PhycocyaninIsolation

I.

Introduction Proteins are composed of amino acids. Side chains of the amino acid residues contain non-polar, neutral, polar, acidic, or basic groups. The primary structure of a protein is determined by the sequence of amino acids and dictates the secondary and tertiary structures of proteins. define their natural or native states, which are often folded. This is called the native conformation and is usually the state in which the protein is most active and functional. Proteins’ native conformations are held by molecular forces such as hydrogen bonds, salt bridges (also called ionic interactions), disulfide bridges, and hydrophobic interactions (Nelson & Cox, 2004).

Protein denaturation is when the unique 3-D structure of proteins is destroyed. It can cause temporary or permanent loss of enzyme activity therefore it can be reversible or irreversible. This change in conformation is not accompanied by disruption of the peptide bonds. The alterations can be cause by various physical and chemical agents such as acids and bases, organic solvents, heat and heavy metals. This alteration can cause decreased in solubility, change in water binding capacity, change in color, destruction of toxins and increased in viscosity.

Phycocyanin which exists as C Phycocyanin is a phycobiliprotein isolated from Spirulina sp., a blue-green algae.C-Phycocyanin is fluorescent, with an extremely high absorbtivity, a high quantum efficiency, a large Stokes shift and excitation and emissionbands at visible wavelengths. It is a stable protein which can be easily linked to antibodies and other proteins by conventional protein cross-linking techniques without

altering its spectral characteristics. (http://www.prozyme.com/documents/pb11.pdf, 2013). The protein has an absorbance at 610-620 nm and a fluoroscence at 647 nm. The fluorescence is only observed when the native conformation is still intact and thus the blue color persists upon denaturation.

In this experiment, phycocyanin will be extracted from Spirulina tablet and the effects of 6M HCl, 2M NaOH, 0.2M lead acetate, 10% trichloroacetic acid, acetone and temperature on protein denaturation will be determined. Also, the concentration and purity of phycocyanin in Spirulina tablet will be determined by reading the absorbance at 650nm, 620nm and 280nm.

II.

Methodology

In this experiment, equal amounts of Spirulina tablet and silica where ground using mortar and pestle to form the smooth paste. The silica was added to disrupt or break the cell walls in order to release the proteins present in the tablet. To the mixture, 28mL of 0.1M phosphate buffer pH 7 was added. Buffer was added to assure that denaturation is not erroneous upon addition of an acid or a base. The resulting blue-red mixture was then stirred and subjected to centrifugation for 2 minute at 10000rpm. The supernatant was collected and transferred to an E-flask. From this solution, around 2-3mL of the solution was obtained. The solution was refrigerated for several hours and the absorbance at 650,620 and 280nm was read.

To determine the effects of different denaturing agents, 0.5mL each of the supernatant solution were placed into 8 test tubes labelled 1-8. One mL of 6.0M HCl, 2M NaOH, 0.2M lead acetate, 10% trichloroacetic acid, and acetone was added to test tubes 1-5 respectively. Test tube 6 was placed in a recently boiled hot water while test tube 7 is placed in a cold water bath. Test tube 8 served as control. The test tube mixtures were left to stand for 5 minutes with shaking and the resulting mixtures were observed.

III.

Results and Discussion

Figure 1. The effects of different agents on denaturation of Phycocyanin.

As seen on test tube 7, in comparison with test tube 8, no visible reaction occurred and thus, the cold temperature did no action on the structures of phycocyanin.

Secondary structure which is composed of α helix sheet and β-pleated sheets was not disturbed. α-helix is stabilized by extensive hydrogen bonding between the peptide-bond carbonyl oxygens and amide hydrogens that are part of the polypeptide backbone and in βpleated, all of the peptide bond components are involved in hydrogen bonding (Nelson &Cox, 2004). These H-bonds were not broken by the cold temperature. Tertiary structure, as well as quaternary structure, was not destroyed. Low temperature has no or little effect on the presence of Disulfide bonds, Hydrogen bonds, hydrophobic interactions and ionic interactions, thus no obvious sign of denaturation occurred. On the other hand, as seen on test tube 6, precipitation and slight change in color occurred. Subjecting phycocyanin to high temperature caused increase in Kinetic energy resulting to vibration of amino acid/protein molecules. The vibrations allowed disruption of weak intermolecular forces, such as Hbonding, and hydrophobic interactions, resulting to unfolding of the tertiary structure and exposure of non-polar residues to water which is seen as the precipitate.

As seen on test tube 1, the color of the mixture changed from blue to bluish accompanied by formation of white precipitate while test tube 2 contained colorless solution with white precipitate. The tinge of blue may have indicated that little amount of phycocyanin is still present. Acids and bases impart slight polarity on amino acids and thus destabilized interactions among the protein fold resulting to denaturation. At the isoelectric pH, there is no net charge on the molecules and so, individual molecules tend to coagulate and precipitate out of solution. At a pH above or below the pI, molecules have a net negative or positive charge, thus when protein molecules approach each other they have the same

overall charge and repulse each other resulting to unfolding. Salt bridges (tertiary structure) is disrupted by the polarity and presence of ions.

As seen on test tube 3, white mixture with relatively large amount of precipitate was derived upon addition of lead acetate. This indicates that lead acetate can be a strong denaturing agent. Salts of metal ions especially heavy metals, such as mercury(II), lead(II), and silver can form strong bonds with sulfhydril groups and carboxylic groups of the acidic amino acids. These result to disruption of disulfide bridges and salt linkages present in the quaternary, secondary and tertiary structure and thus, protein precipitate out of solution as an insoluble metal-protein salt.

Both test tubes 4 and 5 showed signs of precipitation and change in color. Addition of trichloroacteic acid results to color change from aqua blue to faint blue while acetone caused a change of color from blue to colorless. The oxygen of Acetone can interrupt with the hydrogens involve in H-bonding to produce a new H-bond which is more stable as acetone is a smaller molecule compared to the amino acids. Due to the apolar property of trichloroacetic acid, hydrophobic interactions can be disrupted as well as Hbonds( due to C=O). This properties allowed disruption of the tertiary, secondary(acetone) and quaternary structures of phycocyanin resulting to denaturation.

The concentration of phycocyanin was found to be 0.5355mg/ml as determined through absorbance reading. A value of 0.8212 on the ratio of absorbance readings at 620nm

and 280nm renders the Spirilina tablet fit for consumption as it is large less than 4 to be considered analytical grade.

Practical applications of denaturation often involve analytical and clinical procedures. Making of medicines targeting microorganisms revolves on denaturation of proteins in the living system. Agents of denaturation are incorporated in medicines to perform their denaturating ability. Similarly, as denaturation is a part of process of protein purification, proper extraction of proteins by suitable solvent allows study on diseases involving DNAs, enzymes, mutation, etc.

IV.

Sample Calculations

V.

Summary and Conclusion

Protein denaturation is the change in chemical interactions of amino acid residues in a protein resulting in the unfolding of secondary, tertiary and quaternary structures which can be reversible or not. Denaturation can occur by subjecting proteins to various chemical and physical agents. Phycocyanin from Spirulina tablet was treated with HCl, NaOH, lead acetate, trichloroacetic acid, acetone and temperature changes. All of the

agents except low temperature caused denaturation as seen on the the change in color and precipitation that occurred. These agents have different mechanisms and properties that causes disruption of protein’s native conformation. The concentration of phycocyanin was found to be 0.5355mg/ml as determined through absorbance reading. Phycocyanin derived was also food grade according to the ratio of absorbances at 620nm and 280nm.

VI.

Literature cited

Moran, L.A., Horton, R.H. , Scrimgeour K.G. & Perry, M.D.(2012). Principles in Biochemistry (5th ed). Glenview,IL : Pearson Education, Inc.

Nelson, D. L., & Cox, M. M. (2004). Lehninger Principles of Biochemistry (4th ed.). New York, USA: W.H. Freeman and Company

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