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Structural Studies of Phycobiliproteins from Spirulina




To study the effect of denaturation on the structure of phycobiliproteins from spirulina using UV spectroscopy.



The function of a protein is determined by the structure in determining the function of a protein and is of great relevance in protein chemistry. The structure depends on the way in which the protein is folded in a particular conformation.  Non covalent forces and some weak covalent bonds like disulphide linkages   are responsible for stabilizing the protein conformation. These non-covalent forces include hydrogen bonding, electrostatic interactions, van der Waals forces, and hydrophobic interactions.  The destruction of the non covalent forces within a protein may alter the protein's conformation and cause denaturation. Denaturation results in disruption of the tertiary structure of proteins which in turn affects the secondary structure leading to unfolding of the protein .The primary structure of the protein remains unchanged. There are several factors that are responsible for protein denaturation which   includes temperature, pH, addition of organic solvents and chaotropic agents such as urea and potassium thiocyanate.  These denaturing agents increase the solubility of non polar substances in water, and disrupt the hydrophobic interactions that control protein folding. Denaturation can have   both beneficial and hazardous effects. Considering the case in which the milk protein casein is precipitated from milk by adding hydrochloric acid and in other case which involves the use of alcohol to denature the cell wall components and other proteins of bacteria on the wound. Cooking of food to denature the bacterial proteins in the food is another advantage of denaturation. Similarly the hazardous effects include the denaturation of alcohol- ethanol leading to the formation of a highly toxic alcohol- methanol and also many of the diseases like Alzheimer’s and prion disease are resulted due to the improper folding of the protein which occurred due to protein denaturation.    So it is important to study the   folding and unfolding patterns of protein in the presence of certain denaturants like Urea and Potassium thiocyanate. Denaturation by urea occurs by binding   of urea to the protein and stabilizing the denatured state, thus resulting in unfolding. Urea can also act indirectly by altering the solvent environment, thereby reducing the hydrophobic effect and thereby allowing  the exposure of residues in the hydrophobic core. 


Spirulina is a photosynthetic organism which is considered to be a promising source of proteins, is taken as the source of proteins  for studying the structural changes that occur due to denaturation of proteins. It has a photosynthetic membrane which has a light absorbing part called a phycobilisome. The phycobilisome is made up of different classes of proteins, including two phycobiliproteins called allophycocyanin and phycocyanin. Both of these proteins contain a chromophore called phycocyanobilin which is in a planar conformation when the protein is in its native form, but have different maximal absorbance wavelengths due to differences in the protein structure surrounding the pigment. When the protein is denatured, this chromophore undergoes a conformational change, leading to a change in the absorption spectrum which is recorded at 625nm using a UV spectrometer.  When phycocyanobilin is planar, its wavelength of maximum absorbance is high enough to fall in the visible region of the electromagnetic spectrum. When the denaturant is added the protein gets denatured and conformational changes occur in the phycocyanobilin leading to decrease in absorbance at higher wavelength and an increase in absorbance at near UV wavelengths. This experiment examines the structural changes occurring in phycobiliproteins upon denaturation with potential denaturants like urea and potassium thiocyanate, employing UV-Vis spectroscopy. The effect of altering the structure of phycobiliprotein on the ability of the phycocyanobilin to absorb light is also studied. 

UV-Vis Spectroscopy

Ultraviolet-visible (UV-vis) spectroscopy is used to obtain the absorbance spectra of a compound in solution. In UV spectroscopy, the sample is irradiated with the light in the UV-Visible region of electromagnetic spectrum , owing to which there will be transition of electrons from the bonding to anti bonding orbital’s of the molecules present in the sample and the remaining UV light passes through the sample and is absorbed . The energy absorbed against wavelength is plotted and is called as absorption spectrum. UV –Vis spectroscopy provides the structure of details about the structure of molecules present in the sample. UV spectrum depends upon certain factors like the solvent in which the sample is dissolved, the concentration and path length of the solution.


UV-vis spectroscopic data can give qualitative and quantitative information of a given compound or molecule. It is necessary to use a reference cell  to zero the instrument for the solvent the compound is dissolved in. For quantitative information on the compound present in the solution, calibrating the instrument using known concentrations of the compound in a solution with the same solvent as of the sample   to be analyzed would be required.







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