Enzyme inhibitors
Non-competitive inhibitors¹ change the shape of the active site, preventing it from binding with substrates. The inhibitor joins to the enzyme anywhere but the active site, this is how the shape of the active site changes. Therefore the enzyme reaction is stopped.
However, if the inhibitors are competitive² for an active site by increasing the substrate concentration, the substrate can bind with an active site, like it normally does.
Non-competitive inhibitor¹
Competitive inhibitor²
Substrate graph- own source image¹- Google image- ‘non-competitive inhibitor’; image²- ‘competitive inhibitor’
Preliminary
In my preliminary experiment, I took the hydrogen peroxide in a conical flask. I cut a piece of potato and placed it in 15cm³ of hydrogen peroxide. Fizzing occurred and a gas was given off. I tested the gas using a lighted splint and found that the gas given off was oxygen.
I then cut 2 smaller pieces of potato from the same length I used before, and placed it in another 15cm³ of hydrogen peroxide. Doing this I found that there was more gas given off. I continued this with 4 pieces, 12 and then 20. For each experiment I stopped the timer. The results I got from the experiment are:-
Problem
The surface area of the potato can be changed by cutting it. Firstly, cut the potato into half, then into quarters etc. I will use the potato, firstly with 1 piece, then 4, then 8, then 12 and finally 16.
During the experiment there are factors that need to be controlled. This includes the temperature and the concentration and the volume of the hydrogen peroxide. If the temperature is not constant then the experiment would give erroneous results. If there is a higher temperature than the energy given to the enzymes would increase, which then increases the rate of reaction, changing the reliability of the results. If there is low temperature, then the reaction would slow down as the enzyme would have less energy. If the concentration of the hydrogen peroxide is less or more from the accurate lengths, there will be a big difference in the outcome of the results. If there is more of the concentration than the reaction will take place more quickly, and if there is less than the reaction would be slow.
Safety
For the experiment, the safety needed is particularly important when handling the hydrogen peroxide. This solution can cause damage to the skin as it is toxic, so wear gloves and wear goggles when pouring the solution. In addition, if there are spillages they should be cleared straight away, because it could cause damage to skin/clothing. White tile is also needed to cut the potato on. If the potato was cut on the table, the knife will leave marks on the table and if the potato was cut while held by the hand, there is a likely chance of cutting the finger.
Apparatus
Prediction
I predict that as the surface area of the potato increases or as more pieces of potato is being added the rate of reaction will also increase. Another way to describe this is that as the enzyme concentration increases, the initial rate of reaction also increases. If there are more enzymes, then there are more chances of it colliding and joining with substrates. If there are more enzymes added, then there will less substrate left, once all the active sites are used. The graph on the next page shows that at the beginning there is a faster rate of reaction (shown by the steepness of the line) due to the adding of substrate concentration. But as more substrate is being added, eventually the rate of reaction becomes constant.
In the experiment, to prove my prediction I will cut up potatoes into pieces, first one whole piece, then take another potato (cut into the same length), cut it into two. Continue doing this until there at least 20 pieces cut from one long piece of potato. I will then take different boiling tubes and mix the potatoes with the solution of hydrogen peroxide.
The outcome of this experiment is a reaction with bubbles and the gas oxygen being given off. The gas will be measured using a gas syringe.
Refer to page 2 for details about this graph
Method
- Collect all the apparatus and set it up to begin the experiment
- Wear safety goggles as hydrogen peroxide is toxic and use gloves when handling the hydrogen peroxide
- Fill a conical flask with hydrogen peroxide, enough for 15cm³ to be taken each time
- Connect a gas syringe with a wire to a cork. Make sure that the size of the cork is just enough to close the boiling tube tightly and use vaseline to prevent gas from escaping.
- Set up the clamp stand and add boss head to it, then attach the gas syringe to the boss head.
- Cut a potato using a borer to have uniform lengths and place them on a white tile.
- Take three equal lengths of the potato and by using a ruler make sure that the lengths of the potato are all equal.
- The purpose for the three pieces is that the experiment can be repeated three times using the same size of potato from the same potato to avoid variations.
- Measure 15cm³ of hydrogen peroxide using a measuring cylinder. Check the measurement from eye level, while the cylinder is placed on the table.
- Pour the hydrogen peroxide solution into the boiling tube carefully.
- Take a piece of one of the long length of potato and put it into the solution.
- As soon as the potato is placed into the solution, close the boiling tube with the cork immediately and start the timer.
- Record the volume of oxygen given off starting at 0 seconds and every 20 seconds for 3 minutes
- When you have recorded the measurements, take the cork out of the boiling tube, and pour the solution into the sink and throw the potato, as it is no longer needed.
- Wash the boiling tube, so that is it clear enough to pour another 15cm³ of hydrogen peroxide.
- Once you have cleaned the boiling tube, reset the gas syringe to zero.
- Measure another 15cm³ of hydrogen peroxide, and repeat the whole experiment twice again using the same sized potato.
- Repeat the experiment but varying the surface area by cutting it into halves, the quarters; then into eights and finally to 16 pieces by using a knife and measuring the length using a ruler.
- Replicate this experiment twice again for each variable so that each variable will have the results of three experiments.
- Measure the room temperature during every reading during the experiment using a thermometer to make sure there are no fluctuations.
Drawing of the experiment
Implementation
The bold numbers in the table are anomalies; therefore it will not be calculated when working out the average
- Used 15cm³ of hydrogen peroxide
- Total size of potato:-
Length- 4 cm, diameter- 15mm
Table of average time
Table for the rate
Diameter- 1.5cm; length 4cm
Area of length (πd x length)= π(1.5) x 4
= 18.8 cm²
Working out the rate of reaction
Rate of reaction= 2πr²
r= 0.75mm
Trends in the graph
By the results and the graph the experiment has proved that my prediction is correct. In the prediction I stated that as the surface area of the potato increases the rate of reaction will also increase. Graph 1 shows that as the surface area of the potato increased the volume of oxygen gas given off also increases.
The beginning of all 5 lines is slightly steep, which shows the substrate at the start of the reaction. There are a higher number of substrates at the start to collide with enzyme molecules. Gradually, the curve becomes less steep and flattens. This shows that all of the enzyme molecules have joined with substrate and the products have been made; after this the reaction has stopped. The products that were made were the oxygen given off (collected in the gas syringe) and water. The top line (16 pieces) is higher and steeper than the other lines because there is more surface area exposed to the hydrogen peroxide. This can also be defined by saying there are more active sites available in which a substrate can bind with. The bottom line (1 piece) is lower and less steep compared to the other graphs. This is because there is less surface area exposed to the hydrogen peroxide and therefore a slower rate of reaction.
In the graph, there were anomalies which I will describe in the evaluation.
As the graphs were not constant I am going to draw another graph with the rate against the surface area. This line, which is the rate against surface area, will be straight, I have stated in the prediction.
Evaluation
My method was a fair test, which enable me to carry out the investigation more easily and almost accurately. I monitored the room temperature for every testing using a thermometer, to observe whether there are any changes in the environment. If there were any changes in the temperature the test will give me unreliable results. I used a gas syringe to collect the oxygen given off, because it is more accurate and reliable. It had a measurement to the nearest 0.5cm³, which was the reason the experiment gave accurate results. In every test I used 15cm³ of hydrogen peroxide to make the test fairer. Any changes in the concentration of the hydrogen peroxide would cause changes in the results. I used a measuring cylinder because the range is to the nearest 1cm³, which made it easy to work out the total volume of hydrogen peroxide. Finally, I used the same potato for each testing because it would have the same enzyme type.
Anomalies in the graph
In my graph of averages, there were many anomalies which occurred. One of the reasons for this could be the change in the room temperature. There was a slight change in the room temperature; it decreased from 24º- 22º. Due to this change, the rate of reaction slowed down. Therefore on the average graphs, there were anomalies below the line. For example, there were 5 anomalies below the line for 2 pieces of potatoes (one after 60 seconds, then 80, then 100, 140 and after 160 seconds). In all 5 lines, the anomalies were below the line which showed that there was only decrease in the room temperature. Another possible cause of the anomalies could be inaccuracy in the concentration of hydrogen peroxide. As the anomalies are below the lines, this could lead to the conclusion that the concentration of hydrogen peroxide is less than the amount that was required. There was only one anomaly which was higher than the line of 16 pieces at 20 seconds. A possible reason could be that the gas syringe was not set to 0 at the beginning of the test.
From the table above the factor that affected the experiment most is cutting the potato. Using the knife and measuring the size with the ruler will not always give the accurate answer. This is one of the main factors which cause an inaccuracy on the results table. This factor will lead onto ‘overlapping potatoes’. When cutting the potato and putting it into the boiling tube, the potatoes will overlap each other as there is not a lot of space in the boiling tube. Therefore, not all of the surface area is exposed to the hydrogen peroxide solution, causing inaccuracy in the results. Another factor which leads to major errors in the results is the temperature. When there was an increase in the temperature the rate of reaction increase and therefore there was increase in the volume of oxygen given off. When the temperature decreased the rate of reaction slowed down and the volume of oxygen given off became less. Also reading the measurements on the gas syringe would be inaccurate. This is because the readings have the measurements of up to 0.5 cm³. By using a longer and thinner gas syringe, to the nearest 0.2cm³, you can get more accurate answers. Finally, the least important I mentioned is the hydrogen peroxide not covering the potato completely. Not all of the surface area of the potatoes is exposed to the hydrogen peroxide.
Accuracy
Some of the apparatus I used it measured accurately. One of which is the diameter of the potato. When using a cork borer, for each potato piece the diameter is going to be the same. Also, the measuring cylinder is measured to the nearest 0.2cm³ and the gas syringe was to the nearest 0.5cm³
Validity of Conclusion
If I could do the experiment again, by changing or controlling some of the factors, I can get better results. One of the things I can do is to work out what causes changes in the temperature, and work out a solution which will control that factor. Also, if I had more time I can cut the potato in an accurate length and to the same angle.
