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. ____________________________________________________________________________________
Discussion of Data and Results
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. 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. pH 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...