Really struggling on this lab that has to do with protein isolation and characte

Question

Really struggling on this lab that has to do with protein isolation and characterization. A biuret test and coagulation was used in Part A, centrifugation was used in Part B to separate phycobiliproteins from Spirulina, and the isolated phycobiliproteins from part B were exposed to distilled water, 1.0M HCl, 1.0M NaOH, soap, sodium chloride, and 6.0M urea in Part C (Don't really understand how to tell if there was denaturing/when there is supposed to be and why). Any help answering these questions would be greatly appreciated.

b. Discussion of Part A

i. Describe the effect of adding acid on the charge and folded state of casein. (2 pts)

ii. Describe, based on your observations, the effect of protein on the color and max of solutions of copper(II). (3 pts)

c. Discussion of Part B

i. Explain the purpose of grinding Spirulina in a mortar with silica. What occurs microscopically? (2 pts) ii. Explain the purpose of centrifuging the ground and suspended cells. (2 pts)

iii. Use your observations from part B to justify the claim that the liquid extract contained phycobiliproteins. (3 pts)

d. Discussion of Part C

i. Distinguish the chemical environments that caused denaturation (unfolding) from those that did not. Explain how your observations provided evidence of unfolding. (3 pts)

ii. For the acidic (HCl) and basic (NaOH) solutions, if you observed unfolding, explain why it occurred by appealing to the charge state of the protein. (2 pts)

iii. For the soap solution, if you observed unfolding, explain why it occurred by appealing to intermolecular forces. (2 pts)

iv. For the urea solution, if you observed unfolding, explain why it occurred by appealing to intermolecular forces. (2 pts)

v. Explain why heat and mechanical shock lead to unfolding.

Explanation / Answer

1) Effect of acid on casein - Addition of acid decreases the electric charge to that of isoelectric point. Which leads to disruption of its structure and aggregation thereby precipitation of casein micelles.

2) The copper II ion from a violet colored coordination complexes in alkaline solution. The intensity corresponds to the concentration of protein. Hence lambda max at 540 nm can be used to determine the concentration of protein in a sample.

3) Grinding spirulina with silica ruptures the cell wall of the algae and lets the intracellular content out

4) Centrifuging the ground and suspended cells helps to separate cell wall from its intracellular components.

5) The absorption properties of phycobiliproteins depends on the ionic strength and pH of the solution. Hence the absorbance can be recorded at pH 8.0 to determine the presence of phycobiliproteins.

6) Acidic or alkaline conditions which alters the pI of the proteins and presence of surface acting agents such as soap solution or urea cause denaturation of the protein.

7) Because Hcl and NaOH liberates free H+ and OH- ions respectively which reacts with the polar and non polar side chains of amino acid in protein thereby changing the protein confirmation and leads to denaturation.

8) In case of soap solution, it changes the hydrophobicity of the protein

9) Urea bonds with the amide group of amino acid in the protein and is also reported to react with the hydrophobic interactions there by disrupt the protein structure.

10) Heat and mechanical shock breaks the peptide bond and hydrogen bond present in the protein hence it lead to unfolding.

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