Pratical 6 - Concentration and Enzyme activity

At the beginning of the reaction what is the active site of an enzyme like?

It is empty waiting for the substrate to bind.

What happens if the concentration of the substrate is limited?

Limits the rate of reaction and therefore limit the product produced.

What happens if the concentration of the substrate increased?

As the concentration of substrate increases the enzyme active sites are saturated with a substrate then the rate of reaction will no longer increase.

This increases the rate of reaction until it reaches its maximum.

What happens when all active sites are saturated?

When all the enzyme active sites are saturated with a substrate then the rate of reacion will no longer increases.

What would happen if we kept adding more substrate?

As all the avaliable enzymes have been converted to enzyme substrate complex.

If we add more substrate, this will not affect the reaction rate as no more enzyme substrate complexes can be formed.

What does this look like on the graph?

As the substrate concentration increases, so does the rate of reaction. The reaction rate follows this pattern of increasing when the amount of enzyme is fixed.

Before it plateaus, there is a point of active site saturation.

When it is plaiting, the active sites are saturated, and no further addition of substrate will alter the rate of reaction.

What is Hydrogen Peroxide?

Hydrogen Peroxide (H₂O₂) is a by-product of respiration and is made in all living cells.

It is harmful and must be removed as soon as it is produced in the cell.

What is a catalase?

Catalase is an enzyme found in many organisms, including our cells.

(2H₂O₂ —- (catalyse) 2H₂O + O₂)

It can be found in many organisms.

We have it in all our cells as the catalase catalyses the breakdown of Hydrogen Peroxide (toxic) to water and oxygen (non-toxic)

Catalase is found in yeasts (fungus).

We will use yeast (containing catalase) to break down H₂O₂.

What is the aim of this practical?

To investigate the effect of hydrogen peroxide concentration on the rate of activity of catalase by measuring the volume of oxygen produced.

What was the hypothesis?

As the substrate concentration increases the rate of reaction increases.

This will occur whilst the amount of enzyme is fixed until the point of saturation, which means that all the available enzymes have been converted to enzyme substrate comples

So by adding more substrate the reaction rate will not be affected.

What are the independent variables?

Hydrogen peroxide concentration will be changed: 0%, 20%, 40%, 60%, 80%

0,2,4,6, and 8 a.u hydrogen peroxide.

What are the dependent variables?

Quantitative: Volume (cm³) of ocyfen produced.

Qualitiative: Heat released/bubbles.

What are the control variables?

Time (30-second intervals): Start the stop clock after starting the Buchner flask.

25 cm3 of hydrogen peroxide (volume): using a 25 cm3 measuring cylinder.

5 cm3 of yeast (volume): using a 5 cm3 syringe.

5% yeast suspension (concentration)

Keep the temperature of the room the same

Swill the mixture in a conical flask for the same surface area.

What was the Hazard, Risks, Precautions and Emergency Procedure? (H&S)

Hazard - Risks - Precautions - Emergency Procedure

Yeast - Low hazard - Do not ingest - Contact first aid.

Hydrogen Peroxide - Corrosive/irritant - Wear gloves/labcoats/goggles - Rinse with water/first aid

Glassware - Smash/cuts - Place away from the edge of the table/handle with care - Seek first aid for injuries/Contact the teacher for clean up.

Clamp Stand - Heavy, so may cause damage if it falls - Keep away from the bench edge/handle with care - Contact first aid/teacher for clean up.

What was the equipment?

Set up the apparatus by securing the gas syringe in a clamp, connecting it to a Buchner flask with a bung.

5 cm³ syringe - to measure 5 cm³ of the 5% yeast suspension.

25 cm³ measuring cycliner - to measure 25 cm³ of the a.u. hydrogen peroxide.

Buchner flask - to contain hydrogen peroxide and yeast suspension

Gas syringe - to measure the volume of oxygen

Bung - to ensure the oxygen does not escape and enter the gas syringe.

What is the method?

1) Set up the apparatus as shown, securing the gas syringe in a clamp.

2) Using a 5 cm³ syringe, add 5 cm³ if the 5% yeast suspension (ensure that is it stirred first) to the Buchner flask.

3) Ensure the gas syringe plunger is pushed all the way in, and place a black mark where it starts.

4) Use a 25 cm³ measuring cylinder to measure 25 cm³ of 0 au. hydrogen peroxide and add it to the Buchner flask.

5) Secure the bung into the Buchner flask and immediately start the timer.

6) After 30 seconds, draw a black mark on the syringe and record the volume of oxygen collected in your record table.

7) Wash out and thoroughly dry the Buchner flask.

8) Repeats steps 2 to 7 two more times with 2 a.u of hydrogen peroxide to obtain repeats.

9) Repeat steps 2 to 8 with 2,4,6, and 8 a.u hydrogen peroxide.

10) Calculate means and work out the rate of reaction.

How do you improve this experiment?

Do more repeats for the same concentrations to give more relaible results.

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

Use more precise equipment such as

How do you extend this experiment?

Use a different source of catalase e.g. milk, almonds, liver.

Do the experiment at a different temperature e.g. 40 degrees Celsius.