Protein Denaturation

AMINO ACIDS AND PROTEINS

Proteins are one of the main constituents of living cells. The functions of

proteins include building of new cells, the maintenance of cells and the

replacement of old cells. All proteins contain carbon, hydrogen, oxygen and

nitrogen. Most proteins contain sulfur. Elements like phosphorus, iron etc is

present in some special proteins. Some examples of proteins are albumin (present

in eggs), casein (present in milk), collagen (in tissues) and hemoglobin (present in

human blood).

A protein is defined as a polymer made up of several amino acids. An amino

acid is a compound that contains two functional groups, an amino group (-NH2) and

a carboxylic acid group (-COOH). The side chain (R) distinguishes the amino acids.

Some examples of amino acids are alanine, valine, cysteine, and tryptophan.

Glycine is the simplest amino acid known.

Amino

acid

R group

Glycine -H

Alanine - CH3

Valine

Cysteine

Amino acids have both acidic and basic properties. The amino group (-NH2)

lends basic properties whereas the carboxylic acid group (-COOH) lends acidic

properties to the amino acid.

Several amino acids combine to form peptide bonds. Proteins contain

polypeptide units (several peptide units). When a protein is hydrolyzed, it breaks

down into smaller units (tri and dipeptides), eventually forming amino acids.

Structures of proteins can be classified into various levels, primary, secondary,

tertiary and quaternary structure. The sequence of amino acids present in a

protein is known as its primary structure. The regularly repeating order and spatial

arrangements of amino acids in a protein is known as its secondary structure. The

overall three dimensional shape of the protein is called its tertiary structure.

Quaternary structure is the organization among the various polypeptide chains in a

protein. The biochemical function of the protein depends on the three dimensional

structure of the protein.

Denaturation of proteins

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.

Effects Of Denaturation on Proteins

The function principle 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.

1\ If the protein functioned as an enzyme, then denaturation causes it to lose its enzymatic activity.

2\ If the protein was embedded in a cell membrane where it transported ions or molecules through the membrane, then denaturation destroys that ability.

3\ If the protein was an antibody, responsible for recognizing an infectious agent, denaturation will destroy that protective ability.

Consequences Of Denaturation

Loss of Biological Activity

Loss of solubility.

Destruction of toxins.

Improved Digestibility.

Altered Water Binding Capacity.

Factors Of Denaturation

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 below 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 .

Heavy Metal Salts:

Heavy metal salts act to denature proteins in much the same manner as acids and bases. Heavy metal salts usually 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 metals may also disrupt disulfide bonds because of their high affinity and attraction for sulfur and will also lead to the denaturation of proteins.

Definitions

coagulation : The collecting into a mass of minute particles of a solid dispersed throughout a liquid, usually followed by the precipitation or separation of the solid mass from the liquid. The casein in milk is coagulated (curdled) by the addition of acetic acid or citric acid. The albumin in egg white is coagulated by heating. The clotting of blood is another example of coagulation. Coagulation usually involves a chemical reaction.

Aggregation: Aggrigation is a general term that encompasses several types of interactions or characteristics.Protein aggregation is the aggregation of mis-folded

protein and is thought to be responsible for many degenerative dieses such as Alzheimer's

Gelitation:Gelatine is a substantially pure protein food ingredient, obtained by the thermal denaturation of collagen , which is the structural and most common protein in the animal kingdom. Today gelatine is usually available in granular powder form, although in Europe, sheet gelatine is still available.

Other Denaturation Factors

Mechanical Treatments:

Whipping ( Egg) .

Ultraviolet.

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.

Summary

Disruption of secondary, tertiary and quaternary protein structure by

heat/organics

Break apart H bonds and disrupt hydrophobic attractions

acids/ bases

Break H bonds between polar R groups and ionic bonds

heavy metal ions

React with S-S bonds to form solids

agitation

Stretches chains until bonds break

University Of Khartoum

Animal Production Faculty

MSC Dairy Production & Technology (Batch7)

Protein Denaturation

Prepared By:

Ahmed Munir Yousif Elhakim

Somaya Ahmed Kashif A.Kareem

Safa A.Raheem Abdallah

September 2010