Question

Reactants	Color of the mixture after adding Biuret reagent
Tube 1 (egg white + pepsinogen + NaOH)	Blue
Tube 2 (egg white+ pepsinogen + HCl)	Purple
Tube 3 (egg white + NaOH)	Blue
Tube 4 (egg white + HCl)	Blue

a. In which tube(s) did protein digestion occur? How do you know?

b. In which tube(s) protein digestion did not occur? How do you know?

c. What effect does pH have on protein digestion with pepsin? Why?

3.

Reactants	Color	pH (acidic, neutral or alkaline)
Tube 1 (oil + lipase + bile)	Yellow	Acidic
Tube 2 (oil + lipase)	Yellow	Acidic
Tube 3 (oil + bile)	Red/Orange	Neutral
Tube 4 (oil + water)	Yellow	Acidic

a. In which tube(s) did lipid digestion occur? How do you know?

b. In which tube(s) lipid digestion did not occur? How do you know?

c. What effect does bile have on lipid digestion? Why?

Answer 1

2. Biuret test is a test for detecting proteins. Biuret reagent contains hydrated Copper sulphates, potassium hydroxide solution and Potassium sodium tartrate.

Proteins that contain two or more peptide bonds react with copper to form violet-coloured chelate complex in alkaline conditions. Long chain proteins with tertiary and secondary structure do not react and hence, solution remains blue.

a) Protein digestion occurred in tube 2. HCl is required to convert pepsinogen to pepsin in stomach. Pepsin then breaks down the proteins to peptides, which have peptide bonds. These bonds react with Peptide bonds to form purple color.

b) Proteins digestion did not occur in tube 1, 3 and 4. There is no activation of pepsinogen in tube 1 as HCl is absent. In tube 3, there is no HCl and Pepsinogen while in tube 4, despite HCl, there is no pepsinogen present. Hence, there is no peptide formed to give purple color with Biuret reagent.

c) The low pH created by HCl, will convert inactive pepsinogen to pepsin, the active form. Pepsin will then cleave the peptide bonds to form smaller peptides, which have 2 or more peptide bonds. NaOH will create alkaline conditions, which will not activate pepsinogen to pepsin.

3. Bile is known to emulsify the lipids for the lipase to act. Lipase, is the enzyme that acts to break down fats to free fatty acids and triglycerides. Phenol red is the indicator, which turns yellow under acidic condition, which is created by free fatty acids. Phenol red remains red at alkaline conditions at pH greater than 7.4. Hence, a yellow color indicates lipid digestion.

a) Lipid digestion occurred in tubes 1, and 2. In tubes 1 and 2, lipase is present which will break down fats to lipids. This turn phenol red yellow. In tube 4, despite the presence of yellow color, there is no lipid digestion occurring. There is mixing of oil in water. Oil and water separate into two layers as they are immiscible. The surface tension of water is reduced, and the oil will have ends that undergo hydrolysis to produce some free fatty acids, that will reduce the pH. This will decrease the pH and hence, the phenol red becomes yellow.

b) In Tube 3, there is no change in color. Hence, no lipid digestion occurred. This is because bile will only emulsify the lipids. It doesnt break down lipids but only reduces pH to neutral range. In tube 4, oil and water will seperate in 2 layers. There is reduction in pH because of formation of two layers, turning color to yellow due to low pH because of hydrolysis. there is no lipid digestion as there is no enzyme lipase is present. Water is required for lipase to dissolve, which is a water soluble enzyme.

c) Bile will emulsify the lipids and make them miscible in water. The lipids will be broken to smaller particles. This will cause increase activity of lipase.