Practical 7 - Enzyme concentration on trypsin

What happens when the enzyme conentration increases?

As the enzyme concentration increases

More active sites become available, allowing more enzyme-substrate complexes to form.

This increases the reaction rate.

Until it reaches the maximum rate of reaction, which is the point where all the substrate molecules are occupying the enzyme's active sites.

What happens if we continue to increase the concentration of enzymes past that point?

Increasing the concentration of enzymes can not increase the rate of reaction. - it will stay the same.

The maximum number of enzyme-substrate complexes has been reached.

What is the limiting factor?

The limiting factor is something that is stopping the rate of reaction from increasing.

The substrate is said to be the limiting factor, as if we increased the limiting factor, we could increase the rate.

What is trypsin?

It is the enzyme that digests protein.

It is produced by the pancreas and used in the small intestine to break down proteins to amino acids.

It may also be referred to as protease.

What happens to milk and trypsin when added together?

Casein is a protein found in milk and makes it white. It is broken down by protease enzymes.

When mixed casein is hydrolysed by trypsin.

Trypsin breaks down the protein into amino acids which are soluble (they dissolve) in the water.

Milk turns from milky white to clear/translucent.

What is the aim for this practical?

To investigate the effect of enzyme concentration on the rate of trypsin.

What is the hypothesis?

As the concentration of Trypsin increases, the rate of reaction will increase, then plateau.

This is because…

• As the enzyme concentration increases, more active sites become available, allowing more enzyme-substrate complexes to form.

• This will increase the rate of reaction until it reaches its maximum rate, which is the point where all the substrate molecules are occupying the enzyme's active site, so no more can.

What is the independent variable?

The concentration of Trypsin (0.00, 0.25, 0.50, 1.00, 2.00)

What is the dependent variable?

Qualitative observations: Milk turning clear

Quantitative observations: The time it takes in seconds for the milk to turn from white to translucent.

What is the control variable?

Use a measuring syringe to control the volume of milk at 2cm³

Volume of protease: measure 2cm³ using a syringe.

Type of milk: use the same batch/bottle of milk.

Temperature of the room: use a waterbath.

What are the Hazard, Risks, Precaution and Emergency Procedures of this practical? (H&S)

Hazard - Risks - Precautions - Emergency Procedures

Trypsin - Irritant if inhaled to skin/harmful if swallowed - Gloves/Goggles worn - Rinse with water/seek first aid.

Milk Powder Solution - Allergies/Low hazard - Avoid spillage - Clean up spillages.

Glassware - Smash /cuts - Keep away from bench edge/handle with care - Contact first aid/teacher for clean up.

What was the equipment used?

• x5 Test tubes: To hold each concentration of Trypsin.

• 5 cm³ syringe: To measure 2 cm³ of milk powder solution.

• Whiteboard marker: To write an X on each test tube.

• 2 cm³ syringe: To add 2 cm³ of each concentration to the test tubes.

Stop clock: Start immediately after adding the trypsin solution and stop once the X has become visible.

What is the method?

1) Label five test tubes with the Trypsin concentrations 2.00, 1.00, 0.5, 0.25, 0.00%

2) Using a 5 cm³ syringe, measure 2 cm³ milk powder solution into each test tube.

3) Write an X with the whiteboard marker where the milk solution is in the test tube. You should not be able to see the X through the milk solution on the other side.

4) Using a 2 cm³ syringe, add 2 cm³ of 2.00% Trypsin solution to the test tube labelled 2.00% and immediately start the stop watch. (Leave the syringe inside the trypsin beaker)

5) Stop the timer once the X becomes variable (stop after 5 minutes if no change).

6) Record your results in a suitable table. Include a column for the rate of reaction.

7) Repeat steps 3 - 6 two more times for 2% Trypsin.

8) Repeat steps 3 - 7 for the other four concentrations, 1.00%, 0.50%, 0.25%, and 0.00%.

9) Calculate the mean and work out rate (1/time)

How can you improve this experiement?

Do more repeats for the same concentrations to give more reliable reults.

Repeat the experiment with different concentrations e.g. a.u. 1, 2, 3, 4, 5, to extend the range of results.

How can you extend this experiment?

Repeat the experiment with a different temperature e.g. 40^oc

Repeat the experiment with a different concentration of substrate e.g. 2.50,3.00%