Proteins are large polypeptides of defined amino acid structure as defined in its amino acid sequence though it does

not exist as long chains of polypeptide structures but rather as compact, folded structures. Particular structures of a protein in nature determine its function by its conformation or its three-dimensional shape (Garrett, et.al., 2005). Bonds such as peptide bonds in primary structure, Hbonding of carboxyl and amino groups in secondary structures, and H-bonding of the side chains, hydrophobic interaction, salt linkages, and disulfide bonds in tertiary structures contribute to the folding of protein. Interactions such as H-bonding are relatively not as strong as covalent chemical bonding that modest stresses can disrupt the bonds. Since the hydrogen bonding interactions are weak for the secondary and tertiary structures, denaturation occurs, a process where any non-proteolytic modification of the unique structure of native proteins, giving rise to definite changes in chemical, physical, or biological properties (Neurath, et.al., 1944 as cited in R. Gortner, 1949). This also means that denaturation does not involve addition or removal of protons from the native protein. Primary structures are not involved in the breakage of bonds due to the disulfide bridges that covalently link amino acids. The disruption of protein structure is carried by physical and chemical agents called denaturants. Application of mechanical stress such as stirring can disrupt the weak forces i.e., H-bonding in carboxyl and amino groups between protein structures. High temperature increases the kinetic energy between molecules which then produces high-energy vibration, therefore, breaking the bondsprimarily hydrogen bonds. Treatment with acids, bases, salts, alcohol, urea, detergents such as sodium dodecyl sulfate (SDS), CH3(CH2)11- O- SO3Na, reducing agents and other organic solvents induce denaturation (West, 1966). Denaturation of globular protein such as egg albumin leads to an unfolding and uncoiling of peptide chains which is due to the rupture of cross linkages that hold them together (West, 1966). As a result, the globular protein changes to an elongated, fibrous structure. Here, an increase in viscosity is observed. Fluctuations in pH due to the addition of strong acids and bases affect the groups that compose the protein, changing the net charge. These changes or the absence of one or more charge can affect the protein folding by repulsion of charges and the hydrophobic interactions, causing denaturation. In most experiments, strong acids and bases that are used are usually hydrogen chloride or sodium hydroxide respectively. Hydrophobic bonds in globular protein backbones can also be broken by organic solvents like alcohol and acetone. New bonds form between the organic solvent and the non polar protein side chain by disturbing the intramolecular hydrogen bonds. Some reagents disturb the interaction between peptide chains and form hydrogen bonds with the denaturing reagent because the interactions they made are stronger than those in the protein, and thus promote denaturation. Examples of these are urea and guanidino hydrochloride. Sodium dodecyl sulfate (SDS) also acts the same way as urea and guanidine hydrochloride that it disrupts the electrostatic interactions within the protein. Salts, such as NaCl, allow the protein to salt out, which causes precipitation by the binding of the salt ions to the charged side chains. Heavy metal ions form complexes with the ionic bonds in protein. Reducing agents such as -mercaptoethanol reduce disulfide bonds to sulfhydryl groups. Adding
a hydrogen atom forms a thiol group (-SH) thus reducing the protein.

Many proteins are indigestible by the proteolytic enzyme, trypsin (White, et.al., 1959). Example of this protein is the egg albumin. In contrast, when denatured it is easily attacked by the enzyme. Loss of solubility is also observed in the denaturation process. In effect, it exposes the hydrophobic chain and unfolds the protein. This alterations in the structure of the protein, alters their function as illustrated in these examples. Physical appearance such as texture is also observable in denaturation. There are cases that this process is reversible. A process called renaturation.

Figure I. Denaturation Process In the experiment, albumin, a protein from egg was extracted to observe the effects of denaturants through the measurement of its viscosity. Viscosity describes the resistance of a solution to flow. This accounts as an indicator that the native protein takes on its denatured form. Denatured proteins unfold into stretched polypeptide chains that randomly cluster to form aggregates thus increasing the viscosity of the solution. The exposed side chains interact with the viscometer, thus slowing down the rate of flow. Generally, denatured proteins are more viscous than the native protein itself. The denaturant that gives a more viscous solution is considered to be more effective than the one less viscous. The experiment tests the effectivity of six denaturants namely, hydrochloric acid, sodium hydroxide, sodium chloride, urea, SDS, -mercaptoethanol.

RAQUELS PART METHODOLOGY

Ostwald viscometers, also known as glass tube viscometers, are used to measure fluid viscosity. The solution was loaded into the larger tube of the viscometer and allowed to stay in the bulb. The solution was aspirated into the smaller bulb until the upper mark was reached. (Insert variables here)

(Table)
Relative viscosity is the ratio of the viscosity of the native protein and the blank solution. Relative viscosity compares the two values. The greater it is, the greater the difference between the two samples. Since the viscosity has not been calculated, the ratio of time was used instead. This is valid since in dilute solutions, the densities of the sample and the blank are almost equal. Once the to and the t have been obtained, the specific viscosity of the samples may be calculated using the equation

sp = (t /to) 1

(Table of specific viscosity) Specific viscosity is the change in viscosity of the solution, as is expressed as the ratio of the absolute viscosity of the fluid to that of a reference fluid, the protein and protein with denaturant, respectively. (Interpretation of results) Reduced viscosity, however, determines the best denaturant. The reduced viscosity is used to evaluate the extent of denaturation and the reduction of disulfide bonds. The value may be acquired through solving the ratio of the specific viscosity and the protein concentration. The concentration of albumin is 1% (v/v)

Where sp = specific viscosity c= concentration of protein

(Table of Reduced viscosity) Based on the data acquired, 1% SDS is the best denaturant (RAQUEL TO)