Reversible are temporary and when removed the enzyme will resume full activity.
I don’t know what type of inhibitor Copper Sulphate is. Competitive reversible inhibitors are similar in structure to the substrate. It will compete with the substrate for the active site of the enzyme. However, if the concentration of substrate is increased it will reverse the effects of the inhibitor. This is because there will be too many substrate molecules for the inhibitor to compete with and they will gain access to the active site of the enzymes. So in our experiment if the rate of reaction increases after concentration is increased, that would mean that Copper Sulphate in a competitive reversible inhibitor.
In non-competitive inhibitors, the inhibitor permanently joins with the active site of the enzyme. So the substrate will be permanently separated from the active site. This would mean increasing the concentration would not have any effect on the rate of reaction. So if Copper Sulphate is a non-competitive inhibitor the rate of reaction won’t increase again as the concentration of the substrate is increased.
Method
- Set up the equipment as shown in the diagram.
- Using the dilution table, make four samples of Hydrogen Peroxide each of different concentrations. The concentrations that will be made are going to 100%, 75%, 50% and 25%. Place the four different concentrations into four different test tubes.
- Pour the test tube with the 100% concentration of Hydrogen Peroxide in to the side armed test tube and put the bung on the top.
- Fill the water trough with water until it’s about half full. Put the glass delivery tube inside the trough.
- Then fill the measuring cylinder with water until it is full. Put the cylinder inside the trough, over the glass delivery tube, making sure that no air is let in.
- Gently let some air out of the cylinder so that the water is on the one of the unit marks on the cylinder. Take a note of this measurement so that when the oxygen replaces the water you will be able to see exactly how much oxygen is produced.
- Stir the beaker with the yeast in. This is so that all the yeast doesn’t settle at the bottom of the beaker.
-
Collect 2cm3 of the yeast with the syringe and then place the needle on that syringe. Put the needle through the bung.
- Press the syringe so that the yeast goes into the test tube and at the same time start the stopwatch.
- Carry out the experiment for 3 minutes, recording the level of oxygen after every ten seconds.
- Repeat the steps 3-10 but with the different concentrations of Hydrogen Peroxide.
-
Repeat the whole of the experiment but add 2cm3 of Copper Sulphate at the same time as you add the yeast to the substrate. Do this for all of the concentrations.
Below is the Dilution table that I will use:
Reliability of Method
To make sure that our experiment would be reliable we took a number of precautions. First of all we decided that we would repeat the experiment for two of the concentrations. We would have repeated the whole but due to a shortage of lab time we realized that we couldn’t. Also we decided to take a large number of recordings at short intervals, 10 seconds. The measurements should be accurate due to the fact that we will look at the meniscus at eye level. Stirring the yeast will also increase the reliability.
Risk Assessment
The Hydrogen Peroxide will have to be used with care, as it is an irritant. We should also wash hands after the experiment. A lot of the equipment is made with glass, so they will also have to be handled with care. All stools must be tucked in so that people won’t trip over them.
Preliminary Results
Before carrying out the experiment we done some preliminary tests to help us plan the actual experiment. The first test we done, was to carry out the experiment as I have written out before. This was a sort of a trial run to see how the equipment worked and to check if there were any problems.
The first test we did gave us no results. This was due to fact that when we collected the yeast from the beaker, we collected the yeast from the top of the beaker. So actually none of the yeast had actually gone into the syringe, only water, as all of the yeast had fallen down to the bottom of the beaker. After this test we decided to stir the yeast before each of the tests.
The second test we did, gave us results that we needed. The result is summarized in the table below:
At the next reading the amount of oxygen produced had gone off the scale. This was due to the measuring cylinder, at 50ml, being too small. So we decided after this test to use a 100ml-measuring cylinder instead.
Bibliography
Jones, M., Fosberry, R. & Taylor, D.
Cambridge University Press
- www.seps.org
- www.bbc.co.uk/revision
Results
The first table (below) shows the amount of oxygen produced in the reaction without the addition of Copper Sulphate.
I will use this table to draw my first graph.
The table below shows the amount of oxygen produced with the hydrogen peroxide added to the reaction.
I will also draw a graph for this table. After drawing the graphs I will work out the rates of reaction.
Analysis
By carrying out this experiment I have found out that Copper Sulphate is an inhibitor. You can see this by comparing the to tables of results. However I will analyse this further by drawing graphs and calculating rates of reaction and so on.
Graph 1 (Without Copper Sulphate)
After looking at the graph on the previous pages you can see what happened in the experiment. The first graph shows the trend without the addition of Copper Sulphate. We can see from the graph that as the concentration of substrate increases so does the amount of oxygen produced. This means that there is a positive correlation between the amount of oxygen produced and the concentration of the substrate. We can’t yet prove anything that is written in the hypothesis we van only do that by comparing this graph with the second graph (with Copper Sulphate). By comparing the two graphs I will find out if Copper Sulphate is an inhibitor and I might even find out what sort of inhibitor it is, a non-competitive or competitive inhibitor.
There are no anomalous results that can be seen in the graph or the table.
Graph 2 (With Copper Sulphate)
By looking at the second graph you can see that the amount of oxygen produced increases as the concentrations are increased, however it is much less than without Copper Sulphate being added. For example, at 100% concentration for both graphs the amount of oxygen produced in the non-Copper Sulphate experiment was 25ml but it was 16ml when Copper Sulphate was added. That’s a difference of 9ml. The fact that it doesn’t produce as much oxygen proves that Copper Sulphate is in fact an inhibitor.
Again there were no anomalous results that I could spot by looking at the graph or the table. This meant that our experiment was done quite well.
Rates of reaction
To see if I could find out if Copper Sulphate was a competitive or non-competitive inhibitor worked out the rates of reaction for all the concentration for both with and without Copper Sulphate (you can see the table below). I then plotted the points on a graph (which can be seen on the next page).
I worked out the rates of reaction by dividing the total amount of oxygen produced by each graph with the amount of time it took to reach that amount. This gave me the rate of reaction in seconds. I converted this to minutes by multiplying the rates by 60.
After looking at my graph I can only conclude that Copper Sulphate is an inhibitor. I cannot however say if it is a competitive or non-competitive inhibitor yet. By looking at the graph I think that Copper Sulphate is a non-competitive inhibitor as the activity of the inhibitor (to slow down reaction) has not been affected after several concentration increases. At 25% concentration the rate of reaction without Copper Sulphate is 20ml/min while with the inhibitor it is around 4ml/min. At 50% concentration the rate of reaction without Copper Sulphate is 42ml/min while with the inhibitor it is 8ml/min. So you can see that increasing the substrate concentration has affected the activity of the inhibitor.
We did not record the amount of oxygen produced until the end of the reaction, we only recorded oxygen produced until we reached a set time of 3 minutes. So the rate of reaction might have increased towards the end. That would have meant that Copper Sulphate is a competitive inhibitor.
Summary of Results
I know from the results that Copper Sulphate is an inhibitor. It slows down the reaction between the enzyme and the substrate by separating them and preventing them to join and form an enzyme-substrate complex. It does this by bonding to the active site of the enzyme so that the substrate cannot, hence slowing down the rate of reaction.
However, as of yet I don’t know what type of inhibitor Copper Sulphate is. If it is a non-competitive enzyme, as I suspect, it will bind onto the active site of the catalase preventing it to join will the hydrogen peroxide molecules. The Copper Sulphate will change the shape of the active site of the catalase. The Hydrogen Peroxide molecules will then fail to bond with the active site.
If Copper Sulphate is a competitive inhibitor it will compete with the substrate molecule for the active site of the catalase. So if the concentration of the substrate was increased it the Copper Sulphate will have less chance of getting to the active site of the catalase and that would increase the rate of reaction again.
Evaluation
The experimental procedure of my investigation was suitable as it allowed us to produce reliable and accurate results. For the experiment to be suitable we had to record the amount of oxygen at short identical intervals, however it was difficult to record the results exactly on time. This did not affect the results, as all of the readings were acceptable.
The method that we chose must be the correct method as we produced the results that we needed. However, the method did get a bit annoying at times. For example, refilling the measuring cylinder after each experiment. If we used the gas cylinder method it would have been much easier to reset the apparatus after each trial.
We did not have any anomalous results as all of the points were near or on the line of best fit. This also meant that the experiment was done accurately.
My plan for the method was followed very well. This allowed me to produce very accurate and reliable results. So that means that my method was very well suited to this experiment. The results were also accurate; as I made sure that the meniscus was at eye-level.
We did however have limitations in the method. We didn’t continue the experiment until the experiment until the reaction had ended. Next time we shouldn’t run the experiment for a set amount of time, we should only stop when the reaction itself has ended.
The measuring cylinder was too small. Some of the tests made the measuring cylinder fill up with oxygen before the three minutes were up. Next time we should use 200ml or even large measuring cylinder.
If possible we could have used more accurate and reliable equipment instead of the measuring cylinder. The unit marks on the measuring cylinder had a difference of 1ml. To increase the accuracy we could have used measuring cylinders that had unit marks every half a ml. Our experiment would have been more reliable if we used a smooth gas syringe. It a piece of equipment specifically designed for collecting gas and would have been very well suited to this experiment.
I don’t have any sources of error and my experiment was fairly reliable. This means that I have a valid conclusion that, Copper Sulphate is in a fact an inhibitor.