Secondary, tertiary and quaternary structure of proteins can be disrupted by chemicals like urea (6mol/L), 5M guanidinium hydrochloride, heat, high and low pH, detergents such as sodium dodecyl sulphate (1%) and sulfhydryl reagents such as mercaptoethanol.
pH variations alter the ionization states of amino acid side chain, which changes protein charge resulting in the precipitation of proteins.
High temperature disrupts a variety week interactions of proteins, hence, proteins lose the solubility and are then precipitated. An example is the denaturation of proteins caused by heating egg.
Sodium dodecyl sulphate alters the protein structures by interacting with the non-polar residues of proteins thereby interfering with the hydrophobic interactions.
High concentration of water-soluble organic substances such as aliphatic alcohols also interacts with hydrophobic forces.
Solute such as urea can also precipitates proteins by disrupting hydrophobic interactions.
The above conditions overcome the weak forces on which polypeptide chains are folded and disrupt hydrogen bonds causing polypeptides to unfold. Such unfolding is called denaturation. It is accompanied by loss of the normal biological function (e.g. enzyme activity) of the protein.
Denatured proteins are usually not soluble in water, in part because denaturation exposes internal hydrophobic R groups. Denaturation does not break the primary structure of protein.
Renaturation of proteins
If a denatured protein returns to its native state, after the removal of denaturing agent, the process is called as renaturation.
Amphoteric nature
Due to the presence of –NH2 and –COOH group proteins are amphoteric.
Charges on proteins are mainly due to the presence of the side chains of the amino acid residues, the N – terminal amino group and the C – terminal carboxyl group. The other carboxyl and amino group of each amino acids are involved in the peptide bond formation. Therefore, pI of proteins depends on the pH of the solution and pKa of amino acids forming the proteins.
In solutions with pH values above the isoelectric point, the protein will have a net negative charge and at lesser pH values it will be positively charged.
More proteins have pI values below 7(due to presence of aspartate and glutamate), so that most proteins are negatively charged at neutral pH.
Precipitation of proteins
At isoelectric pH the proteins exist as zwitter ion (net charge is ‘0’) at this pH the proteins are easily precipitated and it also shows no migration under an electric field.
Proteins can be easily precipitated from solution by the addition of sulphosalicylic acid and phosphotunstic acid or by the addition of heavy metals like lead, silver and mercury. The principle behind this precipitation is by removing charges on the protein molecules.
Alcohol also precipitates protein by reducing the dielectric constant of water (charges).
Salting out
Solubility of proteins is decreased by increasing the concentration of salt such as (NH4)2SO4. The salts gradually dehydrate proteins by binding to water. The dehydrated proteins aggregate and precipitates.
Salting in
Solubility of some proteins is increased by increasing the concentration of neutral salts. This process is known as salting in.
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