Denaturation of Proteins

2 February 2017

This experiment aimed to study the effect of various denaturants on albumin and casein protein extracts through viscosity measurements. 5 mL samples of native and denatured protein solutions were prepared, using ? -mercaptoethanol, urea and SDS as denaturants for albumin, and NaOH, NaCL, HCL, ? -mercaptoethanol, urea and SDS for casein. 5 mL blank solutions for each denaturant used were also prepared. The viscosity of the solutions were determined using Ostwald viscometer.

Denaturation of proteins is a reversible—and sometimes irreversible—process that 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. Generally, protein denaturation is to be avoided, since proteins are best studied as close to their native state as possible.

Denaturation of Proteins Essay Example

However, denaturation is sometimes done deliberately. For example, in determining the rates of enzyme reactions, proteins are quickly denatured to stop enzyme reactions. Also, to study the detailed nature of the unfolding and refolding of their polypeptide chains, proteins are deliberately denatured. Denaturation of proteins usually result to decreased solubility, altered water binding capacity, loss of biological activity, destruction of toxins, increased intrinsic viscosity, and inability to crystallize.

That way, this process can be a useful way in separating proteins from other classes of biological molecules during purification. In tertiary structures, four types of bonding interactions between “side chains” occur—hydrogen bonding, non-polar hydrophobic interactions, salt bridges, and disulfide bonds. Denaturation occurs because the bonding interactions responsible for the secondary structure and tertiary structure are disrupted. A variety of agents can cause the denaturation of proteins. One of them is heat.

An increase in temperature affects the interactions of the tertiary structure by making the molecule vibrate violently and disrupting the hydrophobic interactions and hydrogen bonds. Microwave radiation and ultraviolet radiation are agents that also operate much like the action of heat. Violent whipping causes molecules in globular shapes to extend to longer lengths and then entangle. Detergents, such as sodium dodecyl sulfate (SDS), have hydrophobic tails which penetrate the interior of the protein and disrupt the hydrophobic interactions of the proteins.

High concentrations of chaotropic agents such as guanidine hydrochloride and urea cause denaturation by forming competing hydrogen bonds with the amino acid residues of the peptide chain, thereby disrupting the internal hydrogen bonding that stabilizes the native structure. Organic solvents such as ethanol or acetone interfere with the hydrogen bonds in the protein by also forming hydrogen bonds and disrupt the hydrophobic interactions of the peptide chain.

These solvents can quickly denature proteins in bacteria, killing them. H is also an agent of denaturation. At either low or high extremes of pH, at least some of the charges of the protein are missing, and so electrostatic interactions that would normally stabilize the native protein are drastically decreased. This can be achieved by adding strong acids or bases, like HCl or NaOH, which disrupt hydrogen bonds and salt bridges. In an acidic environment, acidic groups are protonated and the conformations stabilized by the carboxylate groups are destroyed. In alkaline environments, the amino groups are deprotonated.

Salts of heavy metals such as Hg2+, Ag+, and Pb+ combine with SH groups and form precipitates. Reducing agents like mercaptoethanol or other thiol reagents reduce disulfide bonds to sulfhydryl groups. Urea is usually added to the reacting mixture to facilitate unfolding of the protein and to increase the accessibility of the disulfides to the reducing agent. In the first part of the experiment, 5 mL samples of 1% albumin and 0. 01% casein were prepared. 5 mL samples of blank solution, containing the solvent of the proteins and the denaturant were prepared. mL samples of denatured protein were also prepared.

Denaturants used were ? -mercaptoethanol, urea and SDS for albumin, and NaOH, NaCL, HCL, ? -mercaptoethanol, urea and SDS for casein. Denaturation of proteins can be characterized by measurable physical and chemical changes in the protein. Methods such as circular dichroism, viscometry, nuclear magnetic resonance spectroscopy and flourences can be used for characterization. In this experiment, viscometry was applied to study the effects of several denaturants in protein standard and protein extracts.

In viscometry, the viscosity (resistance of liquid to flow) of the protein sample is studied in order to monitor the denaturation process. Viscosity of a liquid may depend on certain factors such as density, temperature, solute concentration, size and shape of molecules and intermolecular interactions. Generally, a longer fibrous-like protein has a higher viscosity than a globular protein. Denaturation uncoils and changes the globular proteins into fibrous structures with higher frictional coefficient, denaturation tends to increase viscosity. Viscosity measurements were obtained using Ostwald viscometer.

The viscosity of the native protein was taken first. It was aspirated throught the viscometer until the solution level was above the first mark. Bubbles were avoided during aspiration to prevent discrepancies in time measurements. The time interval from when the solution passed the first mark up to when it passed through the second mark was recorded. The same procedure was done for the blank solution, then for the denatured protein.

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