Exer 3 - Protein Denaturation

ASI, Ranelle Janine L. Chemistry 160.1 Section 3L 2015

Date performed: June 22, 2015 Date submitted: June 26,

EXERCISE 3. Protein Denaturation Post-Laboratory Report Proteins are produced as linear chains with different combinations of 20 amino acids, sequenced to form various classes that carry out diverse tasks once functional. A protein’s distinct sequence is its primary structure, formed by peptide bonds between the amino and carboxyl groups of each amino acid (Lodish, et al., 2007). Depending on the primary structure, a protein chain folds into its secondary structure stabilized by hydrogen bonds between certain amino acid residues forming periodic arrangements called alpha helices, beta sheets and U-turns. An alpha helix is formed through hydrogen bonds between the carbonyl oxygen of each peptide bond and the amide hydrogen of an amino acid four residues away. A beta sheet is formed by polypeptide chains that laterally bond to each other forming pleated sheets that project residues on both surfaces. A U-turn is comprised of three to four residues stabilized by hydrogen bonds between end residues (Lodish, et al., 2007). A protein achieves its functional form upon conforming into its tertiary structure. Unlike in secondary structures, these shapes are formed by more than one type of stabilizing force: hydrophobic interactions between nonpolar side chains, hydrogen bonds between polar side chains, disulfide bonds between two cysteine amino acids and ionic bonds between oppositely charged amino acid side chains. These bonds hold helices, sheets and turns together in a compact but not entirely rigid three-dimensional shape (Lodish, et al., 2007). The last level of protein structural organization is quaternary structure, only necessarily achieved by multimeric proteins. Quaternary structures are formed by joining multiple oligomeric proteins and are stabilized by multiple forces similar to those in tertiary structures (Lodish, et al., 2007). A protein adapting its natural conformation based on these levels of organization is said to be in its natural state, which is the most stably folded form of the whole molecule. Denaturation of this state results from various treatments like high heat that break stabilizing bonds, extreme pH that alter charges on amino acid side chains, and chemicals like hydrochloride at high concentrations that can disrupt weak noncovalent bonds. Once a protein loses its native conformation, its chemical properties change and its biological activity is also lost (Cohen, 2004). The protein studied in this exercise is phycocyanin extracted from the cyanobacterium Spirulina sp. Phycocyanin is a chromophoric water-soluble protein comprised of an alpha and beta subunit each containing a cysteine residue. It also contains a prosthetic group called phycocyanobilin covalently attached to the

cysteine of the apoprotein through thioether linkage, and auxiliary proteins devoid of chromophore called linker polypeptides (Britton, 1983). To demonstrate protein denaturation, phycocyanin was mixed with different denaturing reagents and exposed to different temperatures as shown in Table 3.1 and Table 3.2. Table 3.1. Effects Reagent 6 M HCl 6 M NaOH 0.2 M lead acetate 10% trichloroacetic acid 95% ethanol

of known denaturing agents on phycocyanin from Spirulina. Observations Bubbling, solution turned clear light blue, no precipitate Solution turned clear light yellow, no precipitate Formation of white precipitate Bubbling, solution turned clear light blue, no precipitate

Solution turned colorless

Table 3.2. Effect of temperature on phycocyanin from Spirulina. Water bath used Observations hot solution turned clear light blue, no precipitate cold no change An isolated phycocyanin solution is colored dark blue with a tinge of red signalling its functioning biological activity. As observed, all chemical reagents show denaturing effects on phycocyanin as all resulted to loss of color. It is known that reagents that cause denaturation target the abundant hydrogen-bonds present in native proteins. Acidic reagents, in this case HCl, act through protonation of carboxylic groups involved in hydrogen bonding, resulting to loss of the said interactions, and through alteration of pH of the solution, which decreases protein solubility (Lapanje, 1971). Basic reagents like NaOH have similar effects on pH and protein solubility (Chawla, 2014). Addition of organic solvents like alcohol, in this case ethanol, displaces water molecules associated with proteins, causing a decrease in the concentration of water in the solution which then lowers the solubility of the protein leading to denaturation (Chawla, 2014). Addition of heavy metal ions like lead acetate resulted to ionic bonding of the lead cation with negatively charged groups on the proteins causing denaturation and precipitation as metal proteinate (Chawla, 2014). Theoretically, addition of high concentrations of trichloroacetic acid (TCA) should lead to precipitation as negatively-charged TCA ions disrupt native electrostatic interactions in protein, leading to formation of an intermediate form with exposed nonpolar surfaces that promote intermolecular coalescence and precipitation (Rajalingam, et al., 2009).

As most biological compounds are heat-sensitive, exposure to high temperature caused protein denaturation as a result of peptide chain disorganization and breakage of crosslinkages among chains due to increase of conformational entropy (Wu and Wu, 1925). Low temperatures affect protein structure at extremes (hence no change was observed in this experiment) theoretically by promoting interactions of protein nonpolar groups with water leading to unfolding (Privalov, 1990). References: Britton, G. 1983. The Biochemistry of Natural Pigments. Cambridge University Press, Australia. p. 160. Chawla, R. 2014. Practical Clinical Biochemistry: Methods and Interpretations. Jaypee Brothers Medical Publishers, Ltd., India. Cohen, G. N. 2004. Microbial Biochemistry. 1st ed. Springer Science & Business Media, B. V. p. 285. Lodish, H., Berk, A., Kaiser, C.A. and Krieger, M. 2007. Molecular Cell Biology. 6th ed. W.H. Freeman & Co., New York. pp 60-66. Lapanje, S., and Wadso, I. 1971. A Calorimetric Study of the Denaturation of Lysozyme by Guanidine Hydrochloride and Hydrochloric Acid. Lund University, Sweden. Privalov, P. L. 1990. Critical Reviews in Biochemistry and Molecular Biology: Cold Denaturation of Proteins. Volume 25, Issue 4. Retrieved from http://informahealthcare.com/doi/pdf/ 10.3109/10409239009090613 Rajalingam, D., et al. 2009. Trichloroacetic acid-induced protein precipitation involves the reversible association of a stable partially structured intermediate. University of Arkansas, Arkansas. Wu, H., and Wu, D. Y. 1925. Nature of heat denaturation of proteins. Peking Union Medical College, China.