disulfide bonds are formed by oxidation of the sulfhydryl groups on cysteine. Different protein chains or loops within a single chain are held together by the strong covalent disulfide bonds. Both of these examples are exhibited by the insulin in the graphic on the left.If oxidizing agents cause the formation of a disulfide bond, then reducing agents, of course, act on any disulfide bonds to split it apart. Reducing agents add hydrogen atoms to make the thiol group, -SH. The reaction is :Insulin Protein
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