The below mentioned article provides a quick note on Denaturation of Proteins.
Denaturation of Proteins:
Denaturation may be defined as the disruption of the secondary, tertiary and quarternary structure of the native protein resulting in the alterations of the physical, chemical and biological characteristics of the protein by a variety of agents.
The native proteins are said to be the proteins occurring in animal and plant tissues. They possess many characteristic properties such as solubility, viscosity, optical rotation, sedimentation rate, electrophoretic mobility etc. For an oligomeric protein, denaturation may involve dissociation of the protomers with or without subsequent unfolding or with or without undergoing changes in protomer conformation.
1. Physical agents:
Heat, surface action, ultraviolet light, ultrasound, high pressure etc.
2. Chemical agents:
Acids, alkalis, heavy metal salts, urea, ethanol, guanidine detergents etc. Urea and guanidine probably interfere with the hydrogen bonds between peptide linkages. Acids and alkalis probably attack directly the hydrogen bonds in the secondary and tertiary structure of proteins.
Many proteins, especially of the globular type, can be crystallized in the native state. But denatured proteins cannot be crystallized.
The denatured protein is greatly decreased in solubility at its isoelectric point. The chemical groups are exposed to chemical reactions and more readily detected as a result of the unfolding process in denaturation. Among these are sulphydryl group of cysteine, the disulphide group of cystine and the phenolic group of tyrosine.
The digestibility of certain denatured proteins by proteolytic enzymes is increased. Enzymatic or hormonal activity is usually destroyed by denaturation. The antigenic or antibody functions of proteins are frequently altered.
If the denaturation is severe, the protein molecules become insoluble and precipitation results as well as the changes in the properties of the proteins are permanent and “irreversible”. In case of mild denaturation, there is “reversible denaturation” leading to the slight changes in the properties of the protein which can be restored to the native state after suitable treatment.
1. The precipitation of the native protein as a result of denaturation is used to advantage in the clinical laboratory.
2. Blood or serum samples to be analysed for small molecules (e.g., glucose, uric acid, drugs) generally are first treated with acids such as trichloroacetic acid, phosphotungstic acid or phosphomolybdic acid to precipitate most of the proteins present in the sample. This is removed by centrifugation and the protein-free supernatant liquid is then analysed.
3. Denaturation is used to know the enzyme catalysed reaction of an extract at the loss of the enzyme activity when boiled or acidified.
Denaturation and Renaturation of Proteins:
Bovine ribonuclease of single polypeptide chain of 124 amino acid residues with small molecular weight contains four disulphide bonds. When it is treated with β-mercaptoethanol in 8 M. urea, the disulphide bonds are reduced to -SH groups as a result of the denaturation of the enzyme and the enzyme activity is also lost.
Denatured ribonuclease, when freed from urea and β-mercaptoethanol by dialysis, slowly regains enzymic activity as the SH groups are oxidized by oxygen of air to form S-S bonds. But if the reduced ribonuclease in 8 M. urea solution is re-oxidized it loses its enzymic activity almost completely as wrong disulphide bonds are formed resulting in ‘scrumbled’ ribonuclease.
Similarly, when egg albumin is heated till it is coagulated, the denaturation is irreversible and the secondary and tertiary structure of the proteins are completely lost resulting in a mixture of randomly arranged polypeptide chains.