The Effect of Enzyme Concentration on the Rate of Reaction

 gauze and heat resistant mat

• stop clock
• urea solution
• BDH Universal Indicator solution

METHOD

For the first experiment, I wanted to find out the effect of temperature on protease.  This is what I did to do that:-

1. I added 5cm3 of protease to a boiling tube and placed it into a water bath for 5 minutes.
2. I then prepared my film by placing a square of film onto a wooden spill, making sure that I was not touching the film.
3. After 5 minutes, I placed the film spill into the boiling tube containing enzyme that had been in the water bath and was allowed to cool.
4. I then started the stop clock.
5. I recorded the times for how long it took the film to go clear.
6. This was carried out five times at different temperatures, which were 0 C, 20 C, 30 C, 40 C and 50 C.

For the second experiment, protease and the effect of pH, I wanted to see the effect of the different pH on protease.  This was how I carried it out to find out:-

1. I added 5cm3 of protease to a boiling tube, marked A.
2. I then added 5 cm3 of the Buffer solution to a second boiling tube, marked B.
3. Both the tubes were then placed into a water bath, at 40 C, for 5 minutes.
4. While the enzyme and the solution were in the water bath, I prepared a film spill by placing the square film onto a wooden spill, previously carried out in experiment one.
5. After 5 minutes, I mixed the two solutions – the protease and the Buffer solution - and added the film spill.
6. I then started the stop clock.
7. I recorded the times it took the film to go clear.
8. This was experiment was carried out  five times, but at  different pH’s, which were 2, 4, 7, 9 and 10.

For the third experiment, effect of enzyme concentration on the rate of reaction, the following method was used to determine the effect:-

1. A beaker was half-filled with water and gently brought to the boil.  This was used as the water bath.
2. Using a measuring cylinder, 10cm3 of the urease solution was placed into a boiling tube and then placed into the boiling water bath.
3. This was left for at least 5 minutes, and then removed and then allowed to cool.  This sample was referred to as boiled urease.
4. Four test-tubes were labeled from 1-4, and using the measuring cylinder, 5cm3 of urea solution was added.
5. 5 cm3 of acetic (ethanoic acid) solution was also added to the boiling tubes as well as 10 drops of Universal Indicator.
6. To tube 4, 3cm3 of boiled urease was added.
7. To tube 1, 2cm3 of unboiled urease was added.
8. To tube 2, 3cm3 of unboiled urease was added.
9. To tube 3, 5cm3 of unboiled urease was added.
10. I then started the stop clock.
11. At 30 seconds intervals, I shook the tubes and observed the colour of the indicator in each tube.  The observations continued for 5 minutes.
12. At each observation, the pH of each of the mixtures was recorded.

RESULTS

The results of the first experiment of the effect of temperature on protease is as follows, in form of a table:-

As you can see from the results, the experiment of the effect of temperature on protease is quite conclusive.  Before the experiment, it as expected that the higher the temperature the quicker the enzyme would work to make the film spill clear.

The results of the second experiment, protease and the effect of pH is in the following table:-

The results of the second experiment of protease and the effect of pH were inconclusive, as you can see from the above graph.  I expected the higher the pH of the buffer, the quicker the time for the film spill to go clear, however the results I got show that this was not the case and the experiment did not work well as I only managed to obtained two results.

For the third experiment to show the effect of enzyme concentration on the rate of reaction, the following table was the results I got:-

The above table has the pH of the mixture in each tube, for every 30 seconds.  The numbers corresponds with a colour:-

As the results show for the third experiment, the higher the enzyme concentration and the rate of reaction are directly proportional.  The highest peak is the 5cm (pH) of unboiled urease at the longest time of 5 minutes.

HEALTH AND SAFETY PROCEDURES

The safety issues that needed to be taken into consideration were the fact that I was using a Bunsen burner, so there were the obvious fire precautions.  Plus the fact that objects were being heated, so things would be at a high temperature, therefore careful handling of the heated containers.  

DISCUSSIONS

For the first experiment of the effect of temperature on protease, I expected that the higher the temperature went, the less time it would take for the film spill to go clear and from the results I obtained I was right.  There were no anomalous results.

Heating things at a higher temperature will give the molecules more energy therefore making the molecules more active, and causing a quicker rate of reaction – of the film spill going clear.  Working with enzymes, there is an added risk of heating it up above 45 C, as most enzymes tend to denature – destroyed by high temperature – however this did not affect my experiment.  When an enzyme is denatured by heat, the shape of the active site is changed so that the substrate no longer fits.  A change in the pH could have a similar effect if both the conditions are not controlled.

If the conditions, temperature and pH, are controlled it can have a positive effect of speeding things up as two of my experiment shows, however it could go both ways.

Enzymes, which act on proteins, are called proteases.

I did expect the same type of response and trends to protease and the effect of pH, that I got from the effect of temperature on protease, however the results from the experiment was anomalous.  I expected that the higher the pH of the buffer solution, the less time it would take for the enzyme to work and make the film spill clear. How ever, this was not what I obtained.

The result that I got from the third experiment was what I expected.  The larger the volume of unboiled urease, and longer it was left, the higher the pH would be.  There were no anomalous results.  

The above picture illustrates how enzymes work.  Molecules are constantly moving about and bumping into each other.  When a substrate molecule bumps into a molecule of the right enzyme, it its into a depression on the surface of the enzyme molecule.  This depression is called the active site.  The reaction takes place and the molecules of product leave the active site, freeing it for another substrate molecule.

The active site of a particular enzyme has a specific shape into which only one kind of substrate will fit.  The substrate fits into the active site like a lock and key.  This is why enzymes are specific in their actions.