12. Core Practical 4

What happens during this practical?

The primary protein found in milk is called casein and gives milk its characteristic white colour.

Specific enzymes break down the casein in milk and cause the milk to turn white to clear.

Describe the bond that is broken down in proteins:

Peptide bond

Predict the molecules formed when the protein is broken down:

Amino acid

Suggest the type of reaction that occurs:

Hydrolysis

Predict the name of the enzyme that digests proteins:

Protease

Describe what happens to the colour of the enzyme and milk solution over time:

The milk solution turns white to colourless over time.

what happens to the amount of light that would pass through the solution:

When more casein protein is broken down, it will be colour less.

This means more light will pass through when more casein is broken down.

Why do we use a colorimeter in this exp?

As the solution becomes clearer, less light is absorbed by the solution so the absorbancy reading decreases

→ This provides quantitative analysis rather than qualitative so is better than observation

Why do we need to calibrate the equipment?

Data is being compared to a standardised value so the results can then be compared in a valid manner.

What is the aim of the experiment?

Investigate the effect of enzyme concentration on the initial rate of reaction between trypsin and casein solution.

What is the independent variable?

Enzyme concentration (TRYPSIN)

e.g. 0,0.1,0.2,0.3,0.4,0.5 moldm-3

What is the dependent variable?

absorbancy = initial rate of reaction

What is the control variable?

same volume of enzyme solution

same temp volume of trypsin

How do we make the exp reliable?

Repeat each enzyme concentration 5 times and calculate an average.

What is the method?

1. Pipette 2cm² of protein solution into a cuvette.

2. Pipette 2cm² of the protease solution into the cuvette.
   Mix thoroughly & immediately put this cuvette into the colorimeter and start the clock

3. Measure absorbance at suitable time intervals for 5 minutes or until there is little change in reaction

4. Discard the content of the cuvette & rinse with distilled water

5. Plot a graph of absorbance against time.
   Use the graph to determine the initial rate of reaction.
   This is the initial gradient of the graph and should be the steepest part.
   Calculate the initial rate by dividing the change in the y-axis by the change in x-axis values and use whatever units you have plotted on you r- and x-axes.

6. Repeat steps 1-5 of the exp using a range of different enzyme concentrations, ensuring that other conditions are unchanged.
   Plot a separate absorbance against time graph for each enzyme concentration & calculate an initial rate of reaction from each one

7. Present your results in the most appropriate way

8. Identify any trends in your results

9. Explain any trend or patterns, supporting your statements with evidence from your data and using biological knowledge

10. State a clear conclusion to your work, summarising what you gave found out and comment on the validity of your conclusion