Each type of enzyme acts on a specific type of enzyme that the enzyme is fit to do as the shape of its active site has a specific shape that only allows that one type of substrate to fit in. The enzyme may catalyse a reaction causing the substrate molecule to split (2 or more) as shown in the diagram. Alternatively catalysing may cause a joining of two molecules. After this process the molecules leave the unchanged enzyme leaving it for another substrate molecule to go and bond onto it.
There may be one specific substrate that fits into a specific enzymes active site but there is a possibility for another molecule very similar to the enzyme’s substrate which can also fit into the active site. This inhibits the enzyme’s function. This causes competition between the substrate and the enzyme to inhibit the enzyme. If there aren’t many Inhibitors present then the binding of substrate to enzymes won’t be affected but if the substrate was low in concentration compared to the inhibitor then the less likely the substrates will occupy the enzyme’s active site. This is known as competitive inhibition.
In other cases an inhibitor can occupy an enzyme’s site permanently and therefore will not allow a substrate to bind onto the active site. This is known as non-competitive inhibition and is irreversible. Another form of non-competitive inhibition is when a bacteria can bind somewhere else on the enzyme rather than on the active site. This may result in the enzyme becoming mishapened and disrupts the enzyme’s arrangement of hydrogen bonds and hydrophobic interactions.
Using this simple table you can decipher whether or not if an inhibitor is a competitive or non-competitive inhibitor.
Source for background knowledge: Biology 1 (Cambridge, endorsed by OCR).
Preliminary Work
Before I began my actual investigation I had to decipher what apparatus I would need and what the best method of getting a fair test would be. There was an option of using a glass syringe to collect the gas given off or to use a measuring cylinder in a bucket of water. I also had to comprehend how much carrot gratings and hydrogen peroxide I would require.
The method I chose to test out first was the one with the measuring cylinder as I had the notion that not much gas would be given off.
I set up my apparatus as shown in the diagram:
After deciphering how much hydrogen peroxide (ml) and carrot grating (weighed in grams) I would need I carried out the experiment. At first the reaction went hastily so I tried different amounts of carrot grating and hydrogen peroxide and the results turned out to be similar to the first test. There was clearly too much hydrogen peroxide and carrot gratings as substrate molecules were binding onto the enzyme at a quick rate because a lot of oxygen was being given off and also some of the hydrogen peroxide made its way into the bung. After some deliberation I decided to opt for the glass syringe method as it seemed to be more appropriate. So again like before I set up the equipment as shown in the diagram on the following page:
When carrying out the experiment using this method I found that now that there was a larger area for the oxygen to go into, there wasn’t much gas being collected into the glass syringe so this time I had to use more carrot gratings and hydrogen peroxide. I then, after a couple of tries to get an adequate amount of both the hydrogen peroxide and carrot gratings, determined how much of both the carrot gratings and hydrogen peroxide I needed. I found that using 5ml hydrogen peroxide to each gram of carrot gratings would give me a fairly good test. I went with using 4g of carrot gratings and 20ml hydrogen peroxide. This was going to be used as my control for the investigation. I will later add copper sulphate, which acts as an inhibitor.
Also I found that I was using 1% copper sulphate solutions so I decided to use different concentrations in my investigation. I chose to use these concentrations to test inhibition: 1%, 0.8%, 0.6%, 0.4%, 0.2% and 0% as the control (with the addition of water).
Finally I then decided to use 15 second time intervals to take readings on the glass syringe. I will keep doing this for approximately 3 and a half minutes.
I plan to repeat the test for each different concentrations of copper sulphate as this will increase the reliability of my work.
Apparatus
After finding out what method I was going to use I then began to consider about what apparatus I would require and came up with the following list:
- Carrot gratings (4g per test) – Acts as enzyme in investigation.
- Hydrogen peroxide (5ml per gram) – Acts as substrate in investigation.
- Copper sulphate (1% - 0%) – Acts as inhibitor in investigation. I will use different concentration to test inhibition.
- 2 Measuring cylinders – to measure the amount of hydrogen peroxide and copper sulphate
- Petri dish – to hold 4g carrot gratings in ready for using in the investigation for each test.
- Weighing pan – to weigh carrot gratings (4g approx)
- Glass syringe – will collect gas given off by the reaction between the enzyme and substrate (carrot gratings and hydrogen peroxide).
- Clamp stand – to hold glass syringe to stop it from rolling around and breaking.
- 2 Beakers – to hold hydrogen peroxide and copper sulphate close to apparatus.
- Test tube – To put carrot gratings, hydrogen peroxide and copper sulphate in for reaction to take place.
- Bung and rubber tube – Bung will go over test tube and the rubber tube will be connected to the glass syringe where oxygen gas given off will be collected.
- Test tube rack – simply to keep test tube in one place and stable so it doesn’t go anywhere and end up breaking.
- Stopwatch – to know when to take readings from the glass syringe (15 second intervals).
- Water – to dilute copper sulphate
Risk assessment
As usual the lab is a place of work and working with chemicals can be harmful. I will make sure of the following to reduce the risk of an accident:
- Wear goggles – keep eyes from being damaged
- Wear lab coat – any staining chemicals and corrosive chemicals will go onto lab coat and not on clothes.
- Stay near apparatus – make sure that nothing wrong happens during the investigation or nothing breaks which will put others at risk.
- Put bags on side benches and stools out of the way – stop people from tripping over.
There may be other risks to take on board and I will try to make sure that I follow the rules of working in a lab.
Variables
There are a few variables in this investigation that I have to take account for. There are things where I will be changing concentrations or amounts of and things where I will be changing nothing at all and keeping it constant throughout the investigation. My variables are water, weight of carrot gratings, hydrogen peroxide, copper sulphate and time intervals.
The variables I will be changing are water and copper sulphate. Both will change according to what concentration of solution is needed, for example for a 0.8% concentrated solution I could use 8ml 1% copper sulphate and 2ml water.
The variables which I will be keeping the same are hydrogen peroxide, time intervals and weight of carrot gratings. The carrot gratings will be 4g per test and I will take a reading off the glass syringe every 15 seconds. I will be keeping the amount of hydrogen peroxide the same (20ml).
Method
Firstly I will collect my apparatus and set it up as follows (similar to diagram in preliminary work section):
These are the steps I will take in doing my investigation:
- Weigh grated carrot – 4g per test done.
- Measure the amount of hydrogen peroxide needed, e.g. if testing a 0.8% concentration of copper sulphate I will need to dilute the 1% copper sulphate with water the add it to 10ml of hydrogen peroxide with 4g carrot grating in.
- Get required amount of copper sulphate depending on concentration. I will be doing the following concentrations: 1%, 0.8%, 0.6%, 0.4%, 0.2% and 0% which is the control (20ml of hydrogen peroxide). To do the 0.6%, for example, I would dilute 6ml of 1% copper sulphate with 4ml water and so on.
- I will then put in the hydrogen peroxide and the copper sulphate solutions into a test tube.
- Then I will put in the carrot gratings and quickly put the bung over the test tube which is connected to the glass syringe.
- I will then take a reading every 15 seconds for 3 and a half minutes. This is so that there is enough time for the reaction to take place and for it to slow down as more and more enzymes are inhibited or reacted with the substrate.
- I will then repeat this for using different concentrations of copper sulphate to test inhibition.
- I will also try to repeat the steps another 2 times for each concentration for reliability of results if I can.
Results
These are the results I came out with:
As you can see I didn’t repeat any tests for the different concentrations as there were some difficulties (mentioned in evaluation).
Conclusion
To begin with I can by just looking at just my results table that my prediction was correct. It’s easy to see that as the concentration of copper sulphate (inhibitor) the reaction between the carrot gratings and hydrogen peroxide slows down. This is even shown on my graphs I have done, there is a general trend shown. This happens because, as mentioned in my background knowledge in detail, when there aren’t many inhibitors present the reaction between the enzyme and substrate isn’t affected much as there are more substrate molecules than inhibitors. But as the concentration of the copper sulphate increases the reaction between the substrate and enzymes slows down as more inhibitor molecules are present which makes it so there is competition between the inhibitor and substrate to get to an enzymes active site. This means that the inhibitor is a competitive inhibitor because as you increase the amount of inhibitors the competition increases as more inhibitors can bind onto an active site.
There is a general trend shown on my graphs, the reaction begins quickly but as more and more substrate molecules or inhibitors bind onto an enzyme the reaction slows down which makes it harder inhibition to take place, this is why the line on my graph begins to level off.
Overall there is a pattern shown that the higher concentration of inhibitors makes the reaction slow between the enzymes and substrates.
Evaluation
There are quite a few things that could be improved and could be taken account for and they are:
- Anomalous results – the rate of reaction may have been affected in that the bung wasn’t put over the test tube quick enough.
- Some misreadings may have occurred; this is due to the available apparatus. The investigation could be done with more accurate apparatus.
- The anomalous results on my initial rate of reaction graph may be my error of reading the gradients wrong on the other graphs and if done correctly the graph may show a pattern.
Things I have to account for if the investigation was to be repeated:
- Make sure I know how to do initial rate of reaction graphs because I did the one in my investigation on what I thought was correct.
- More sources for information relating to investigation giving a wider and more understanding of the investigation.
- If possible use more accurate apparatus to stop some human errors.
Accuracy of results:
I think that my results were accurate but not very. They showed a pattern or trend that told me that as the copper sulphate concentration increased the reaction (release of oxygen gas) became slower due to inhibition taking place.
The accuracy of the apparatus is ok but I think that even more accurate apparatus could have been used to get correct amount of liquids or solutions. The gas syringe readings were pretty accurate but there were times when a decision had to be made about the results because the reading may have been between points, e.g. in between 12ml and 13ml.
The results aren’t very reliable as I didn’t repeat any of the tests. I only have one set of results which makes the results unreliable as I don’t know if I would get different values which are completely different to the original ones and have large differences.
